Biomarkers in Systemic Juvenile Idiopathic Arthritis, Macrophage Activation Syndrome and Their Importance in COVID Era

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This review identifies current biomarkers to help distinguish Systemic Juvenile Idiopathic Arthritis (sJIA) and Macrophage Activation Syndrome (sJIA-MAS) from similar conditions, crucial for correct diagnosis and targeted therapy initiation.
Biomarkers play a key role due to the non-specific clinical signs and symptoms of sJIA at onset, such as fever, headache, rash, or arthralgia, highlighting the need for accessible and sensitive biomarkers for accurate recognition.
sJIA and sJIA-MAS share characteristics with cytokine storm syndromes (CSSs) in the COVID era, necessitating careful biomarker selection to improve the comprehension of sJIA and CSS pathophysiology for homogeneous patient classification.

Citation: Ailioaie, L.M.; Ailioaie, C.;
Litscher, G. Biomarkers in Systemic
Juvenile Idiopathic Arthritis,
Macrophage Activation Syndrome and
Their Importance in COVID Era. Int. J.
Mol. Sci. 2022, 23, 12757. https://
doi.org/10.3390/ijms232112757
Academic Editor: Hyoun-Ah Kim
Received: 13 September 2022
Accepted: 19 October 2022
Published: 22 October 2022
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International Journal of
Molecular Sciences
Review
Biomarkers in Systemic Juvenile Idiopathic Arthritis, Macrophage
Activation Syndrome and Their Importance in COVID Era
Laura Marinela Ailioaie 1, Constantin Ailioaie 1 and Gerhard Litscher 2,*
1 Department of Medical Physics, Alexandru Ioan Cuza University, 11 Carol I Boulevard, 700506 Iasi, Romania
2 Research Unit of Biomedical Engineering in Anesthesia and Intensive Care Medicine, Research Unit for
Complementary and Integrative Laser Medicine, Traditional Chinese Medicine (TCM) Research Center Graz,
Department of Anesthesiology and Intensive Care Medicine, Medical University of Graz,
Auenbruggerplatz 39, 8036 Graz, Austria
* Correspondence: gerhard.litscher@medunigraz.at; Tel.: +43-316-385-83907
Abstract: Systemic juvenile idiopathic arthritis (sJIA) and its complication, macrophage activation
syndrome (sJIA-MAS), are rare but sometimes very serious or even critical diseases of childhood
that can occasionally be characterized by nonspecific clinical signs and symptoms at onset—such
as non-remitting high fever, headache, rash, or arthralgia—and are biologically accompanied by
an increase in acute-phase reactants. For a correct positive diagnosis, it is necessary to rule out
bacterial or viral infections, neoplasia, and other immune-mediated inflammatory diseases. Delays in
diagnosis will result in late initiation of targeted therapy. A set of biomarkers is useful to distinguish
sJIA or sJIA-MAS from similar clinical entities, especially when arthritis is absent. Biomarkers should
be accessible to many patients, with convenient production and acquisition prices for pediatric
medical laboratories, as well as being easy to determine, having high sensitivity and specificity,
and correlating with pathophysiological disease pathways. The aim of this review was to identify
the newest and most powerful biomarkers and their synergistic interaction for easy and accurate
recognition of sJIA and sJIA-MAS, so as to immediately guide clinicians in correct diagnosis and
in predicting disease outcomes, the response to treatment, and the risk of relapses. Biomarkers
constitute an exciting field of research, especially due to the heterogeneous nature of cytokine storm
syndromes (CSSs) in the COVID era. They must be selected with utmost care—a fact supported
by the increasingly improved genetic and pathophysiological comprehension of sJIA, but also of
CSS—so that new classification systems may soon be developed to define homogeneous groups of
patients, although each with a distinct disease.
Keywords: chemokines; cytokines; cytokine storm syndrome; ferritin; hub genes; innate immunity;
natural killer; neutrophils; platelets; S100 proteins; biomarkers; systemic juvenile idiopathic arthritis;
COVID-19
1. Introduction
Juvenile idiopathic arthritis (JIA) is a set of chronic childhood disorders that usually
cause inflammation and pain, swelling, and stiffness in the joints of children under the
age of 16 years, which may last from months to the entire life of the patient, with a risk
of locomotor and ocular disabilities. The unparalleled therapeutic advances since the
beginning of this century have turned the remission or minimization of disease activity
from an ideal goal to an achievable one for most JIA patients, with fewer joint and extraarticular injuries and reduced incidence of long-term physical disabilities. However, with
all current therapeutic means of conventional synthetic disease-modifying anti-rheumatic
drugs (csDMARDs) and/or biological DMARDs (bDMARDs), long-term remission is
difficult to achieve for some patients [1–3].
Nevertheless, there are scientific reports indicating that after 5 years from diagnosis,
less than half of JIA patients who received contemporary medication went into remission.
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At the same time, prolonged anti-inflammatory treatment is associated with high financial
costs, high stress for the patient and their family, and potential side effects [4,5].
Current diagnostic guidelines for patients with JIA are not currently accompanied by
recommendations for the use of predictive molecular biomarkers for relapses, complications, disease progression, and prognosis.
Action plans based on predictive biomarkers are promising tools in decision-making
as to whether to withdraw or discontinue medication if the inflammation is no longer
severe enough to show clear clinical symptoms, and could be very beneficial to patients [6].
Systemic juvenile idiopathic arthritis (sJIA) is a subtype manifested by arthritis or
arthralgia, in addition to extra-articular symptoms, which include the following: high
daily and prolonged fever of unknown origin, persisting for at least 3 consecutive days
and recurring for at least 2 weeks; transient rash; and at least one of the following clinical
characteristics: enlarged lymph nodes; hepatosplenomegaly; or pleural, pericardial, or
peritoneal effusion, sometimes associated with pneumonitis and/or signs of central nervous
system damage. sJIA, previously thought to be an autoimmune disease, is now more
commonly classified as an autoinflammatory disease, because the genetic abnormalities
of the major histocompatibility complex (MHC) and the innate immune system (such as
natural killer (NK) cells, polymorphonuclear neutrophils (PMNs), and macrophages (MP))—
with the participation of interleukins 1 (IL-1), 6 (IL-6), and 18 (IL-18)—have been shown to
be involved. The prognosis and evolution of the disease are unpredictable, ranging from a
mild single-phase evolution to a severe chronic cyclic polyarticular disease complicated by
extra-articular manifestations that can induce significant morbidity and mortality [7–11].
sJIA has many characteristics of an autoinflammatory pathology, associated with an
increased synthesis of pro-inflammatory interleukins such as IL-1, IL-6, and IL-18, as well
as S100 proteins; at the same time, in its evolution, sJIA can be complicated by macrophage
activation syndrome (MAS). This term is used by rheumatologists to describe the aggressive, life-threatening complications in patients with sJIA or AOSD, and is phenotypically
associated with secondary hemophagocytic lymphohistiocytosis (sec-HLH), in which IFN-γ
and CXCL9 play essential roles as biomarkers. MAS is manifested by high fever, cytopenia,
cytokine storm (CS), severe liver dysfunction, consumptive coagulopathy, seizures, coma,
and even death. During the evolution of sJIA as active disease, MAS may occur (sJIA-MAS)
in 7–17% of cases, while subclinical cases are even more frequent, found in 30–40% of
patients. The criteria for the classification of sJIA-MAS were advanced in 2016 [12–16].
Biomarkers have a high potential as indispensable tools for pediatric rheumatologists
in the early differentiation of cytokine storm syndromes (CSSs).
Currently, due to the diversity of cases and the increasingly complicated pathologies
that appear after infection with SARS-CoV-2—such as multisystem inflammatory syndrome
in children (MIS-C)—the need to refine and maximize these biomarkers has become more
and more important, in order to move from the laboratory to the patient’s bedside as soon
as possible, to apply the results of research in as many clinical laboratories as possible for
sampling the analyses. Based on this approach, a successful positive diagnosis and the
early initiation of the appropriate individualized treatment for the respective patient could
save their life in the event of CSS-aggravating circumstances.
The aim of this review was to identify the newest and most powerful biomarkers
and their synergistic interactions for easy and accurate recognition of sJIA, as well as its
complication sJIA-MAS, to immediately guide clinicians in the correct diagnosis and in
predicting the outcomes of the disease, the response to treatment, and the risk of relapses.
Biomarkers constitute an exciting field of research, especially due to the heterogeneous
nature of CSS in the COVID-19 era. They must be selected with the utmost care—a fact
supported by the increasingly improved genetic and pathophysiological comprehension
of sJIA, but also of CSS—so that new classification systems can be developed to define
homogeneous groups of patients, each with a distinct disease.
The discovery of new biomarkers with the best possible diagnostic/prognostic value
could increase the speed of reaching the final goal, i.e., the introduction of new targeted

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therapies and personalized interventions, improving disease remission percentages while
minimizing all undesirable pathophysiological effects of the disease.
2. Biomarkers for Diagnosis, Treatment, and Disease Activity in sJIA Complicated
by MAS
A positive diagnosis of sJIA is initially very difficult, especially when arthritis is absent
as a defining symptom. In this case, it is necessary to extend the investigations by making a
differential diagnosis with different entities that are caused by viral or bacterial infections,
neoplasms, and other inflammatory diseases. It is precisely these issues that generate the
urgent need to find biomarkers to help exclude the various other entities for the early
diagnosis of sJIA. These biomarkers must have high sensitivity and specificity, be easy to
determine at low cost, and be found in the pathogenic mechanisms of the disease.
The absence of arthritis at the beginning of the disease is an impediment for clinicians,
as there are currently no reliable diagnostic criteria, and this could lead to a delay in early
positive diagnosis, which may have consequences for the initiation of targeted treatment,
as well as patients’ outcomes and prognosis [17,18].
The discovery of the roles of IL-1 and IL-6 in the pathogenesis of sJIA had the effect of
introducing an extremely effective therapy—especially if administered at the onset of the
disease—by targeting these cytokines. However, 20–30% of cases do not respond to initial
therapy with anti-IL-1 or anti-IL-6 biological agents, leading to the urgent need to discover
new biomarkers for diagnosis, develop a more effective management plan, and accurately
predict the course and prognosis of the disease [7,19].
2.1. Genetic Biomarkers
Perception of the genetic hazard posed by sJIA—which, albeit less frequent than
the other subtypes of JIA and with an unusual clinical expression at the first visit, can
evolve in a severe manner that is sometimes complicated by MAS—has initiated important
research to determine the genetic susceptibility to sJIA, as a challenge in a neglected
area of investigation, so as to be able to more easily identify genetic biomarkers for early
diagnosis, establishing a targeted treatment from the beginning and accurately predicting
the long-term response to therapy for these patients.
For 136 sJIA patients at onset, De Benedetti et al. assessed the association of the
MIF-173 polymorphism with patients’ long-term response, observed for more than 5 years.
The levels of MIF-173 single-nucleotide G-to-C polymorphism of the macrophage migration
inhibitory factor (MIF) gene in the serum and synovial fluid of patients with sJIA were
studied in correlation with the required amount of glucocorticoids and the outcomes in
sJIA patients. Subjects carrying the MIF-173*C allele had significantly higher serum and
synovial concentrations of MIF than those with the GG genotype; therefore, the former
needed a much longer treatment with glucocorticoids than in MIF-173 GG homozygous
patients and had a shorter duration of clinical response to intraarticular injections. Finally,
the number of joints with active arthritis, the questionnaires scores, and the number of joints
with limited range of motion were significantly higher in patients carrying the MIF-173*C
allele, so this could predict poor outcomes of sJIA at onset [20].
Starting from the fact that the polymorphisms in the interferon regulatory factor 5 (IRF5)
gene have been linked with susceptibility to autoimmune diseases (ADs), Yanagimachi et al.
intended to evaluate the associations of IRF5 gene polymorphisms with the vulnerability to
sJIA and MAS. Using TaqMan assays, the authors genotyped three IRF5 single-nucleotide
polymorphisms (rs729302, rs2004640, and rs2280714) in 81 patients with sJIA (33 with MAS,
48 without) and 190 controls. A significant association of the rs2004640 T allele with the
vulnerability to MAS was highlighted. The IRF5 haplotype (rs729302 A, rs2004640 T, and
rs2280714 T), known to be an increased risk factor in systemic lupus erythematosus (SLE),
was also significantly associated with susceptibility to MAS in sJIA. IRF5 gene polymorphism influences susceptibility to MAS in sJIA, so IRF5 could trigger MAS in sJIA [21].

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At the level of the innate immune system, inflammasomes receive and respond to the
signals of the pathological factors, as well to the injuries produced through the activation
of caspase-1, the secretion of IL-1β and IL-18, and pyroptosis of macrophages; as a result,
Canna et al. found a de novo missense mutation, c.1009A > T, p.Thr337Ser, in the nucleotidebinding domain of the NLRC4 inflammasome that causes early-onset relapsing fever and
MAS. Operational analyses shed light on the spontaneous generation of the inflammasome,
along with the production of IL-1β- and IL-18-dependent cytokines, revealing a new
monoallelic inflammasome defect that broadens the field of monogenic ADs—including
MAS—and opens the possibility of new targets in therapy [22].
Ombrello et al. investigated whether genetic variation in the MHC locus on chromosome 6 influenced the susceptibility to the onset of sJIA in a group of 982 children with sJIA
and 8010 healthy control subjects from nine countries. The authors used a meta-analysis
of directly observed and imputed single-nucleotide polymorphism (SNP) genotypes and
imputed classical HLA types, finding that the strongest SNP associated with sJIA was
rs151043342 from the HLA class II region. Meta-analysis of the imputed classical HLAtype associations showed that HLA-DRB1*11 (conferring at least a twofold increase in
disease risk in each population studied) and its defining amino acid residue—glutamate
58—were strongly associated with sJIA, as were the HLA-DRB1*11 haplotypes, i.e., HLADQA1*05–HLA-DQB1*03. This research demonstrated the relationship between the HLA
class II region and sJIA, as well as the participation of adaptive immune molecules in the
etiopathogenesis of sJIA [23].
In the etiopathogenesis of JIA, the genetic predisposition mainly concerns HLA class
II molecules (e.g., HLA-DRB1, HLA-DPB1), although HLA class I molecules and non-HLA
genes have also been found to be involved. A meta-analysis of selected papers for the
evaluation of the HLA-DRB1 genetic background of patients with JIA compared to healthy
controls, conducted by De Silvestri et al., confirmed that HLA-DRB1*04 has a predisposing
role in sJIA [24].
Arthur et al., with the largest cohort at the time, investigated whether susceptibility
loci previously determined through studies on candidate genes in JIA had a provable
association with sJIA. The authors studied SNPs for risk loci in sJIA by association in nine
populations, which incorporated 770 patients with sJIA and 6947 controls. The results
demonstrated that among the 26 previously reported SNPs there was only one notable association: the promoter region of IL1RN. The IL1RN gene encodes the interleukin-1 receptor
antagonist (IL-1Ra). sJIA-associated SNPs are well-correlated with IL1RN expression in
lymphoblastoid cell lines (LCLs), with an inverse correlation between sJIA risk and IL1RN
expression. The high expression of homozygous IL1RN alleles is strongly correlated with
the lack of response to anakinra therapy, paving the way for personalized treatments in sJIA
guided by homozygous high-expression alleles with the attributes of ideal biomarkers [25].
Recent advances in “omics” technologies (e.g., genomics, transcriptomics, proteomics,
and metabolomics) have made innovative contributions to human genome sequencing,
bioinformatics, and analytical tools, significantly facilitating knowledge of the molecular
mechanisms of JIA and providing the potential to discover new therapeutic agents. In a
review on the multi-omics architecture of JIA, Hou et al. identified several human leukocyte
antigen (HLA) alleles (including both HLA class I and class II genes) and 23 non-HLA
genetic loci in association with different JIA subtypes. HLA class II alleles are remarkably
associated with sJIA. HLA-DRB1*11 has a strong association, while the SNP rs151043342
has the strongest association with sJIA [26].
Zhou et al. attempted to identify key modules and hub genes in the pathophysiology
of sJIA, using two datasets on GSE7753 and GSE13501, to accomplish a weighted gene
co-expression network analysis (WGCNA). The authors identified a total of 5414 genes
for WGCNA, of which the highly correlated ones were divided into 17 modules, where
the red module had the highest correlation with sJIA, while the green–yellow one was not
related to sJIA. The red module was enriched in the activation of immune responses, infection, nucleosomes, and erythrocyte differentiation, including the ALAS2, AHSP, TRIM10,

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TRIM58, and KLF1 genes, while the green–yellow module was enriched in inflammation
and immune responses, such as the KLRB1, KLRF1, CD160, and KIR genes. These results
may deepen the comprehension of the pathophysiology of sJIA, and hub genes may play a
role as biomarkers and future treatment targets for sJIA [27].
Due to the fact that sJIA is a severe disease, with an as-yet undetermined etiology and
atypical manifestations at the onset, it requires diagnosis and effective treatment from the
beginning. In this regard, Zhang et al. aimed to identify diagnostic biomarkers, immune
cells, and pathways involved in the pathogenesis of sJIA, as well as possible treatment
targets. The authors checked the gene expression profiles of GSE17590, GSE80060, and
GSE112057 from the Gene Expression Omnibus (GEO) database to identify differentially
expressed genes (DEGs) between sJIA and healthy controls; they analyzed whole-blood
samples from 10 subjects, including 5 patients with sJIA (1 very recently diagnosed and 4
already undergoing treatment) and 5 healthy controls perfectly matched for age, race, and
sex. The results identified 73 common DEGs, indicating the enrichment of neutrophil and
platelet functions as well as the MAPK pathway in the pathogenesis of sJIA. Six hub genes
were identified, three of which had high diagnostic sensitivity and specificity; ARG1 and
PGLYRP1 were validated by quantitative reverse-transcription polymerase chain reaction
(qRT-PCR) and microarray data from the GSE8361 dataset as potential markers for the early
diagnosis of JIA, revealing new pathways and molecular targets for sJIA. Further studies
with larger sample sizes and in-depth analysis are needed to confirm these results [28].
Ren et al. analyzed 70 active sJIA patients compared with 55 healthy controls to
highlight differentially expressed genes (DEGs), hub gene sets, and patterns of immune
system cell organization. The results showed 118 DEGs, of which 94 were upregulated
(most: CD177, OLFM4, ARHGEF12, MMP8, PLOD2, CEACAM6, and CEACAM8) and 24
were downregulated (most: TCL1A, ALOX15 and HLA-DQB). A hub gene set of eight
upregulated genes (ARG1, DEFA4, HP, MMP8, MMP9, MPO, OLFM4, and PGLYRP1) was
found, and none were downregulated. Functional enrichment analysis clearly showed
that these eight hub genes were mainly connected with the complex tasks of neutrophils,
playing a decisive role in the pathogenesis of sJIA, while macrophages, CD8+ T cells, and
naïve B cells were relevant drivers of disease progression. TPM2 and GZMB were identified
as possible markers of positive short-term response to canakinumab treatment [29].
A summary of the abovementioned published studies on genetic biomarkers is shown
in Table 1.
Table 1. Genetic biomarkers proposed for the diagnosis, activity, and prognosis of sJIA and MAS.
Reference/Year Predictive Effects in sJIA and MAS
[20]. De Benedetti, F.; Meazza, C.; Vivarelli, M.; Rossi, F.; Pistorio, A.;
Lamb, R.; Lunt, M.; Thomson, W.; Ravelli, A.; Donn, R.; et al. Functional
and prognostic relevance of the −173 polymorphism of the macrophage
migration inhibitory factor gene in systemic-onset juvenile idiopathic
arthritis. Arthritis Rheum. 2003, 48, 1398–407.
https://doi.org/10.1002/art.10882
Patients with sJIA onset with MIF-173*C-allele-associated persistent
active disease have higher concentrations of MIF in serum and synovial
fluid, as well as a weaker response to glucocorticoid therapy. The
MIF-173*C-positive allele in patients with sJIA is a predictor of poor
steroid therapy outcomes.
[21]. Yanagimachi, M.; Naruto, T.; Miyamae, T.; Hara, T.; Kikuchi, M.;
Hara, R.; Imagawa, T.; Mori, M.; Sato, H.; Goto, H.; Yokota, S.
Association of IRF5 polymorphisms with susceptibility to macrophage
activation syndrome in patients with juvenile idiopathic arthritis. J.
Rheumatol. 2011, 38, 769–774. https://doi.org/10.3899/jrheum.100655
IRF5 gene polymorphism influences the susceptibility to MAS in sJIA,
so IRF5 could trigger MAS in sJIA.
[22]. Canna, S.W.; de Jesus, A.A.; Gouni, S.; Brooks, S.R.; Marrero, B.;
Liu, Y.; DiMattia, M.A.; Zaal, K.J.; Sanchez, G.A.; Kim, H.; et al. An
activating NLRC4 inflammasome mutation causes autoinflammation
with recurrent macrophage activation syndrome. Nat. Genet. 2014, 46,
1140–1146. https://doi.org/10.1038/ng.3089
An activating de novo mutation (c.1009A > T, p.Thr337Ser) in the
nucleotide-binding domain of inflammasome component NLRC4
generates early-onset repeated episodes of fever, as well as MAS.

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Table 1. Cont.
Reference/Year Predictive Effects in sJIA and MAS
[23]. Ombrello, M.J.; Remmers, E.F.; Tachmazidou, I.; Grom, A.; Foell,
D.; Haas, J.P.; Martini, A.; Gattorno, M.; Özen, S.; Prahalad, S. et al.
International Childhood Arthritis Genetics (INCHARGE) Consortium.
HLA-DRB1*11 and variants of the MHC class II locus are strong risk
factors for systemic juvenile idiopathic arthritis. Proc. Natl. Acad. Sci.
USA 2015, 112, 15970–15975. https://doi.org/10.1073/pnas.1520779112
HLA-DRB1*11 and its defining amino acid residue, glutamate 58, were
strongly associated with sJIA, as well as the HLA-DRB1*11 haplotypes
HLA-DQA1*05–HLA-DQB1*03.
[24]. De Silvestri, A.; Capittini, C.; Poddighe, D.; Marseglia, G.L.;
Mascaretti, L.; Bevilacqua, E.; Scotti, V.; Rebuffi, C.; Pasi, A.; Martinetti,
M.; et al. HLA-DRB1 alleles and juvenile idiopathic arthritis: Diagnostic
clues emerging from a meta-analysis. Autoimmun. Rev. 2017, 16,
1230–1236. https://doi.org/10.1016/j.autrev.2017.10.007
HLA-DRB1*04 was confirmed to play a predisposing role in sJIA.
[25]. Arthur, V.L.; Shuldiner, E.; Remmers, E.F.; Hinks, A.; Grom, A.A.;
Foell, D.; Martini, A.; Gattorno, M.; Özen, S.; Prahalad, S.; et al. IL1RN
Variation Influences Both Disease Susceptibility and Response to
Recombinant Human Interleukin-1 Receptor Antagonist Therapy in
Systemic Juvenile Idiopathic Arthritis. Arthritis Rheumatol. 2018, 70,
1319–1330. https://doi.org/10.1002/art.40498
sJIA-associated SNPs were interdependent with IL1RN expression in
LCLs, with an inverse correlation between sJIA risk and IL1RN
expression. Homozygous high-expression alleles predicted the failure of
anakinra therapy, making them ideal candidate biomarkers to manage
the treatment of sJIA.
[26]. Hou, X.; Qu, H.; Zhang, S.; Qi, X.; Hakonarson, H.; Xia, Q.; Li, J.
The Multi-Omics Architecture of Juvenile Idiopathic Arthritis. Cells
2020, 9, 2301. https://doi.org/10.3390/cells9102301
Several HLA alleles (both HLA class I and class II genes) and
23 non-HLA genetic loci were associated with different JIA subtypes.
HLA class II alleles were significantly associated with sJIA.
HLA-DRB1*11 had a strong association and SNP rs151043342 had the
strongest association with sJIA.
Nuclear factor interleukin-3-regulated gene (NFIL3) mutations drove
elevated IL-1β, sensitizing patients to arthritis and rewiring the innate
immune system for overproduction of IL-1.
The laccase (multicopper oxidoreductase) domain-containing 1 (LACC1)
gene regulated inflammation (i.e., macrophages, dendritic cells, and
TNF-α levels).
The Unc-13 homolog D (UNC13D) gene disrupted transcription factor
binding.
[27]. Zhou, M.; Guo, R.; Wang, Y.F.; Yang, W.; Li, R.; Lu, L. Application
of Weighted Gene Coexpression Network Analysis to Identify Key
Modules and Hub Genes in Systemic Juvenile Idiopathic Arthritis.
BioMed Res. Int. 2021, 2021, 957569.
https://doi.org/10.1155/2021/9957569
The ALAS2, AHSP, TRIM10, TRIM58, and KLF1 genes were associated
with sJIA, but also with the differentiation of red blood cells, which may
be related to anemia or MAS. The KLRB1, KLRF1, CD160 and KIR genes
might have a relationship with the dysregulation of NK cell function in
sJIA. These results may deepen the comprehension of the
pathophysiology of sJIA, and the hub genes may play the part of future
biomarkers and treatment targets for sJIA.
[28]. Zhang, M.; Dai, R.; Zhao, Q.; Zhou, L.; An, Y.; Tang, X.; Zhao, X.
Identification of Key Biomarkers and Immune Infiltration in Systemic
Juvenile Idiopathic Arthritis by Integrated Bioinformatic Analysis. Front.
Mol. Biosci. 2021, 8, 681526. https://doi.org/10.3389/fmolb.2021.681526
The hub genes ARG1 and PGLYRP1 are potential biomarkers for the
early diagnosis of sJIA.
[29]. Ren, Y.; Labinsky, H.; Palmowski, A.; Bäcker, H.; Müller, M.;
Kienzle, A. Altered molecular pathways and prognostic markers in
active systemic juvenile idiopathic arthritis: integrated bioinformatic
analysis. Bosn. J. Basic Med. Sci. 2022, 22, 247–260.
https://doi.org/10.17305/bjbms.2021.6016
A total of 118 DEGs were identified, of which 94 were upregulated
(most: CD177, OLFM4, ARHGEF12, MMP8, PLOD2, CEACAM6, and
CEACAM8) and 24 were downregulated (most: TCL1A, ALOX15, and
HLA-DQB) in active sJIA. A hub gene set of eight upregulated genes
(ARG1, DEFA4, HP, MMP8, MMP9, MPO, OLFM4, and PGLYRP1) and
none downregulated was also identified.
TPM2 and GZMB were identified as possible markers of positive
short-term responses to canakinumab.
2.2. Specific Cellular Biomarkers in sJIA and MAS
2.2.1. NK Cell Dysfunction in sJIA and MAS
Natural killer cells are large granular lymphocytes with cytotoxic activity that are
crucial to the innate immune system, being a member of the rapidly growing innate
lymphoid cell (ILC) family, which constitutes 5–20% of all circulating lymphocytes in
humans and mediates antiviral and antitumor responses [30].
Innate lymphoid cells (ILCs) are the latest family of innate immune cells to be discovered from common lymphoid progenitors (CLPs). ILCs secrete signaling molecules
and regulate both innate and adaptive immune cells. ILCs are mainly tissue-resident cells
found in both lymphoid and non-lymphoid tissues, and not often in the blood [31].

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ILCs are deeply embedded lymphocytes in tissues that do not express specialized
antigen receptors for T and B cells. The study of ILCs has opened new perspectives for
understanding immune regulation and maintenance of tissue homeostasis by the immune
system. ILCs produce cytokines for various immune pathways expressed in relation to
commensal agents and pathogens in mucosal barriers, while also stimulating adaptive
immunity and adjusting inflammation in tissues, contributing to metabolic homeostasis,
morphogenesis, remodeling, and tissue regeneration in direct association with the nervous
system [32].
NK cells originate from hematopoietic stem cells that have recently been classified
as ILCs in group 1 (ILC1s), comprising conventional NK (cNK) cells and subsets of “unconventional” NK cells. ILC1s are found in tissues and are also known as tissue-resident
NK (trNK) cells, which are found in abundance in organs such as the liver, lungs, and
uterus, whereas cNK cells circulate through the blood vessels. Although cNKs and ILC1s
share interferon-gamma (IFN-γ) release when activated, these two categories of cells
developed from different progenitors and have diverse tissue distributions and specific
functions [32,33].
cNK cells have a much higher cytotoxic capacity and higher perforin expression
compared to ILC1s, which have a low cytotoxic capacity but can release large amounts
of IFN-γ, tumor necrosis factor alpha (TNF-α), and granulocyte–macrophage colonystimulating factor (GM-CSF) [34,35].
NK cells’ numbers, growth, multiplication, and function are modulated and monitored
by two pro-inflammatory cytokines: IL-6 and IL-18 [36,37].
Experimental studies showed that in mice with MAS, IL-6 levels were elevated in
plasma and led to decreased perforin levels and granzyme B expression in NK cells, but
without degranulation—phenomena that improved after targeted treatment with anti-IL-6.
Similar attributes were found in patients with sJIA. Thus, IL-6 increases the inflammatory
response and disrupts the function of NK cells, favoring the occurrence of MAS [38,39].
Among the first studies performed to inspect the functions of NK cells and their
cytotoxicity in patients with sJIA complicated by MAS, Grom et al. used flow cytometry to investigate the expression of perforin in NK cells (CD56+/TCRαβ−), NK T cells
(CD56+/TCRαβ+), and CD8+ cells. NK cells’ cytotoxic activity was quantified as measured
after co-incubation with an NK-sensitive K562 cell line. The authors found two important
categories of immunological dysfunctions: Some patients had reduced NK cell counts
and activity, but slightly increased perforin expression in CD8+ and CD56+ cytotoxic cells.
Other patients with sJIA and MAS had reduced NK activities associated with decreased
perforin expression in all cytotoxic cells—a pattern similar to the perforin deficiency found
in patients with familial hemophagocytic lymphohistiocytosis (fHLH). The authors’ conclusion was that in sJIA-MAS there is an NK cell dysfunction analogous to that in fHLH,
which requires further investigation [40].
MAS has been described in association with many rheumatic disorders, but most
often with sJIA. Clinical manifestations in MAS are similar to those of hemophagocytic
lymphohistiocytosis (HLH)—a genetic disease accompanied by NK cell dysfunction. Villanueva et al. explored global NK cell dysfunction in JIA by studying peripheral blood
mononuclear cells (PBMCs) from 40 patients with pauciarticular (n = 4), polyarticular
(n = 16), and systemic (n = 20) subtypes of JIA. NK cells’ cytolytic function was determined
after co-incubation of PBMCs with the NK-sensitive K562 cell line. NK cells (CD56+/T
cell receptor [TCR]-αβ-), NK T cells (CD56+/TCR-αβ+), and CD8+ T cells were studied
for perforin and granzyme B expression by flow cytometry. The results showed that NK
cells’ cytolytic activity was much lower in sJIA patients than in other JIA groups or controls.
The authors concluded that the subgroup of JIA patients who had not yet experienced any
MAS event had low NK function and a lack of circulating CD56bright cells, similar to defects
found in patients with MAS and HLH. These aspects were particularly prevalent in the
sJIA subtype, which is known to be strongly associated with MAS [41].

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(NF-κB) p65 in NK cells after being boosted by in vitro recombinant IL-18 (rIL-18) in
31 patients with sJIA and 6 healthy controls. The authors investigated the connections
between clinical symptoms, serum IL-18 levels, and the strength of phosphorylation in NK
cells. Patients were distributed in conformity with their disease activity into four groups:
systemic characteristics (n = 8), chronic arthritis (n = 7), remission on medication (n = 10),
and remission off medication (n = 6). The results showed that the phosphorylation of
MAPK p38 and NF-κB p65 was more intense in the group of healthy patients than in those
in remission without therapy, in remission on drugs, or with chronic arthritis, while there
was a complete defect in phosphorylation in those with systemic manifestations. The serum
concentration of IL-18 was highest in the group with systemic manifestations, followed by
those with chronic arthritis, those in remission on medication, those in remission without
medication and, finally, the healthy group. The authors concluded that the increased levels
of IL-18 induce the phosphorylation defects of MAPK and NF-κB in NK cells, and that the
inappropriate signaling of IL-18 in NK cells is directly related to the activity of sJIA [45].
2.2.2. Macrophages in sJIA and MAS
Macrophages are known as cells equipped for the detection, phagocytosis, and destruction of bacterial agents, as well as other microorganisms that are harmful to the body.
They are antigen-presenting cells to T lymphocytes and produce molecules (cytokines)
that develop the inflammatory process by activating other cells. Two types of activated
macrophages are known: classically activated macrophages, i.e., macrophages that encourage inflammation (M1); and macrophages alternately activated by phages, i.e., macrophages
that decrease inflammation and encourage tissue repair (M2). The activated or polarized
type (M1) secrete interleukins with pro-inflammatory action (e.g., TNF-α, IL-1 beta, IL-6,
IL-12), chemokine (C-X-C motif) ligands 9 and 10 (CXCL9, CXCL10, etc.), and low amounts
of IL-10, which promotes and modulates the immune response of Th1 helper cells (CD4+
cells or CD4-positive cells), i.e., Th1 lymphocytes [46].
Feng et al. investigated the roles played by different macrophage subtypes in the
evolutionary stages of sJIA. The study included 22 children diagnosed with sJIA, divided
into two groups: 12 patients with active disease, recently diagnosed and still without
treatment, as well as 10 with inactive disease, who were monitored for two years. The
control was a group of 10 children with orthostatic proteinuria. For all subjects, peripheral
blood was analyzed by flow cytometry to highlight the following macrophage subtypes:
M1 (CD14+CD86+CD80+), M2a (CD14+CD206+CD301+), M2b (CD14+CD206+CD86+), and
M2c (CD14+CD206+CD163+), as well as IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17, INF-α,
INF-γ, and TNF-α. The authors found the M1 phenotype marker CD80, M2 phenotype
marker CD163, and CD301 to be strongly expressed in children with active sJIA—a group
in which most macrophages were M1 and M2a. In contrast, in the group with inactive
disease, M2 was polarized predominantly into M2b and M2c. IL-6 had high levels in the
group with active disease, while in the other group IL-4, IL-10, and IL-17 had high values.
The authors concluded that M1 induces inflammation in active sJIA, while M2a almost
simultaneously triggers inflammation inhibition, whereas M2b and M2c play major roles
in inhibiting inflammation in inactive disease [47].
Macrophage polarization is a biological phenomenon through which these cells are
able to follow certain functional programs as they are guided by signals from the microenvironment. Polarization can be caused by chemical stimulation or by stiffness substrates on
which the macrophage is grown [48].
M2 macrophages can be polarized by the action of the cytokines IL-10, IL-13, and IL-4
which, thus activated, can inhibit inflammation and promote tissue repair, angiogenesis,
and fibrosis. Recent studies have shown that M2 macrophages can be divided into different
subtypes, such as M2a, M2b, M2c, and M2d, which have activities specific to the stage
of the disease. M2a macrophages are polarized by IL-4 and IL-13, lowering the levels of
IL-10 and transforming growth factor beta (TGF-β), participating in tissue regeneration
and incorporation of pro-inflammatory molecules, thereby preventing the inflammatory

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found to have non-inflammatory disorders. The results indicated a higher percentage
of CD16+CD62Llo neutrophils compared to controls. This subset of neutrophils was not
observed in patients with CID or in those with active arthritis but without systemic features. CD16+CD62Llo neutrophils from sJIA-MAS patients showed increased nuclear
hypersegmentation compared with CD16+CD62L+ neutrophils. Serum concentrations of
S100A8/A9 and S100A12 were correlated very strongly with peripheral blood neutrophil
counts. Whole-transcriptome analysis of highly purified neutrophils from children with active sJIA identified 214 differentially expressed genes (DEGs) compared with controls. In all
samples, regardless of disease activity, increased inflammatory gene expression was found,
including inflammasome components and S100A8. The authors’ conclusion was that in the
case of activation of neutrophils in both active and inactive sJIA, a pro-inflammatory gene
expression signature can be identified, indicating prolonged innate immune activation [60].
Known to be key phagocytic cells belonging to the innate immune system, neutrophils
are endowed with the potential for oxidative and non-oxidative defense against pathogens.
Their complex decision-shaping function, by which they instruct other leukocytes to adjust
innate and adaptive immune responses, has recently been discovered. Under physiological
conditions, they have a short life and are permanently set free by the bone marrow, starting
from undifferentiated stem cells and going through multiple stages until maturation, in
order to finally develop accelerated and intense immune responses. The human body
needs many such cells during systemic inflammation in order to generate emergency granulopoiesis, but in excess they can cause tissue damage, as seen in sJIA. It is fundamental
to better understand the activity and the types of neutrophils in the joints and blood of
patients with sJIA. NET activation and release (NETosis) is a unique form of cell death in
which neutrophils remove decondensed deoxyribonucleic acid (DNA), histones, cytokines,
and granular proteins, and has never been studied directly in patients with sJIA. However,
high serum histone levels in active sJIA, compared to inactive or healthy control patients,
suggest increased NETosis, being well-correlated with sJIA disease activity. Increased
high-mobility group box-1 (HMGB1) levels in patients with sJIA are associated with intensified NETosis, forming a positive feedback loop. Serum HMGB1 concentrations of NET
molecules (including DNA without cells, myeloperoxidase (MPO)–DNA complexes, and
α-defensin) were found to be increased in patients with adult-onset Still’s disease (AOSD)
and sJIA, compared to healthy controls. NET molecules induce the release of HMGB1 and
the production of IL-1β by monocytes, and as ligands of TLR4 they could be involved in
activating the TLR signaling pathway in systemic arthritis [61–63].
In a very recent review, Kim et al. described the roles of neutrophils and the formation
of NETs, which participate in the aggravation of inflammation in the pathogenesis of sJIA
and AOSD. Activation of neutrophils and their surface receptors (including Fc receptors)
by pathogen-associated molecular pattern (PAMP) or damage-associated molecular pattern
(DAMP) molecules leads to their displacement in inflamed tissues and, subsequently, to
the release of cytokines and chemokines with pro-inflammatory potential. Activated neutrophils and expelled mediators form a positive feedback loop that increases the recruitment
of new neutrophils and exacerbates the inflammatory phenomena, on which the pathogenic
mechanisms of AOSD and sJIA can be focused. Simultaneously, the activated neutrophils
produce and release NETs and participate in the activation of macrophages in the inflamed
area. At the same time, the levels of other DAMPs (e.g., HMGB-1, S100 proteins, LL-37,
a human antimicrobial peptide from the cathelicidin group, and myeloperoxidase–DNA
(MPO–DNA) complexes) increase in patients’ blood, accelerating systemic inflammation
even more intensively. These novel results deepen the comprehension of the pathophysiological mechanisms involved in sJIA and AOSD, and can accelerate the patenting of
neutrophil-associated biomarkers to confirm the diagnosis and likely course, as well as the
development of original drugs to address the neutrophils and NETs [64].

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2.2.4. Platelets
In 2016, a group of experts analyzed 115 sJIA-MAS patient profiles, starting from
laboratory data before the onset of MAS, followed by those corresponding to the onset
of MAS, with a particular focus on the changes between the two key moments. They
selected and ordered the most important laboratory tests with the most dramatic changes
for the timely diagnosis of sJIA-MAS, the relevance of which was debated and cataloged
at an expert consensus conference. At the end of the analysis, platelet count, ferritin level,
and aspartate aminotransferase (AST), followed by white blood cell count, neutrophil
count, fibrinogen (Fg), and erythrocyte sedimentation rate (ESR), were declared to be the
most valuable laboratory parameters with the most important changes at the onset of
sJIA-MAS [65].
In a recent review, Crayne et al. noted that laboratory features of MAS include
pancytopenia, hyperferritinemia, fibrinolytic coagulopathy, and liver dysfunction [66].
At the 2020 American College of Rheumatology Congress, De Matteis et al. highlighted
platelet count and ferritin as two significant parameters with high specificity and sensibility,
respectively, to diagnose MAS-sJIA. The authors stipulated that these biomarkers could be
practical prognosticators of the clinical outcomes and the effectiveness of the administered
therapy [67].
The important roles of platelets and the MAPK pathway in the pathogenesis of sJIA
provide a new perspective for exploring potential molecular targets for the treatment of
sJIA, as Zhang et al. revealed by integrated bioinformatics analysis [28].
2.2.5. Complete Blood Cell Count
Currently, there is a consensus that sJIA and AOSD are similar diseases but emerge at
different ages [68].
Children with sJIA are particularly susceptible to developing MAS. In a multinational,
multicenter study that included 95 pediatric rheumatologists and hemato-oncologists
from 33 countries, data collected from 362 patients were retrospectively investigated and
included 22% with MAS at the onset of sJIA. Clinical manifestations in order of frequency
were as follows: fever (96%), hepatomegaly (70%), splenomegaly (58%), central nervous
system dysfunction (35%), and hemorrhages (20%). In terms of laboratory data, platelet
counts and levels of liver transaminases, ferritin, LDH, triglycerides, and D-dimers were
the only laboratory biomarkers showing a change of more than 50% between the preMAS consultation and the onset of MAS. Macrophagic hemophagocytosis was detected by
bone marrow aspiration in 60% of patients. Approximately one-third of patients required
admission to the intensive care unit (ICU), and the mortality rate was 8%. MAS remains a
grave complication, as an important percentage of children require mandatory admission
to an ICU or will die [69].
A group of experts statistically analyzed 982 potential criteria for 428 profiles of
patients with MAS-sJIA and obtained the best results for 37 of them, analyzed in comparison
with eight criteria from the literature. With a consensus of 82%, they obtained the final MAS
classification criteria, validated with a sensitivity of 0.73 and a specificity of 0.99. These
points of reference could contribute to a better insight into MAS in sJIA, to the discovery
of new active and safe therapies, and to a better selection of patients for future clinical
studies [15].
The fact that systemic inflammation in sJIA includes elevated ESR, C-reactive protein
(CRP), white blood cell count, platelet count, ferritin, transaminases, aldolases, and Ddimers may help define disease activity; according to Lee et al., primary care must closely
monitor patients with sJIA to detect early complications and unexpected drug reactions [7].
Some of the studies on cellular biomarkers discussed above are presented in Table 2.

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Table 2. Cont.
Reference/Year Cellular Biomarkers Predictive Effects in sJIA and MAS
[59]. Ter Haar, N.M.; Tak, T.; Mokry, M.; Scholman
R.C.; Meerding, J.M.; de Jagerz, W.; Verwoerd, A.;
Foell, D.; Vogl, T.; Roth, J. et al. Reversal of
Sepsis-Like Features of Neutrophils by
Interleukin-1 Blockade in Patients with
Systemic-Onset Juvenile Idiopathic Arthritis.
Arthritis Rheumatol. 2018, 70, 943–956.
https://doi.org/10.1002/art.40442
Neutrophils
Neutrophils play an important role in sJIA,
especially in the early inflammatory phase of the
disease, and the neutrophil counts and the
inflammatory activity in sJIA are both susceptible
to IL-1 blockade.
[60]. Brown, R.A., Henderlight, M., Do, T., Yasin,
S., Grom, A.A., DeLay, M., Thornton, S., Schulert,
G.S. Neutrophils from Children with Systemic
Juvenile Idiopathic Arthritis Exhibit Persistent
Proinflammatory Activation Despite
Long-Standing Clinically Inactive Disease. Front.
Immunol. 2018, 9, 2995.
https://doi.org/10.3389/fimmu.2018.02995
The results showed a higher percentage of the
CD16+CD62Llo neutrophil population in active
sJIA than in the control group. Serum
concentrations of S100 alarm proteins (i.e.,
S100A8/A9 and S100A12) were strongly correlated
with the number of neutrophils in the peripheral
blood, and analysis of the entire neutrophil
transcriptome identified 214 differentially
expressed (SD) genes compared to the neutrophils
from controls. Neutrophil activation in patients
with active sJIA or CID, together with the
pro-inflammatory gene expression blueprint,
demonstrated prolonged activation of innate
immunity.
[64]. Kim, J.-W.; Ahn, M.-H.; Jung, J.-Y.; Suh, C.-H.;
Kim, H.-A. An Update on the Pathogenic Role of
Neutrophils in Systemic Juvenile Idiopathic
Arthritis and Adult-Onset Still’s Disease. Int. J.
Mol. Sci. 2021, 22, 13038.
https://doi.org/10.3390/ijms222313038
Neutrophils, including NETs, play a pivotal role in
the pathogenesis of sJIA and AOSD as future
clinical biomarkers for monitoring and prognosis.
[65]. Ravelli, A.; Minoia, F.; Davì, S.; Horne, A.;
Bovis, F.; Pistorio, A.; Aricò, M.; Avcin, T.; Behrens,
E.M.; De Benedetti, F.; et al. Expert consensus on
dynamics of laboratory tests for diagnosis of
macrophage activation syndrome complicating
systemic juvenile idiopathic arthritis. RMD Open
2016, 2, e000161.
https://doi.org/10.1136/rmdopen-2015-000161
Platelets
Platelet counts, followed by ferritin levels, AST,
white blood cell counts, neutrophil counts,
fibrinogen, and ESR, were selected for the early
diagnosis of MAS in sJIA.
[67]. De Matteis, A.; Pires Marafon, D.; Caiello, I.;
Pardeo, M.; Marucci, G.; Sacco, E.; Prencipe, G.; De
Benedetti, F.; Bracaglia, C. Traditional Laboratory
Parameters and New Biomarkers in Macrophage
Activation Syndrome and Secondary
Hemophagocytic Lymphohistiocytosis [abstract].
Arthritis Rheumatol. 2020, 72 (Suppl. S4).
Available online:
https://acrabstracts.org/abstract/traditionallaboratory-parameters-and-new-biomarkers-inmacrophage-activation-syndrome-andsecondary-hemophagocytic-lymphohistiocytosis/
(accessed on 21 August 2022).
Platelet count and ferritin are two relevant
laboratory parameters, with high specificity and
sensitivity, respectively, for the diagnosis of MAS
in the context of sJIA.

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Table 2. Cont.
Reference/Year Cellular Biomarkers Predictive Effects in sJIA and MAS
[69]. Minoia, F.; Davì, S.; Horne, A.; Demirkaya, E.;
Bovis, F.; Li, C.; Lehmberg, K.; Weitzman, S.;
Insalaco, A.; Wouters, C.; et al. Clinical features,
treatment, and outcome of macrophage activation
syndrome complicating systemic juvenile
idiopathic arthritis: a multinational, multicenter
study of 362 patients. Arthritis Rheumatol. 2014, 66,
3160–3169. https://doi.org/10.1002/art.38802
Complete blood cell
count
Typical laboratory features of sJIA include
microcytic anemia; leukocytosis, thrombocytosis;
elevated immunoglobulins; elevated ESR, CRP,
and fibrinogen; and hypoalbuminemia.
[15]. Ravelli, A., Minoia, F.; Davì, S.; Horne, A.;
Bovis, F.; Pistorio, A.; Aricò, M.; Avcin, T.; Behrens,
E.M.; De Benedetti, F.; et al. 2016 Classification
Criteria for Macrophage Activation Syndrome
Complicating Systemic Juvenile Idiopathic
Arthritis: A European League Against
Rheumatism/American College of
Rheumatology/Paediatric Rheumatology
International Trials Organisation Collaborative
Initiative. Arthritis Rheumatol. 2016, 68, 566–576.
https://doi.org/10.1002/art.39332
MAS includes pancytopenia; increased levels of
ferritin, liver enzymes (e.g., aspartate and alanine
transaminases), triglycerides, and D-dimers; and
hypofibrinogenemia.
2.3. Cytokines, Chemokines, and Other Biomarkers in sJIA and MAS
2.3.1. IL-1
The role of innate immune system involvement and the aberrant responses in the
pathophysiology of sJIA have been documented in numerous clinical studies and publications [70].
The role of IL-1 in the pathogenesis of sJIA was first discovered by Virginia Pascual in
2005 [71]; since then, evidence has shown many other aspects of the involvement of this
cytokine. Additional evidence includes clinical observations after the administration of
biological agents blocking the IL-1 pathway via rIL-1Ra (anakinra) or prolonged blocking
of IL-1 by the biological agent canakinumab [71–75].
Ling et al. highlighted that the flare versus quiescent signature in sJIA attests to the
key role of IL-1 in the onset of the disease [76].
Several genetic alterations or mutations associated with dysregulated IL-1 activity
and autoinflammatory disorders have been identified, and these findings have led to the
successful use of IL-1 inhibitors in rheumatic diseases that were previously considered to
be untreatable [77].
In a recent review on the role of IL-1 in general pathology, Kaneko et al. identified
IL-1 as a key cytokine not only for cell-damage-related inflammation, but also for cell,
tissue, and organ homeostasis in terms of overall pathology; thus, IL-1 has become an
important molecular target for treating a wide range of diseases, such as autoinflammatory,
autoimmune, and infectious diseases, malignant tumors, etc. [78].
Particular attention should be devoted to patients with recent-onset sJIA or pediatric
Still’s disease—a syndrome with several clinical forms that may benefit from a treat-totarget strategy with a key role of IL-1 inhibition. Due to the heterogeneous and often
difficult-to-treat nature of sJIA, the experience of expert teams is recommended [79].
At the onset of sJIA, it has been demonstrated that an early and correct treatment in the
so-called “window of opportunity” by administering the use of IL-1 inhibitors—especially
in steroid-naïve patients—can enable the interruption of the pathogenic cycle of the disease
and induce a prompt and full remission. Ter Haar et al. conducted a prospective study of
42 patients diagnosed with sJIA at onset, but without an adequate response to non-steroidal
anti-inflammatory drugs (NSAIDs), compared with 12 patients who did not have arthritis
at onset, and both groups were followed for a mean period of 5.8 years. The first group
received rIL-1Ra as monotherapy, as a targeted treatment. The results showed that the

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disease generally became inactive after 33 days, and after one year 76% of the patients
had inactive disease, 52% of whom had not received any drug(s). It was observed that
there was a correlation between the increased number of neutrophils at the beginning,
the positive response after one month of treatment with rIL-1Ra, and the disappearance
of symptoms one year after the initiation of the study. The monitoring of patients over a
period of 5 years showed that 96% no longer had active disease, and 75% achieved inactive
disease status without any medication. Articular and extra-articular damages were found
in a small percentage (less than 5%) of cases, and only one-third of the patients required
the administration of glucocorticoids. The authors also noted the effectiveness of therapy
with rIL-1Ra in children with sJIA without arthritis at the onset. The use of rIL-1Ra as a
first-line, short-term monotherapy may be a valuable option to interrupt the pathogenic
cycle of sJIA at onset, in which IL-1 is the central component to be targeted [9].
Saccomanno et al. retrospectively analyzed the response to treatment with an IL-1
inhibitor (anakinra) for 62 patients diagnosed with sJIA over a 14-year period, including
demographic, clinical, and laboratory data, as well as previous or concomitant therapies
administered. The results of anakinra management were estimated by univariate and
multivariable statistical analyses after one year of treatment, outlining the clinical patterns
of patients with sJIA for whom IL-1 blockade may be a successful treatment. The authors
recommend future in-depth studies on the efficacy of early therapy with IL-1 inhibitors,
but also the detection of biomarkers that accurately predict the response to IL-1 or IL-6
antagonists [80].
Lainka et al. undertook a retrospective study on 202 patients diagnosed with sJIA and
included in the German AID registry from 17 centers, of which 111 were children treated
with IL-1 inhibitors, i.e., anakinra (ANA) (n = 84, 39 f) and/or canakinumab (CANA)
(n = 27, 15 f). In the first year of therapy, 75/111 (ANA 55, CANA 20) patients were
evaluated according to the Wallace criteria, and disease inactivation was achieved in 28/55
and 17/20, respectively, while remission over 6 months under medication was achieved
in 13/55 and 7/20 patients, respectively. The clinical response to biological medication
was maintained in most patients (ANA 54/80, CANA 20/27). The study acknowledged
positive clinical results with both IL-1 inhibitors (anakinra and canakinumab), which were
well-tolerated by patients and showed reasonable safety and efficiency when tested in a
true clinical framework [81].
2.3.2. IL-1β
Pro-inflammatory cytokines in the innate immune system—such as IL-1, IL-6, IL-18,
and TNF-α—are directly involved in the inflammatory process of sJIA. For example, IL1β—a pleiotropic pro-inflammatory cytokine—is excessively released by mononuclear
cells in the blood and adjusts not only its own transcription, but also that of IL-6. The
fundamental role of IL-1β in the pathogenesis of sJIA and the perpetuation of chronic
inflammation has been demonstrated by stopping its activity after the administration of
biological agents [82–86].
In recent decades, special efforts have been made to establish complex consensusbased plans for the early diagnosis and management of sJIA as an autoinflammatory
disease. These strategies also involve the implementation of expensive treatment methods
that directly target the main initiating and supporting cytokines (i.e., IL-1 and IL-6) of the
chronic inflammatory process [81,87].
It has already been shown that if the initial autoinflammation with the disturbance of
innate immunity is not stopped, the phenotype changes with the disruption of the elements
of adaptive immunity will have evolutionary consequences in the form of destructive
arthritis. If biological treatment is applied early—that is, when the “window of opportunity”
is open—and there is financial possibility of long-term continuation, patients with sJIA will
be more likely to be diagnosed with inactive disease and even go into remission.
It is believed that the mutation in the nucleotide-binding oligomerization domain-like
receptor pyrin domain-containing protein 3 (NLRP3) inflammasome leads to overexpres-

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$Int. J. Mol. Sci. 2022, 23, 12757 17 of 61 sion of the cytokine IL-1β. The NLRP3 gene codifies the multimeric protein complex cryopyrin, which monitors the activation of caspase-1 which, in turn, catalyzes the cleavage of pro-IL-1β into IL-1β [88]. Inflammasomes are cytosolic multiprotein oligomers responsible for the activation of inflammatory responses as important parts of the innate immune system. NLRP3 inflammasome activation during infections requires two signals: one priming signal, and one activation signal. Once assembled, the NLRP3 inflammasome activation leads to the auto-cleavage of pro-caspase-1, which mediates the proteolytic process of pro-IL-1β, pro-IL-18, and the propyroptotic factor gasdermin D [89,90]. Studies on the pathogenesis of sJIA have shown the biphasic nature of events—at the beginning of systemic manifestations, the elements of innate immunity participate with the key role of the cytokine IL-1β; and in the second phase, chronic arthritis interacts with the adaptive immunity through the cytokine IL-17A. On this basis, IL-1 blocking treatment was initiated in the early stages of sJIA [91]. Brunner et al. investigated the long-term benefits and safety of canakinumab and the predictability of responses in 123 patients with sJIA with or without fever (70 with fever, and 52 without fever) at the start of therapy. Only 84 subjects (68.3%) remained in the study to completion—a mean period of 1.8 years. Clinical–biological sJIA-ACR 50 (American College of Rheumatology score = at least 50% improvement in disease activity) responses occurring by day 15 were the most representative predictor of clinical remission, according to the JADAS (Juvenile Arthritis Disease Activity Score), as well as of steroid discontinuation. The biological agent, as a monoclonal antibody that inhibits IL-1β and can decrease the levels of IL-6 and the hepatic synthesis of CRP and Fg, rapidly and persistently improved the clinical condition of patients with active sJIA, regardless of whether or not they had fever at the initiation of therapy [92]. MAS, as a potentially fatal complication of sJIA, may not respond to conventional doses of biological cytokine inhibitors requiring dose escalation. It has been shown that when MAS-sJIA is triggered, it can be treated with anakinra—i.e., with IL-1Ra—and increasing the dose can be beneficial for the patient. Another IL-1 inhibitor, canakinumab—which is a monoclonal antibody of IL-1β—has been reported to effectively treat MAS-sJIA that is refractory to conventional treatment. Kostik et al. retrospectively analyzed the data from the electronic medical records of an academic center with respect to the use of canakinumab in eight children (five girls) diagnosed with MAS-sJIA, with an average age of 8.5 years, during the period 2011–2020. Among these patients, five children developed MAS at the onset of sJIA, while three developed it during the treatment with canakinumab. MAS remitted in all children included in the research, but when the dose of canakinumab was not sufficient, or when MAS developed during the canakinumab therapy, the dose of the administered drug was increased in the short term (2–3 times the normal dose), without observed side effects, attesting to the efficacy and safety of increased doses of canakinumab in MAS-sJIA, but posing the need for additional studies [93]. 2.3.3. IL-6 IL-6 plays a special role in autoinflammatory diseases, and especially in sJIA, where it contributes to fever; increased acute-phase reactants (e.g., ESR, CRP, alpha-1 and alpha-2 globulins, Fg, haptoglobin, etc.), serum amyloid A (SAA), white blood cells, and platelets; and decreased red blood cells with chronic anemia. Elevated serum IL-6 concentrations have been found in several inflammatory diseases, such as AOSD and sJIA. IL-6 acts on B and T lymphocytes, hematopoietic progenitor cells, megakaryocytes, macrophages, hepatocytes, and neuronal cells [94]. In early research on the involvement of IL-6 in the pathophysiological mechanisms of sJIA, de Benedetti et al. evaluated serum IL-6 levels in 25 patients with systemiconset juvenile arthritis using the B9 hybridoma cell line, finding significantly increased concentrations during active disease—correlated with the extent and severity of joint$

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sJIA. The authors’ results contribute to a deeper understanding of the immune mechanisms
in active sJIA, for new diagnostic and therapeutic plans of action [104].
2.3.4. IL-10
Interleukin 10 is a pleiotropic cytokine with various effects in immunoregulation
and inflammation, secreted by almost all cells participating in immune defense, such as
macrophages, mast cells, neutrophils, basophils, eosinophils, dendritic cells, B cells, and
the various subtypes of T cells [105].
The main functions of IL-10 are performed through autocrine and paracrine mechanisms, which primarily give it an anti-inflammatory, inhibitory, or autoregulatory role
through a negative feedback on inflammatory processes. IL-10 suppresses harmful inflammatory responses by preventing antigen presentation by dendritic cells and inhibiting
the stimulation and penetration of macrophages in the lesion area, with the subsidiary
consequence of reducing the expression of pro-inflammatory cytokines. Expanding the
research areas for new cell-based therapies that benefit from the knowledge of IL-10 signaling pathways could bring important benefits to patients with chronic inflammatory
phenomena and fibrotic consequences [106].
In a translational study using a comparative murine model of sJIA and samples from
patients diagnosed with sJIA, Imbrechts et al. investigated whether the pathophysiological mechanisms of sJIA could be caused by defects in IL-10—a cytokine known to
be crucial in controlling inflammation. The authors used a translational approach with
an sJIA-like murine model, compared with samples from patients with sJIA. Freund’s
complete adjuvant (CFA) containing heat-killed mycobacteria (1.5 mg/mL) was injected
into IFN-γ-deficient BALB/c mice, as the corresponding wild-type (WT) mice are known
to develop only a mild and transient inflammatory reaction. Diseased IFN-γ-deficient
mice showed defective IL-10 production in CD4+regulatory T cells, CD19+ B cells, and
CD3-CD122+CD49b+NK cells, with B cells as the main IL-10 generators. Neutralization of
IL-10 in WT mice generated a chronic immune inflammatory condition with an identical
clinical and hematological picture to sJIA. The authors found plasma levels of IL-10 to
be remarkably low compared to pro-inflammatory mediators in patients with sJIA, and
CD19+ B cells from these patients showed decreased production of IL-10 both ex vivo and
after stimulation in vitro. The authors concluded that neutralization of IL-10 in WT mice
by CFA transformed a transient inflammatory reaction into a chronic disease and found
cell-specific IL-10 imperfections in both mice and patients with sJIA, along with insufficient
production of IL-10 to counterbalance the pro-inflammatory cytokines, indicating that the
faulty production of IL-10 could contribute to the pathogenesis of sJIA [107].
Peng et al. investigated IL-10 concentrations in 21 patients with sJIA, compared with
35 patients with febrile illnesses suspected to be sJIA. The results showed that subjects with
confirmed sJIA had significantly higher levels of IL-10 compared to those with other febrile
illnesses; at the same time, serum concentrations of IL-10 were much higher in patients
with active sJIA compared to those with inactive sJIA, with a direct relationship with other
activity markers such as ESR, CRP, serum ferritin, and IL-6. Concomitantly, serum IL-10
levels at the time of diagnosis were observed to be much higher in sJIA patients with
a non-monocyclic course than in those with a monocyclic pattern. IL-10 concentrations
were more valuable in distinguishing monocyclic from non-monocyclic patterns in sJIA
outcomes, in comparison with CRP, ESR, serum ferritin, and IL-6. The study showed
that IL-10 levels were much higher in sJIA, leading to differentiation from other febrile
diseases and a correct diagnosis, and were associated with the type of activity and disease
progression. The authors proposed that serum IL-10 should be considered a reliable clinical
marker in the diagnosis and prognosis of sJIA [108].
2.3.5. IL-18
IL-18, which belongs to the IL-1 cytokine family, is initially inactive and is presented
as an integral membrane protein, and then under the action of caspase-1 it transforms into

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motion some inflammatory mediators (e.g., adhesion molecules, chemokines, and Fas
ligand (FasL)). IL-18, as a pleiotropic cytokine, plays an important role in various infectious,
metabolic, and inflammatory diseases. IL-18 flows in the range of tens of nanograms/mL.
Particularly high IL-18 concentrations in the sera of patients with active sJIA have been
reported in several studies and correlated with elevated serum ferritin levels, predictive of
MAS. sJIA and AOSD are characterized by high serum IL-18 concentrations and could be
treated by IL-18BP [114].
Kudela et al. provided additional evidence in a study of 30 adult patients diagnosed
with AOSD and 20 children diagnosed with sJIA with serum IL-18 levels up to 1000-fold
higher during active disease compared to other rheumatic subtypes, proposing IL-18 as a
biomarker for disease activity [115].
Yasin et al. investigated the total levels of IL-18, CXCL9, and S100 proteins in
40 patients with sJIA, comparing them between subjects to detect disease activity and
history of MAS. Total IL-18 was greatly increased in patients with active sJIA and remained persistently elevated even in the vast majority of patients with inactive disease.
Subjects with a history of MAS presented much higher concentrations of IL-18 compared
to those who had never had MAS. Total IL-18 could predict disease activity and history of
MAS. A moderate correlation was noted between IL-18 and CXCL9, as well as between
S100A8/A9 and S100A12, being more pronounced for ferritin and, generally, for those with
active disease. Elevated IL-18 may signal increased sJIA activity or the development of
MAS [116].
Krei et al. conducted a systemic review of a total of 14 studies that included all HLH
subtypes (and MAS subtypes) in both children and adults, investigating the role of IL-18
as a potential biomarker for the diagnosis and monitoring of HLH/MAS. Serum IL-18
was found to be elevated in both primary and secondary HLH (>1000 pg/mL), with a
significantly higher level in MAS (IL-18 > 10,000 pg/mL) compared to other inflammatory
conditions and healthy patients; therefore, serum IL-18 levels may differentiate between
HLH subtypes and other inflammatory conditions. The authors concluded that IL-18 is
a potential biomarker for HLH/MAS but is not currently part of the diagnostic criteria.
IL-18 may help to differentiate between HLH subtypes and other inflammatory conditions. The potential of IL-18 to distinguish MAS from sJIA is more ambiguous, as IL-18
levels > 100,000 pg/mL were described in sJIA patients both with and without MAS [117].
Mizuta et al. investigated the significance of serum IL-18 concentrations for the
diagnosis and prediction of sJIA’s progression; 116 patients with sJIA, 151 with other
diseases, and 20 healthy controls were studied. IL-18 was measured in 41 patients with sJIA
from the active period until remission. The minimum serum IL-18 concentration required
for distinction from other diseases was 4800 pg/mL. IL-18 was steadily decreased during
inactivity and remission in patients with monocyclic evolution. In contrast, in patients with
polycyclic evolution, serum IL-18 concentrations were increased during the active period
and normalized during the inactive phase. The cutoff value of serum IL-18 for remission
in sJIA was 595 pg/mL. The authors noted that serum IL-18 levels > 4800 pg/mL may be
useful in differentiating between sJIA and other autoimmune/autoinflammatory diseases;
additionally, serum IL-18 levels may be useful in predicting progression and remission in
sJIA [118].
Other recent studies have reported IL-18 as a predictive biomarker for the occurrence
of MAS, along with the correlation of its high serum levels with the clinical parameters of
MAS activity in patients treated with TCZ [103,119].
IL-18BP is abundant in the bloodstream under normal conditions and in many pathological disorders, stopping the negative systemic pro-inflammatory impact of IL-18. Severe
clinical manifestations of sJIA are dependent on the levels of free IL-18 being elevated in
the bloodstream, along with the imbalance between IL-18 and IL-18BP. The disequilibrium
of IL-18/IL-18BP in favor of free-circulating IL-18 is known for its important role in the
pathogenesis of sJIA, AOSD, and the severe complication of MAS. The use of recombinant

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$Int. J. Mol. Sci. 2022, 23, 12757 22 of 61 IL-18BP in patients with AOSD or sJIA with MAS has shown promising results, making free IL-18 a biomarker and a therapeutic target in these diseases [120]. Recent findings support a pattern in which patients with high serum IL-18 levels (associated with pro-inflammatory activated neutrophils in the bloodstream) but low/normal CXCL9 levels are likely to respond well to IL-1 blockade. However, patients with sJIA who have low IL-18 (i.e., weak neutrophil marks) and low CXCL9 levels are unlikely to respond to IL-1 blockade, while patients with high IL-18 but also high CXCL9—reflecting the activation of IFN-γ (i.e., IL-18/CXCL9 ratio remains lower)—have a high risk of both MAS and failure of therapy [121]. Several studies have indicated that CXCL9, IL-18, neopterin, and the soluble TNF receptor (sTNFR) type II (sTNFR-II) could be useful in predicting MAS in patients with active sJIA. IFN-γ and the IFN-induced mediators are elevated during active MAS, and high blood levels of CXCL9 are predictive of future MAS. However, recent research has shown the opposite, where most patients with sJIA, despite significant increases in CXCL9, never developed MAS; this can be explained by the multiplex platform used in the laboratory determinations. Therefore, given these uncertainties, the use of CXCL9 and IL-18 to predict the outcome of IL-1 blocking treatment requires further study. 2.3.6. IFN-γ IFN-γ is mainly regulated by NK cells and natural killer T (NKT) cells as a reaction of the innate immune system, and by CD4+Th1 and CD8+cytotoxic T lymphocyte (CTL) effector T cells after the antigen-specific (i.e., adaptive) immunity develops [122]. IFN-γ attaches to and stimulates the specific receptors of antigen-presenting histiocytes and dendritic cells in tissues, causing the synthesis and release of CXCL9 and CXCL10 chemokines, which bind CTLs in the peripheral blood; when activated, these CTLs move to the site of inflammation, where they release more CTL-derived IFN-γ which, in turn, increase the inflammatory response [123]. On the other hand, IFN-γ, as a pro-inflammatory cytokine, plays an important role in regulating hematopoietic stem cells (HSCs) in physiological conditions. IFN-γ actively plays a special pathogenic role in various diseases that harm hematopoiesis, including HLH, aplastic anemia, liver cirrhosis, etc. Increased serum IFN-γ levels have been shown to negatively influence HSC balance by overstimulating differentiation and self-renewal until exhaustion of HSCs [124]. Antigen-presenting cells activate T lymphocytes and NK cells, which secrete IFN-γ which, in turn, activates monocytes and macrophages. IFN-γ-activated M1-type macrophages secrete large amounts of pro-inflammatory cytokines, including IL-6, IL-12, and IL-23, as well as IP-10 chemokines and monokines that recruit polarized Th1 lymphocytes and NK cells. At the same time, the continuous stimulation produced by IFN-γ activates the macrophages that participate in the phenomenon of hemophagocytosis [14]. There are studies that show that IFN-γ plays a key role in triggering primary hemophagocytic lymphohistiocytosis (pHLH) and MAS, because serum levels of IFN-γ and of the IFNγ-induced chemokines are significantly elevated. Thus, the clinical manifestations of pHLH and MAS can be stopped by therapy with monoclonal antibodies directed against IFN-γ. The use of emapalumab—an anti-IFN-γ monoclonal antibody—for the treatment of patients with pHLH symptoms and disease that is refractory, recurrent, progressive, or intolerant to conventional therapy has been approved by the Food and Drug Administration (FDA) [125–127]. Put et al. undertook research that aimed to determine the role of IFN-γ in the pathogenesis of sJIA and HLH by discovering a profile of IFN-γ and its connection with other cytokines; 10 patients with active sJIA, 10 patients with inactive sJIA, and 5 patients with HLH—of whom 3 had associated sJIA-MAS—were included in the study, along with 16 healthy controls. The authors investigated the cytokines, the IFN-γ-induced genes, and the proteins from patients’ plasma and peripheral blood mononuclear cells (PBMCs), as well as the lymph node biopsies taken from a patient during two sJIA and MAS episodes. IFN-γ responses were also investigated in healthy donors’ PBMCs, primary fibroblasts,$

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of the study showed that the concentrations of CXCL9, CXCL10, and neopterin at T0
were much higher in patients with MAS and sec-HLH compared to those with sJIA, while
they were progressively reduced at T1 and normalized at T2. A more rapid decrease was
observed for CXCL9 than for neopterin, similar to the drop in routine laboratory data.
The study confirmed that platelet count and ferritin were the most distinctive laboratory
parameters with high specificity and sensitivity for sJIA-MAS. At the same time, elevated
LDH levels, which are not included in the 2016 classification criteria for sJIA-MAS, may be
useful in predicting the occurrence of MAS. The results revealed increased levels of IFNγ-related biomarkers in patients with MAS and sec-HLH, which could be useful together
with traditional laboratory parameters for diagnosis, clinical follow-up, and response to
therapy [67].
Guo et al. performed a retrospective cohort study of 149 patients with sJIA, of whom
27 had 31 episodes of MAS. Clinical and laboratory parameters of patients with sJIA-MAS
were investigated and compared with those without MAS. Clinically, a high percentage
of patients with MAS at onset did not have fever, arthritis, or central nervous system
dysfunction; 35% of MAS patients had hypotension without shock, and 22.6% of patients
had gastrointestinal symptoms. Subjects with MAS and hypotension had a higher rate of
ICU hospitalization; they presented more frequently with arthritis, serositis, pneumonia,
and gastrointestinal symptoms, accompanied by higher white blood cell and neutrophil
counts, superior serum bilirubin concentrations, and lower total serum protein levels. The
platelet count, LDH, and aspartate aminotransferase (AST), together with the ferritin/ESR
ratio of approximately 20.0, had high sensitivity and specificity in the early diagnosis of
MAS. The association between IFN-γ > 17.1 pg/mL and IL-10 > 7.8 pg/mL appears to be a
valuable cytokine prototype to confirm the onset of MAS.
The sudden onset of arterial hypotension, the ferritin/ESR ratio, and the pattern of
elevated IFN-γ and IL-10 cytokines are important parameters in predicting the early onset
of sJIA-MAS [131].
2.3.7. TNF-α
TNF-α, recognized as TNF superfamily member 2 (TNFSF2), is a multifunctional
cytokine with a well-determined role in innate and adaptive immunity, as well as in the
normal physiology of the immune system. Abnormally high and sustained production
of TNF-α is correlated with pathological manifestations of inflammatory diseases such
as sepsis and chronic ADs. Physiologically, TNF-α is a crucial component for a normal
immune response and can be considered a key factor in the development of infectious and
autoimmune pathologies [132].
In the pathogenesis of MAS, it has been proposed that macrophages produce several
cytokines, including TNF-α and pro-inflammatory interleukins (e.g., IL-6, IL-1, and IL-18),
which unleash a cascade of inflammatory pathways and, ultimately, create a CS. TNF-α can
be considered a key factor in the development of infectious and autoimmune pathologies.
With advances in the development of many diagnostic tools to identify MAS in sJIA as well
as other forms of MAS/sHLH, early management with cytokine-targeted therapies will
likely save the lives of many patients. TNF-α, when produced in excess, participates in the
spread of inflammatory phenomena by activating and concentrating fibroblasts, causing
joint erosion, fibrosis, and joint deformity in rheumatic diseases. There are already five
TNF-α receptor blockers (i.e., infliximab, adalimumab, etanercept, certolizumab pegol, and
golimumab) currently used in the treatment of several inflammatory diseases, including in
children [133,134].
Hügle et al. conducted a retrospective study of 32 patients for a period of 6 years,
considering demographic and clinical data as well as the presence and titers of antinuclear
antibodies (ANA) and rheumatoid factor (RF) at diagnosis, from the AID-Net database—a
German registry and biobank that gathers information and biomaterials from patients with
autoinflammatory syndromes, including periodic fever syndromes and sJIA; 8/32 patients
had ANA titers ≥ 1:80 at diagnosis, 22/32 patients had rising ANA titers with titers ≥ 1:80

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at their last visit, and 10/32 patients had a positive RF at least once during follow-up,
compared to 0/32 at diagnosis. The authors concluded that a proportion of patients with
sJIA developed increased ANA titers and positive RF over time, indicating the potential role
of lymphocyte activation and autoimmune processes in this primarily autoinflammatory
condition, independent of TNF antagonist treatment in sJIA [135].
Shimizu et al. measured the ratio between the levels of soluble TNF receptor (sTNFR)
type II (sTNFR-II) and type I (sTNFR-I)—i.e., (sTNFR-II/I)—in 117 patients with sJIA,
including 29 patients with MAS, 15 with EBV-HLH, 15 with KD, and 28 healthy controls, in
correlation with the activity and severity of the disease. At the same time, they determined
the serum levels of IL-18 in patients with EBV-HLH compared to MAS. The results showed
that the ratio of sTNFR-II/I had a significantly higher level in patients with MAS and
EBV-HLH compared to patients with active sJIA and KD. Serum IL-18 concentrations were
also significantly increased in patients with MAS in comparison with EBV-HLH. Serum
IL-18 and sTNFR-II/I could be used as biomarkers for the diagnosis of MAS and the
differentiation between MAS and EBV-HLH [136].
Irabu et al. studied the serum concentrations of neopterin, IL-18, chemokine (C-X-C
motif) ligand 9 (CXCL9), sTNFR-I, and sTNFR-II in a cohort of 36 patients with sJIA-MAS,
including 12 patients under management with TCZ. The serum ratio of sTNFR-II/I was
compared with the clinical characteristics of MAS. The results showed that the levels
of all serum cytokines studied at the diagnosis of MAS were much lower in the TCZ
group compared to those without this treatment, even if the serum sTNFR-II/I ratio at the
diagnosis of MAS was similar between the compared groups. The serum ratio of sTNFRII/I, which was elevated in sJIA-MAS patients, was positively correlated with disease
activity, with cutoff values of 0.9722 and 4.71, respectively. The authors concluded that the
serum sTNFR-II/I ratio could be a useful biomarker for assessing disease activity in sJIAMAS, but also a predictive biomarker for the onset of MAS in the sJIA active phase—even
in patients under TCZ [137].
Mizuta et al. comparatively studied the cytokines involved in the onset of MAS in
order to detect the serum biomarkers for early prediction of MAS in different childhood
rheumatic pathologies. Serum concentrations of neopterin, IL-6, IL-18, sTNFR-I, and
sTNFR-II were investigated via the ELISA technique in 12 patients with SLE, including
5 with MAS; 12 patients with juvenile dermatomyositis (JDM), including 4 with MAS;
75 patients with KD, including 6 with MAS; and 179 patients with sJIA, including 43 with
MAS. The results were analyzed in the context of the clinical symptoms of MAS. It was
observed that the serum concentrations of neopterin, IL-18, and sTNFR-II were much
higher during MAS compared to the active phase in all patients and all conditions studied.
Serum concentrations of sTNFR-I were much higher during MAS compared to the active
phase only in patients with SLE, KD, and sJIA. Depending on the disease, the curve analysis
revealed the following cytokines at the highest levels: serum sTNFR-I for SLE, serum IL-18
for JDM, and serum sTNFR-II for KD and sJIA, which were positively correlated with the
levels of serum ferritin. The authors assumed that the overproduction of IFN-γ, IL-18, and
TNF-α could highlight the initiation of MAS, and that the serum concentrations of sTNFR-I
for SLE, IL-18 for JDM, and sTNFR-II for KD and sJIA could be important biomarkers in
the active phase of these diseases for the prediction of MAS onset [138].
The scientific studies discussed above to provide insight into the utility important
cytokines as biomarkers are summarized in Table 3.

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Table 3. Cont.
Reference/Year Cytokines as Biomarkers Predictive Effects in sJIA and MAS
[95]. de Benedetti, F.; Massa, M.; Robbioni, P.;
Ravelli, A.; Burgio, G.R.; Martini, A. Correlation
of serum interleukin-6 levels with joint
involvement and thrombocytosis in systemic
juvenile rheumatoid arthritis. Arthritis Rheum.
1991, 34, 1158–1163.
https://doi.org/10.1002/art.1780340912
IL-6
High serum IL-6 levels were correlated with the
extent and severity of joint involvement, and
these data suggest that this cytokine plays a
significant role in the pathogenesis of sJIA.
[98]. Brachat, A.H.; Grom, A.A.; Wulffraat, N.;
Brunner, H.I.; Quartier, P.; Brik, R.; McCann, L.;
Ozdogan, H.; Rutkowska-Sak, L.; Schneider, R.
et al. Pediatric Rheumatology International
Trials Organization (PRINTO) and the Pediatric
Rheumatology Collaborative Study Group
(PRCSG). Early changes in gene expression and
inflammatory proteins in systemic juvenile
idiopathic arthritis patients on canakinumab
therapy. Arthritis Res. Ther. 2017, 19, 13.
https://doi.org/10.1186/s13075-016-1212-x
Analysis of IL-1β, IL-1 (IL1-R1 and IL1-R2), IL-1
receptor accessory protein (IL1-RAP), and IL-6
expression profiles in patients with sJIA after
initiation of treatment with canakinumab found
that the best clinical response was in patients
with higher initial expression of disordered
genes and a strong transcriptional response on
day 3. Treatment interrupted a positive feedback
loop between IL-1β signaling and IL-1β
production. Canakinumab applied in sJIA
patients resulted in downregulation of innate
immune response genes and decreased IL-6 and
clinical symptoms.
[103]. Shimizu, M.; Mizuta, M.; Okamoto, N.;
Yasumi, T.; Iwata, N.; Umebayashi, H.; Okura, Y.;
Kinjo, N.; Kubota, T.; Nakagishi, Y.; et al.
Tocilizumab modifies clinical and laboratory
features of macrophage activation syndrome
complicating systemic juvenile idiopathic
arthritis. Pediatr. Rheumatol. 2020, 18, 2.
https://doi.org/10.1186/s12969-020-0399-1
TCZ, a humanized anti-IL-6 receptor monoclonal
antibody, could modify the clinical and
laboratory features of sJIA-MAS.
[104]. Qu, H.; Sundberg, E.; Aulin, C.; Neog, M.;
Palmblad, K.; Horne, A.C.; Granath, F.; Ek, A.;
Melén, E.; Olsson, M.; Harris, H.E.
Immunoprofiling of active and inactive systemic
juvenile idiopathic arthritis reveals distinct
biomarkers: a single-center study. Pediatr.
Rheumatol. 2021, 19, 173.
https://doi.org/10.1186/s12969-021-00660-9
The biomarkers IL-6, IL-18, and S100A12 were
confirmed to be increased during active sJIA as
compared to healthy controls, and IL-18 was the
only one with elevated levels in inactive sJIA.
HMGB1 was found to be higher in active than in
inactive sJIA.
CASP8, CCL23, CD6, CXCL1, CXCL11, CXCL5,
EIF4EBP1, KITLG, MMP1, OSM, SIRT2,
SULT1A1, and TNFSF11 were found to be
differentially expressed in active and/or inactive
sJIA compared to the control group.
[107]. Imbrechts, M.; Avau, A.; Vandenhaute, J.;
Malengier-Devlies, B.; Put, K.; Mitera, T.;
Berghmans, N.; Burton, O.; Junius, S.; Liston, A.
et al. Insufficient IL-10 Production as a
Mechanism Underlying the Pathogenesis of
Systemic Juvenile Idiopathic Arthritis. J.
Immunol. 2018, 201, 2654–2663.
https://doi.org/10.4049/jimmunol.1800468
IL-10
IL-10 was shown to be an immunosuppressive
interleukin that counteracts IFN-γ activity, and
disruption of normal IL-10 concentration led to
the overproduction of IFN-γ, which participates
in the pathogenesis of MAS.
This study indicates that defective IL-10
production contributes to the pathogenesis of
sJIA.
[108]. Peng, Y.; Liu, X.; Duan, Z.; Duan, J.; Zhou,
Y. The Association of Serum IL-10 Levels with
the Disease Activity in Systemic-Onset Juvenile
Idiopathic Arthritis Patients. Mediators Inflamm.
2021, 2021, 6650928.
https://doi.org/10.1155/2021/6650928
Patients with sJIA had higher serum
concentrations of IL-10 compared to patients
with other febrile illnesses. Serum IL-10 levels
were much higher in active sJIA compared with
inactive disease and were consistent with ESR,
CRP, ferritin, and IL-6 levels. Serum IL-10 could
be a valuable marker of sJIA activity.

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Table 3. Cont.
Reference/Year Cytokines as Biomarkers Predictive Effects in sJIA and MAS
[135]. Hügle, B.; Hinze, C.; Lainka, E.; Fischer, N.;
Haas, J.P. Development of positive antinuclear
antibodies and rheumatoid factor in systemic
juvenile idiopathic arthritis points toward an
autoimmune phenotype later in the disease
course. Pediatr. Rheumatol. Online J. 2014, 12, 28.
https://doi.org/10.1186/1546-0096-12-28
TNF-α
Many patients developed positive ANA or
positive RF, independent of the treatment with
anti-TNF drugs, suggesting an autoimmune
phenotype rather than an autoinflammatory one
in the course of sJIA, probably involving the
activation of B cells during the inflammatory
process.
[136]. Shimizu, M.; Inoue, N.; Mizuta, M.;
Nakagishi, Y.; Yachie, A. Characteristic elevation
of soluble TNF receptor II:I ratio in macrophage
activation syndrome with systemic juvenile
idiopathic arthritis. Clin. Exp. Immunol. 2018,
191, 349–355. https://doi.org/10.1111/cei.13026
sTNFR-II/I had significantly higher levels in
patients with sJIA-MAS.
Serum IL-18 levels were elevated significantly in
MAS patients.
Higher serum IL-18 and sTNFR-II/I could be
used as biomarkers for the diagnosis of
sJIA-MAS, as well as for the differentiation
between MAS and EBV-HLH.
[137]. Irabu, H.; Shimizu, M.; Kaneko, S.; et al.
Comparison of serum biomarkers for the
diagnosis of macrophage activation syndrome
complicating systemic juvenile idiopathic
arthritis during tocilizumab therapy. Pediatr. Res.
2020, 88, 934–939.
https://doi.org/10.1038/s41390-020-0843-4
Serum sTNFR-II/I ratio could be a useful
biomarker for assessing disease activity in
sJIA-MAS, but also a predictive biomarker for
the onset of MAS in the active phase of sJIA,
even in patients under TCZ therapy.
[138]. Mizuta, M.; Shimizu, M.; Irabu, H.; Usami,
M.; Inoue, N.; Nakagishi, Y.; Wada, T.; Yachie, A.
Comparison of serum cytokine profiles in
macrophage activation syndrome complicating
different background rheumatic diseases in
children. Rheumatology 2021, 60, 231–238. https:
//doi.org/10.1093/rheumatology/keaa299
Serum levels of sTNFR-I for SLE, IL-18 for JDM,
and sTNFR-II for KD and sJIA could be useful
diagnostic biomarkers for the transition from the
active phase to MAS. Overproduction of IFN-γ,
IL-18, and TNF-α might be closely related to the
onset of MAS.
2.3.8. CXCL9 Chemokine
Chemokines are a family of low-molecular-weight proteins that participate in inflammation as mediators, signaling molecules, migration stimulators and macrophage
activators. There are four groups of ligands (XC, CC, CXC, and CX3C) for chemokines and
over 20 rhodopsin-like receptors—7 of which are transmembrane—including CC receptors,
CXC receptors, and receptors that exhibit non-G-protein-related signaling.
Chemokines act on macrophage differentiation and polarization in physiological and
pathological conditions, especially in inflammatory diseases. M1-type macrophages expose
CXCL9 and CXCL10 chemokines, resulting from the activation of the cytokines by IFN-γ
and TNFα, which attract Th1 lymphocytes. At the same time, the chemokines CXCL9 and
CXCL10 work as attractants of macrophages in various inflammatory diseases (stomach
burns, joints, etc.). It has been shown that osteoclast precursors exhibit the chemokine
CXCR3, which can cross-react with the chemokine ligand CXCL9 and stimulate migration
and differentiation of osteoclasts [136,139–145].
To define the cytokines and serum biomarkers involved in the onset of sJIA-MAS,
Mizuta et al. used a special antibody array that simultaneously detects 174 cytokines
to investigate samples from 15 sJIA patients, including 5 patients on TCZ therapy. Concentrations of five cytokines were highly increased in MAS compared to active sJIA. The
chemokine CXCL9 showed the greatest increase after the onset of sJIA-MAS. Serum levels
of CXCL9 were measured comparatively in 56 patients with sJIA, including 20 with MAS,
to highlight the clinical value of CXCL9 as a biomarker of sJIA-MAS. The authors proved
that CXCL9 was positively correlated with disease activity and that monitoring the serum
levels of this chemokine could enable surveillance of sJIA-MAS [146].

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Hinze et al. aimed to examine the associations of baseline serum biomarkers with the
responses and outcomes of 54 active sJIA patients without recent MAS after canakinumab
therapy. Most biomarkers were elevated at baseline in canakinumab-naïve patients compared to healthy controls, and some rapidly decreased by day 15 (i.e., IL-1Ra, IL-6, IL-18,
and S100A12). Patients who responded to treatment had higher IL-18 and IFN-γ levels and
lower CXCL9 levels at baseline, as documented by IL-18/CXCL9 and IFN-γ/CXCL9 ratios,
which had significant accuracy in predicting treatment responses. The authors observed a
differential regulation of the IL-18–IFN-γ–CXCL9 axis in patients with sJIA, and the higher
the IL-18/CXCL9 and IFN-γ/CXCL9 ratios, the better the clinical response to canakinumab
in sJIA [147].
2.3.9. sCD163 and sCD25
In order to investigate MAS, which is a huge cascade of inflammatory phenomena
triggered by the very intense activity of T cells and hemophagocytic macrophages, Bleesing
et al. analyzed the concentrations of sIL2RA—also called sCD25 and soluble cluster of
differentiation 163 (sCD163)—which reflected the levels of stimulation and expansion of
these cells, for samples taken from 7 patients with sJIA-MAS compared with 16 patients
with systemic sJIA at baseline without therapy, correlating these tests with serum ferritin
levels and clinical symptoms. The mean level of sCD163 was 23,000 ng/mL in MAS,
compared to only 5480 ng/mL in patients with active sJIA, while the mean level of sIL2RA
was 19,646 pg/mL in MAS, compared to only 3787 pg/mL in active disease. In 5 out of
16 patients with sJIA, the serum concentrations of sIL2RA or sCD163 were similar in order
of magnitude to those in MAS. It was concluded that sIL2RA and sCD163 could constitute
sensitive biomarkers for the diagnosis of MAS, even in subclinical cases, and could also
help identify patients with subclinical MAS [148].
Clinical and traditional laboratory markers of MAS, together with sCD25 and sCD163,
were investigated by Reddy et al. in 33 patients with sJIA, 11 patients with active polyarticular JIA (polyJIA), and 2 patients with sJIA-MAS. Subjects with MAS had thrombocytopenia,
leukopenia, and hypofibrinemia. The sCD25 value > 7500 pg/mL observed in MAS was
also found in eight patients with active sJIA, who also had other highly altered laboratory
values suggestive of MAS. Elevated levels of sCD63 (>1800 ng/mL) were detected in four
patients with sJIA, and it was concluded that sCD25 > 7500 pg/mL could be a predictive
biomarker of subclinical MAS [149].
Sakumura et al. conducted a study of 63 patients with sJIA, 4 with EBV-HLH, and
7 with KD, compared to 14 healthy controls, for whom the authors measured the serum
concentrations of sCD163, neopterin, IL-18, and IL-6, and correlated them with the patients’
clinical symptoms. sCD163 was highly elevated in patients with sJIA-MAS and EBVHLH compared to patients with only sJIA or KD in an acute phase. sCD163 increased
dramatically with the evolution of MAS, being positively dependent on sJIA activity, even
in patients under TCZ therapy. sCD163 showed a significant decrease in the inactive
phase and later normalized, representing a potential diagnostic parameter for clinical
remission in sJIA and an exclusive biomarker for the evaluation of disease activity, but also
of MAS-sJIA [150].
Verweyen et al. conducted a study on gene expression in whole-blood samples taken
from patients with sJIA at study entry and after 3 days of canakinumab therapy, compared
to healthy controls. The authors found a distinct gene expression signature in patients with
a very good clinical response to canakinumab compared to non-responders, mediated by
upregulation of neutrophil-associated genes and IL-1, as well as an upregulated CD163
expression signature for the non-responders to biological therapy with canakinumab [151].
In a recent published review, Dik et al. concluded that in immune-mediated diseases
where T-lymphocyte responses play an important role in the pathophysiological mechanisms, sCD25 could be a useful marker for prognosis or for monitoring the efficacy of
immunosuppressive management [152].

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2.3.10. Neopterin
In a recent study, Takakura et al. comparatively investigated the serum concentrations
of neopterin, IL-18, CXCL9, and sTNFR-I and II in 78 children diagnosed with sJIA, including 21 with MAS, to draw conclusions regarding the determination of the most accurate
biomarkers for establishing the diagnosis of sJIA-MAS. From the performed analyses, the
authors finally indicated neopterin as having the highest concentrations in sJIA-MAS and
being positively correlated with transition to MAS from active sJIA; thus, neopterin could
be a valuable future biomarker in sJIA-MAS [143].
2.3.11. S100 Proteins
Various biomarkers are currently being researched for the diagnosis, monitoring of
disease activity, predicting response to treatment, or the risk of recurrence in sJIA, AOSD,
MAS, and other autoinflammatory diseases [57].
S100 proteins are a subgroup of the Ca2+-binding protein family; they are engaged
in cell signal transduction, cell differentiation, motility regulation, transcription, and cell
cycle progression, and extracellularly they perform a similar role to cytokines. The proteins
S100A8/A9 (MRP8/14 or calprotectin) and S100A12 (calgranulin C) are secreted by phagocytic cells and may act as endogenous agonists of the toll-like receptor-4 and stimulate
the innate immune response by releasing alarmins. These proteins have been identified as
being more valuable than ESR and CRP as inflammatory biomarkers of the activity levels
of rheumatic diseases. It is already known that the proteins S100A8/S100A9 and IL-1β
play a special role in the pathogenesis of sJIA by activating phagocytic cells via two major
signaling pathways of innate immunity [153–155].
S100 proteins, after being released by activated cells of the innate immune system
(i.e., monocytes or neutrophils), play a special role in extending inflammation by binding
to TLR4 and RAGE. TLR4 is known as a multiligand receptor for PAMPs of pathogenic
bacteria, as well as DAMPs, while RAGE is a multiligand member of the cell surface
molecule superfamily and interacts with various other molecules involved in homeostasis
and inflammation. Both TLR4 and RAGE are expressed on the surface of immunological
cells, endothelial cells, and other extravascular cells, so they participate in the onset and
spread of systemic diseases during infection or inflammation [156–158].
Calprotectin, or CP—also known as MRP-8/MRP-14 or S100A8/A9 heterocomplex—
released primarily by activated neutrophils, has been investigated in feces and used as a
biomarker of inflammatory bowel disease and chronic rheumatic diseases in adults and children, including sJIA. CP functions through a mechanism termed “nutritional immunity”, by
retaining transition metals essential for growth and vitality. Overaccumulation of CP could
play a crucial role in inflammatory diseases, different types of arthritis, metastatic cancer,
and septicemia. S100A8/A9 and S100A12 are valuable biomarkers for diagnosis, treatment
response predictions, recurrence risk, and disease activity surveillance in sJIA [159–162].
Upon activation of neutrophils or monocyte endothelial adhesion, CP is secreted by an
alternative microtubule-mediated, calcium-dependent pathway, along with protein kinase
C (PKC), necrotic cells, and NETs, and may act as a marker for mononuclear phagocyte
crowding at the site of inflammation [163].
Since CP is released at the site of inflammation, plasma CP concentrations have been
proposed as a biomarker that represents the local activity of inflammatory disease, as
opposed to CRP, which is produced mainly by hepatocytes during the nonspecific systemic
inflammatory process. Being a relatively stable product, CP is more advantageous than
IL-6, TNF-α, or IL-1β [164].
Altobelli et al. carried out a systematic review and meta-analysis of the role of CP as a
potential biomarker of inflammation in JIA. The results showed a statistically significant
difference between patients with the active systemic subtype compared to other subtypes of
JIA, inactive disease, and healthy controls; the mean values of ESR and CRP were not found
to be statistically significant. In conclusion, the evaluation of serum PC could provide us

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with data on the more accurate stratification of disease activity and the administration of
more tailored and appropriate therapy in JIA [165].
Wittkowski et al. investigated the serum concentrations of the phagocytic proinflammatory protein S100A12 in serum samples taken from 240 patients with various
pathologies with fever of unknown origin (FUO), diagnosed as follows: 60 with sJIA,
17 with familial Mediterranean fever (FMF), 18 with neonatal-onset multisystem inflammatory disease, 17 with Muckle–Wells syndrome, 40 with acute lymphoblastic leukemia,
5 with acute myeloblastic leukemia, and 83 with systemic infections, compared with
45 healthy controls. The samples were taken at the introduction to the study, before starting any kind of therapy. The results demonstrated a sensitivity of 66% and a specificity
of 94% for S100A12 in differentiating sJIA from infections. The authors concluded that
S100A12, as a biomarker of granulocyte activation, is highly overexpressed in patients
with sJIA or FMF and can be a very valuable parameter in the evaluation and differential
diagnosis of FUO, helping the doctor to make the correct decision to treat the patient with
antibiotics or immunosuppressive agents [166].
Holzinger et al. studied the serum concentrations of myeloid-related protein complex
8 and 14 (MRP8/14) in 52 patients with sJIA to monitor disease activity and attempt a
classification of patients at risk of relapse. The results indicated highly elevated serum
concentrations of MRP8/14 in active disease and significantly reduced concentrations in
the case of positive-response therapies. MRP8/14 faithfully reflected the clinical activity of
sJIA, along with its inactivity or relapses, representing a valuable parameter for monitoring
the response to treatment, being the first predictive biomarker for the subclinical activity of
the disease, and allowing the stratification of patients at risk of relapse during periods of
clinical inactivity of sJIA [167].
Starting from the hypothesis that the expression of programmed death ligand-1 (PDL1)—an important immunoregulator (i.e., a glycoprotein expressed on antigen-presenting
cells to limit T-lymphocyte activation during an inflammatory response)—is deregulated in
sJIA monocytes, Shenoi et al. analyzed it in a pilot study on 20 patients (10 with active sJIA
and 10 febrile non-sJIA). The authors compared PD-L1 expression in myeloid cells with
other possible predictive biomarkers of sJIA, such as: CRP, ESR, leukocyte counts, S100A12,
S100A8, S100A9, calprotectin, and procalcitonin, each used as a predictor independent of
the other parameters in the analysis. The results highlighted that PD-L1 was significantly
lower in sJIA, while S100 proteins were significantly increased with >80% sensitivity and
>90% specificity in sJIA. The other investigated biomarkers were not found to be specific to
sJIA, and the exploratory gene analysis indicated 106 genes as being important for sJIA,
several of which were associated with immune response pathways. Although performed in
a small cohort, S100 proteins stood out as specific diagnostic biomarkers for febrile patients
with sJIA, and the decreased PD-L1 expression on the surface of circulating myeloid cells
in sJIA could indicate a mechanism of loss of regulation of peripheral immunity [168].
Aljaberi et al. retrospectively investigated the usefulness of S100 protein testing in
sJIA and other autoinflammatory and fever syndromes, in a pediatric clinical setting, to
monitor the activity of each disease and support the diagnosis in 136 patients with the
following pathologies: sJIA, non-systemic JIA (nsJIA), other defined autoinflammatory
diseases (AIDs), and systemic undifferentiated recurring fever syndromes (SURFSs). In the
case of sJIA, the concentrations of S100A8/9 and S100A12 were higher in active disease
compared to inactive disease and had much higher values compared to all other categories
included in the research; by comparison with CRP, they proved to be clearly superior
in differentiating sJIA from AID and SURFS, and they were not associated with disease
activity in nsJIA, AIDs, or SURFSs. The authors concluded that the significantly higher
S100A8/9 and S100A12 protein levels in sJIA can support the correct diagnosis in this
pathology, compared to other autoinflammatory and febrile diseases, and are superior to
CRP in establishing the positive diagnosis of sJIA compared to other diagnoses, as well as
when monitoring sJIA activity [169].

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Because the differential diagnosis of sJIA from other infectious pathologies is sometimes very difficult in cases of prolonged fever in children, Park et al. investigated which
would be the most useful biomarkers in diagnosis and tried to determine the usefulness
of myeloid protein 8/14 (MRP8/14) measurements in order to transfer them from the
laboratory to the bedside in clinical practice, in order to develop new methods of diagnosis for sJIA patients. Laboratory data from 1110 children and adolescents were divided
into two cohorts: group A, to validate the performance of the MRP8/14 test by experimental ELISA, commercial ELISA, and an innovative lateral flow immunoassay (LFIA);
and group B, to validate the accuracy of the diagnosis only with the last two tests. The
authors found that MRP8/14 was increased in group A (n = 940) in sJIA compared to
other conditions—including infections and autoinflammatory diseases—regardless of fever
or the anti-inflammatory treatment received by the patient. In children and adolescents
with fever, but without treatment (n = 195), serum concentrations of MRP8/14 in sJIA
(19,740 ± 5080 ng/mL) were much higher compared to other diagnoses, having the highest
precision compared to all other parameters measured—such as ferritin, IL-18, ESR, soluble
IL-2 receptor, and procalcitonin. The authors highlighted that serum tests of MRP8/14 can
be useful for the positive diagnosis of sJIA in febrile patients, and commercial ELISA and
LFIA allow accurate testing for rapid diagnostic screening at the point of care [18].
The lack of highly accurate biomarkers for monitoring disease activity—and as valuable tools for classifying and prognosticating treatment response or risk of flares in JIA
patients—led La et al. to investigate serum calprotectin (sCP) concentrations in a multicenter cohort of 81 JIA patients, compared with 11 healthy controls. As compared to the
controls, the researchers found that sCP values increased 8-fold in active sJIA and 2-fold
in inactive disease, with greater specificity than CRP values and even stronger than ESH,
allowing the differentiation of forms such as oligoarthritis, polyarthritis, and systemic
forms, and recommending the future applications of sCP as a biomarker not only in the
diagnosis and follow-up of JIA, but also for predicting relapses after stopping the treatment
with non-steroidal anti-inflammatory drugs, methotrexate, or etanercept; thus, sCP should
be implemented as soon as possible in clinical settings [170].
2.3.12. Procalcitonin
First identified in the 1970s [171] as a precursor of calcitonin (a hormone involved
in calcium homeostasis), procalcitonin (PCT) is a 116-amino-acid peptide generated by
the parafollicular cells of the thyroid and the neuroendocrine cells of the intestine and the
lungs. Plasma concentrations in healthy subjects are below the detection limit (0.01 µg/L)
of clinical tests [172], increasing in response to pro-inflammatory stimuli—especially in
bacterial infections—and considered to be an acute-phase reactant. In pediatric pathologies
with high fever of unknown origin, an elevated plasma PCT level at the threshold of
0.5 ng/mL has a sensitivity and specificity of over 80% for the diagnosis of sepsis in infants
and children [173].
Ninety-two subjects aged between 6 months and 18 years with untreated active JIA,
inactive JIA, and healthy elective preoperative patients were included in a cohort study
by Trachtman et al. to test the hypothesis that serum procalcitonin (sPCT) levels would
differ between active JIA, inactive JIA, and healthy controls, by measuring their serum
PCT and other common laboratory parameters of inflammation. The results indicated
increased values for ESR, CRP, and PCT in patients with bacteremia compared to the other
groups, with high rates of overlap between groups for ESR and CRP. The PCT concentration
was equal to or greater than 0.15 µg/mL in only 1 out of 59 patients with JIA, whereas
it was equal to or less than 0.15 µg/mL in only 1 patient with bacteremia. The authors’
conclusions were that although ESR, CRP, and PCT may all be biomarkers of choice for
distinguishing JIA at presentation from serious bacterial infection (SBI), the most suitable
biomarker appeared to be PCT, which was the most accurate in distinguishing between
patients with infection and those with non-infectious inflammatory arthritis, which can
greatly help clinicians to initiate the proper management immediately [174].

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2.3.13. sRAGE
Receptor for advanced glycation end products (RAGE; UniProtKB-Q15109) is a multiligand transmembrane receptor that is part of the immunoglobulin gene superfamily;
35 kD soluble cleaved RAGE—or soluble receptor for advanced glycation end products
(sRAGE)—can be quantified in plasma or serum, and it has been found that high levels
are associated with an increased risk of chronic diseases such as diabetes, atherosclerosis,
coronary heart disease, chronic lung disease, etc. Increased sRAGE is positively associated
with other pro-inflammatory advanced glycation end products (AGEs), and negatively
with leukocytosis and increased levels of CRP and Fg [175–178].
Geroldi et al. presented an overview of the state of the art in the use of sRAGE as
a biomarker and discussed its therapeutic potential for inflammatory diseases such as
Alzheimer’s disease, atherosclerosis, and arthritis [179].
Elevated plasma levels of sRAGE may account for the potential anti-inflammatory
and atheroprotective properties of this biomarker. sRAGE levels are inversely correlated
with white blood cell counts and high-sensitivity C-reactive protein (hsCRP), and could
indicate a slower rate of progression and a better prognosis of carotid artery disease [180].
In a recently published study, Prasad claimed that a low serum percentage of sRAGE
cannot be considered a universal biomarker for patients with different pathologies compared to healthy control subjects. However, evidence supports the validity of a high
AGE/sRAGE ratio as a universal biomarker/risk marker for diseases [181].
In an experimental murine model of cardiac ischemia/reperfusion (I/R), Zhang et al.
demonstrated that sRAGE protected the heart from I/R lesions that could be induced
by infiltration and proliferation of M1-type macrophages, which release IFN-γ following
activation of the NF-κB signaling pathway by sRAGE in the differentiated M1 macrophages.
The recruitment and proliferation of macrophages increased significantly in the presence of
sRAGE [182].
2.3.14. HMGB1
The high-mobility group box-1 (HMGB1; also called HMG1 or amphoterin) protein is
the most plentiful member of the HMG family of DNA-binding proteins—a prototype of
alarmins or DAMPs that are released into the extracellular space by activated or necrotic
cells in viral and bacterial infections, as well as in several diseases with acute or chronic
inflammation, such as cancer and ADs, and that work as key molecules connecting tissue
damage and stress with innate immunity. In addition to its pro-inflammatory functions,
HMGB1 also has effects in repairing and regenerating tissues. HMGB1 could be a therapeutic target in inflammatory chronic diseases. Recently generated HMGB1-specific inhibitors
have been proposed as an interesting therapeutic target in inflammatory conditions that
currently lack efficient therapies [183–185].
Together with extrinsic factors, intrinsic components can trigger an unpredictable
immune response in sJIA. HMGB1 takes part in the pathogenesis of inflammatory diseases
through interactions with pivotal transmembrane receptors, including RAGE and toll-like
receptor-4 (TLR4). Macrophages and monocytes that are highly activated, along with
disrupted TLR signaling and its undertrained ligands, are implicated in sJIA and AOSD.
Extracellular HMGB1 has the ability to amplify inflammation and modulate immune
responses in conjunction with RAGE. HMGB1 is an extracellular DAMP that binds to
RAGE and TLR4 and spreads inflammatory signals. HMGB1 is involved in systemic
inflammation from septicemic infections, arthritis, SLE, and liver damage [186–189].
The role of extracellular HMGB1 was discovered in 1999 as an important secreted
protein in inflammation and infection. Extracellular HMGB1 transporting nucleic acids
binds to RAGE and is internalized via endocytosis, so that the transferred nucleic acids interact with intracellular receptors and generate interferon and cytokine responses. HMGB1,
as a pro-inflammatory mediator, binds several pattern-recognition receptors (PRRs); in
addition to RAGE, members of the Toll-like receptor (TLR) family—i.e., TLR2, TLR4, and
TLR9—as well as the CXC chemokine receptor 4 (CXCR4) and T-cell immunoglobulin

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mucin-3 (TIM-3) are released during apoptosis or necrosis, acting as DAMPs, while the
inflammatory cytokines (i.e., TNF-α and IFN-γ) improve their release. When the concentration of the HMGB1 ligand increases, transmembrane RAGE is overexpressed in monocytes
and macrophages and triggers an inflammatory immune response. sRAGE was shown to
decrease the pro-inflammatory effects of HMGB1 [63,190–192].
Extracellular HMGB1 levels are increased in the inflamed joints of JIA patients and
could be a biomarker of inflammatory activity and a target for therapy [193].
Bobek et al. investigated HMGB1 and sRAGE in a total of 144 enrolled children
(97 with different subtypes of JIA, including sJIA; 19 with juvenile SLE; and 28 healthy
controls), collecting consecutive samples of peripheral blood as well as synovial fluid
(SF) at the time of diagnosis—prior to the initiation of treatment—and studying them in
correlation with other common acute-phase reactants and clinical markers. The obtained
results indicated much higher serum concentrations of HMGB1 and lower concentrations
of sRAGE for children with JIA and those with SLE, respectively, compared to healthy
controls, with positive correlations between HMGB1 and ESR, CRP, and α2 globulin, while
the concentrations of sRAGE were inversely proportional to the same inflammatory markers
in children with JIA. The median serum HMGB1 concentration in sJIA (17,402 pg/mL)
was 15-fold higher than that in the control group (1149 pg/mL) and was also much higher
than those in oligoarticular (3552 pg/mL), polyarticular (4374 pg/mL), and SLE patients
(8523 pg/mL). Serum concentrations of HMGB1 were correlated with hepatosplenomegaly
or serositis in sJIA. All JIA subgroups, as well as the SLE group, were characterized by
lower serum levels of sRAGE, but the difference was significant only in patients with sJIA
and in those with SLE. The inverse relationship between HMGB1 and its receptor sRAGE
in blood and SF reflected that the inflammatory processes triggered by alarmins could
play an important role in both investigated pathogeneses, and that HMGB1 could be an
inflammatory biomarker to track in these patients as a potential target in the framework of
biological therapy and for monitoring the activity of the disease [194].
Xu et al. conducted a prospective longitudinal study of 64 children with a mean age of
9.25 years and a mean duration of illness of 2.38 months, of whom 48.4% were female, and
collected blood samples at the first visit and after 1 month, 3 months, and 6 months, for
comparison to samples from healthy controls as well as from patients with reactive arthritis
at the first visit. HMGB1 levels, routine laboratory data, and clinical disease characteristics
were analyzed according to the protocol. The results at the first visit indicated significantly
increased serum concentrations of HMGB1 in patients with sJIA compared to the other
groups, as well as in enthesitis-associated arthritis compared to healthy controls. They also
highlighted important correlations at the first visit between HMGB1 levels and disease
duration, C-reactive protein, neutrophil percentage, and ferritin, suggesting that serum
HMGB1 could be a sensitive inflammatory biomarker in sJIA. The authors recommended
that further large-scale studies would be useful to explore the full range of HMGB1-related
properties in JIA [195].
2.3.15. Key Laboratory Data in sJIA-MAS
In order to confront the concentrations of CRP and ESR in children with JIA, and to
trace their influence on diagnostic variables and prognostic factors, Wu et al. investigated
107 patients, of whom 18 had sJIA. At the time of diagnosis, the prevalence of ESR was much
higher than that of CRP (86.8% vs. 47.2%) and was positively correlated with good response
to therapy. On the other hand, the high concentrations of CRP at the beginning of the
treatment were correlated with an inadequate response to the therapy and generally proved
to be a predictive parameter of the failure of the first remission. This study demonstrated
that ESR is useful in the diagnosis of sJIA—much more valuable than CRP—whereas a
high initial CRP value may predict failure of remission [196].
Bloom et al. investigated 31 children with sJIA who were at high risk of developing
severe disease based on their first measured D-dimer concentration. Ten children who
initially had their risk category classified as “high” developed a severe outcome. The

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authors proved the paradigm that the risk of severe disease indicated by persistently
elevated levels of fibrin D-dimer could be a prognostic parameter indicating a poor longerterm outcome in sJIA [197].
Follistatin-like protein 1 (FSTL-1) is known as a glycoprotein that is overexpressed
in certain inflammatory diseases. Gorelik et al. investigated FSTL-1 concentrations in
28 patients with sJIA, including 7 patients who developed MAS, and compared them to
30 healthy controls, in correlation with already known biomarkers for the evaluation of
sJIA activity. FSTL-1 gene expression concentrations were investigated in peripheral blood
mononuclear cells (PBMCs) in correlation with ESR, ferritin, and sIL2RA. Serum concentrations of FSTL-1 were high in patients with sJIA at the beginning of the study, and they
returned to normal over the following 24 months. The same high concentrations of FSTL-1
were also found in patients with acute MAS, which normalized after treatment. FSTL-1
was correlated with several serum markers of inflammation, including sIL2RA and ferritin.
The ferritin/ESR ratio was superior to ferritin, sIL2RA, and FSTL-1 in differentiating MAS
from new-onset sJIA. High serum concentrations of FSTL-1 detected before sJIA therapy are
accompanied by dysregulated gene expression that may predict the presence of hidden MAS
and progression to observable MAS. It has been shown that the serum ferritin/ESR ratio
may be more valuable than ferritin alone in differentiating MAS from new-onset sJIA [198].
Minoia et al. conducted extensive research within a multinational collaborative project
on 766 patients, of whom 362 had sJIA-MAS and 404 had active sJIA without MAS, in
an attempt to develop and validate a score for the diagnosis of sJIA-MAS. The score for
sJIA-MAS, named MS score, comprised seven variables: central nervous system (CNS)
dysfunction, hemorrhagic manifestations, active arthritis, platelet count, Fg, LDH, and
ferritin. The cutoff value ≥ −2.1 was the most reliable in differentiating MAS from active
sJIA, with a sensitivity of 0.85 and a specificity of 0.95. The MS score could be a valuable
practical instrument to be used by clinicians in the early diagnosis of sJIA-MAS [199].
Eloseily et al. investigated the value of the serum ferritin/ESR ratio for the diagnosis
of sJIA-MAS and ferritin alone as a screening parameter for detecting MAS of other febrile
etiologies. For sJIA patients with MAS (n = 362), without MAS (n = 404), and subjects
(n = 345) hospitalized with systemic infection (SI), the ferritin/ESR ratio and ferritin alone
were investigated to detect MAS among sJIA patients.
Meanwhile, the same parameters were investigated on a smaller number of cases
with MAS of various causes, but also in patients hospitalized for fever. The ferritin/ESR
ratio of 21.5 was 82% sensitive and 78% specific for the diagnosis of sJIA-MAS versus
active sJIA without MAS. Only ferritin as a single laboratory parameter had a sensitivity of
95% (screening tool) and a specificity of 89.3% for differentiating patients with MAS from
subjects with other febrile conditions. It was concluded that the serum ferritin/ESR ratio is
a valuable parameter for the diagnosis of sJIA-MAS, and that the serum ferritin level alone
can be used in screening to detect MAS among febrile patients [200].
In a multicenter study of 80 patients with MAS, Zou et al. included 53 cases of
sJIA-MAS, 10 cases of KD-MAS, and 17 cases of connective tissue disease (CTD-MAS) and
investigated the clinical symptoms and laboratory data before (pre-), at the onset, and during
the complete phase of MAS. Among all patients, 51.2% presented MAS for the first time at
the diagnosis of the underlying disease. Among patients with sJIA, 22.6% had hypotension
before the onset of sJIA-MAS; in these patients, an increase in aspartate aminotransferase
(AST) and lactate dehydrogenase (LDH) was observed, along with a decrease in serum
albumin. Serum ferritin levels and ferritin/ESR ratios were greatly increased in subjects
with active sJIA-MAS compared to the pre-MAS period. At the same time, a very high
serum ferritin concentration and a significantly increased ferritin/ESR ratio were found in
sJIA subjects compared to KD and CTD patients. In conclusion, almost half of the cases of
MAS occurred when sJIA, KD, and CTD were present at first diagnosis. Hypotension was
present in almost 30% of cases of MAS at onset, which could constitute a predictive aspect
of the diagnosis. Increased serum ferritin, ferritin/ESR ratio, AST, and LDH, along with
decreased serum albumin, could be predictive elements of the emergence of sJIA-MAS [201].

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Ganeva et al. investigated biomarkers and their relationships with disease progression
in the first year after diagnosis in a cohort of 266 JIA patients. Clinical symptoms and
laboratory data were recorded quarterly in the first year and semiannually thereafter. The
investigated biomarkers were increased in most JIA patients compared to healthy controls.
In 10 subjects with sJIA, high concentrations of CRP, ESR, IL-18, S100A8/A9, and S100A12
were recorded compared to subjects with other JIA subtypes. Higher levels of ESR, G-CSF,
IL-6, IL-17A, and TNF at baseline were predictive of a high risk of uninterrupted disease
still active after 12 months. Elevated serum levels of CRP, S100A8/A9, and S100A12, as
well as high ESR at baseline, were correlated with the necessity of intensified management
with biological DMARDs in the first year of follow-up [202].
2.3.16. Serum Amyloid A
Serum amyloid A is an acute-phase protein belonging to the group of apolipoproteins,
synthesized by macrophages, adipocytes, synoviocytes, and especially hepatocytes. During
the acute inflammatory process, it can increase significantly and has a chemotactic capacity
for neutrophils and mast cells. SAA participates in the release of TNF-α, IL-1, IL-6, and
granulocyte colony-stimulating factor (G-CSF); it stimulates angiogenesis, matrix metalloproteinases, and tissue factors. It is validated as a biomarker of the activity of diseases such
as RA, JIA, rheumatic polymyalgia, ankylosing spondylitis, and sarcoidosis [203–205].
The main role of SAA in the pathogenesis of rheumatic inflammatory diseases is
recognized by contemporary studies demonstrating its involvement in activating the
inflammasome cascade and attracting IL-17-producing T-helper lymphocytes. From the
earliest findings, it was clear that SAA could be used to monitor and assess the severity of
disease activity in patients with rheumatoid arthritis and secondary amyloidosis. SAA has
been observed to be a valuable biomarker in the immune pathology of rheumatic diseases,
especially in the case of subclinical inflammation. Unlike other biomarkers, recent research
has recognized the benefits of SAA monitoring in biological immunotherapy, especially
after the implementation of proteomic techniques. Modern techniques encourage the
research of SAA and its isoforms as sensitive biomarkers in rheumatic diseases—and
even the development of therapeutic agents with SAA as a target. Another argument for
recognizing its potential is given by recent findings that have shown that SAA is a very
useful biomarker in monitoring severe coronavirus disease [206–208].
The clinical utility of SAA in both predicting and monitoring the response to therapy
in patients with JIA has been less explored in recent decades. Dev et al. conducted a study
on 50 newly diagnosed cases of JIA of all subtypes, along with 40 healthy controls, to find
out whether SAA could be a biomarker of disease activity in JIA. The results showed that
SAA concentrations were significantly higher in patients with JIA compared to healthy
controls. Elevated SAA levels were positively correlated with disease activity in JIA, but
not with CRP or ESR levels. The study showed that SAA is a more sensitive laboratory
marker than ESR and CRP for assessing the presence of active joints [209].
2.3.17. Leucine-Rich α2-Glycoprotein
Leucine-rich α2-glycoprotein (LRG) is a plasma glycoprotein whose physiological role
is being studied. Recent data have highlighted the role of LRG in the differentiation and
proliferation of Th17 lymphocytes and in the process of neovascularization by acting on
the TGF-β in endothelial cells [210,211].
LRG is synthesized by the hepatocytes, and its concentration increases during inflammation under the action of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, making
it useful in diagnosing a multitude of inflammatory diseases (e.g., intestinal, rheumatoid
arthritis, AOSD, sJIA, etc.). As part of the family of acute-phase proteins, similar to CRP
and SAA, proteomic studies have shown that LRG is a sensitive biomarker in patients
with rheumatoid arthritis, which increases during disease activity and can be reduced after
anti-TNF-α therapy [212–214].

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Leucine-rich α-2 glycoprotein 1 (LRG1) is an important member of the leucine-rich
repetitive sequence protein family and is implicated in many human conditions, including
cancer, diabetes, cardiovascular disease, neurological disease, inflammatory disorders, etc.
It has recently been proven that LRG1 is compacted into the granules of human neutrophils
and secreted upon the neutrophils’ stimulation to adjust the micro-medium. Serum LRG1
has been recognized as a practical biomarker for monitoring disease activity in patients
with rheumatoid arthritis, lupus nephritis, adult-onset Still’s disease, and vasculitis. LRG1
is a recently detected important upstream signaling molecule of TGF-β that influences
multiple pathophysiological mechanisms through the TGF-β signaling pathway [215–217].
In an early study, Shimizu et al. investigated the attributes of LRG as a biomarker for
controlling sJIA activity during IL-6-targeting therapy. Serum LRG concentrations were
investigated in four JIA patients treated with TCZ and correlated with clinical parameters
and pro-inflammatory cytokines, including IL-18, IL-6, neopterin, and TNF-α receptor
types I and II. Serum LRG concentrations increased simultaneously with the progression
of active sJIA and the onset of MAS. Even when the disease was no longer active, serum
LRG concentrations remained higher than normal values. A significant positive reciprocity
was observed between serum levels of LRG and pro-inflammatory cytokines. Serum
concentrations of LRG could probably be used as a unique biomarker for monitoring the
progress of sJIA activity during IL-6 blocking therapy [218].
In another study, Shimizu et al. explored serum levels of LRG, IL-6, IL-18, sTNFR-I,
and sTNFR-II in 59 patients with sJIA, 15 with other subtypes of JIA, 7 with KD, 7 with
influenza A infection, 7 with infection with enterohemorrhagic Escherichia coli (EHEC), and
20 healthy controls (HCs) to whom the clinical symptoms were also compared. The serum
LRG concentrations in active sJIA were much higher compared to other subtypes of JIA,
EHEC, influenza patients, and HCs. Serum LRG values were normalized in patients with
inactive sJIA after treatment and were positively correlated with those of serum CRP and
serum ferritin. The authors claimed that serum LRG concentrations reflected the level of
sJIA activity and could be useful as a biomarker for monitoring sJIA disease activity [219].
2.3.18. Adenosine Deaminase 2
Lee et al. evaluated the serum levels of adenosine deaminase 2 (ADA2)—a protein
with as-yet unknown function released mainly by monocytes and macrophages—as a novel
biomarker of MAS in 324 healthy children and adults, and compared these serum levels
with those of 173 children with inflammatory and immune-mediated diseases, including
sJIA and nsJIA, KD, SLE, and JDM. The results showed increased levels of ADA2 in the
peripheral blood of patients with sJIA and MAS; the activity of this protein was strongly
correlated with the levels of ferritin, IL-18, and CXCL9 inducible by IFN-γ, but inconsistent
with inflammatory markers (i.e., CRP and ESR). IL-18 and IFN-γ generated increased
production of ADA2 by peripheral blood mononuclear cells, and ADA2 was abundant
in MAS hemophagocytes. In conclusion, the authors noted the usefulness of research on
serum ADA2 levels as a future biomarker for the diagnosis of sJIA-MAS [220].
The chemokines and other biomarkers discussed above are highlighted in Table 4.
Table 4. Chemokines and other biomarkers for the diagnosis, activity, and prognosis of sJIA and
MAS.
Reference/Year Chemokines and Other Biomarkers Predictive Effects in sJIA and MAS
[146]. Mizuta, M.; Shimizu, M.; Inoue, N.;
Nakagishi, Y.; Yachie. A. Clinical
Significance of Serum CXCL9 Levels as a
Biomarker for Systemic Juvenile Idiopathic
Arthritis Associated Macrophage Activation
Syndrome. Cytokine 2019, 119, 182–187.
https://doi.org/10.1016/j.cyto.2019.03.018
CXCL9
CXCL9 showed the most significant
increase following the onset of sJIA-MAS
and was positively correlated with disease
activity. Monitoring the serum levels of this
chemokine would enable surveillance of
sJIA-MAS.

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Table 4. Cont.
Reference/Year Chemokines and Other Biomarkers Predictive Effects in sJIA and MAS
[147]. Hinze, T.; Kessel, C.; Hinze, C.H.;
Seibert, J.; Gram, H.; Foell, D. A
dysregulated
interleukin-18-interferon-γ-CXCL9 axis
impacts treatment response to
canakinumab in systemic juvenile
idiopathic arthritis. Rheumatology 2021, 60,
5165–5174. https://doi.org/10.1093/
rheumatology/keab113
There was a differential regulation of the
IL-18–IFN-γ–CXCL9 axis in patients with
sJIA, and the higher the IL-18/CXCL9 and
IFN-γ/CXCL9 ratios, the better the clinical
outcome in response to canakinumab in
sJIA.
[148]. Bleesing, J.; Prada, A.; Siegel, D.M.;
Villanueva, J.; Olson, J.; Ilowite, N.T.;
Brunner, H.I.; Griffin, T.; Graham, T.B.;
Sherry, D.D.; et al. The diagnostic
significance of soluble CD163 and soluble
interleukin-2 receptor alpha-chain in
macrophage activation syndrome and
untreated new-onset systemic juvenile
idiopathic arthritis. Arthritis Rheum. 2007,
56, 965–971.
https://doi.org/10.1002/art.22416
sCD163 and
soluble IL-2Rα (sCD25)
Elevated serum concentrations of sCD25
and sCD163 may be predictive biomarkers
of MAS and help detect patients with
subclinical MAS.
[149]. Reddy, V.V.; Myles, A.; Cheekatla,
S.S.; Singh, S.; Aggarwal, A. Soluble CD25
in serum: a potential marker for subclinical
macrophage activation syndrome in
patients with active systemic onset juvenile
idiopathic arthritis. Int. J. Rheum. Dis. 2014,
17, 261–267.
https://doi.org/10.1111/1756-185X.12196
The presence of sCD25 levels > 7500 pg/mL
can be considered a useful biomarker in the
detection of patients with subclinical MAS.
[150]. Sakumura, N.; Shimizu, M.; Mizuta,
M.; Inoue, N.; Nakagishi, Y.; Yachie, A.
Soluble CD163, a unique biomarker to
evaluate the disease activity, exhibits
macrophage activation in systemic juvenile
idiopathic arthritis. Cytokine 2018, 110,
459–465. https:
//doi.org/10.1016/j.cyto.2018.05.017
sCD163 might be a potential indicator of
the disease activity and remission in sJIA
and sJIA-MAS, as well as for patients
receiving TCZ.
[151]. Verweyen, E.L.; Pickering, A.; Grom,
A.A.; Schulert, G.S. Distinct Gene
Expression Signatures Characterize Strong
Clinical Responders Versus Nonresponders
to Canakinumab in Children With Systemic
Juvenile Idiopathic Arthritis. Arthritis
Rheumatol. 2021, 73, 1334–1340.
https://doi.org/10.1002/art.41640
A signature including upregulated CD163
expression was associated with
non-response to canakinumab.
[143]. Takakura, M.; Shimizu, M.; Irabu, H.;
Sakumura, N.; Inoue, N.; Mizuta, M.;
Nakagishi, Y.; Yachie, A. Comparison of
serum biomarkers for the diagnosis of
macrophage activation syndrome
complicating systemic juvenile idiopathic
arthritis. Clin. Immunol. 2019, 208, 108252.
https:
//doi.org/10.1016/j.clim.2019.108252
Neopterin
Serum neopterin levels may be used as an
indicator of disease activity in sJIA and
MAS, as well as for evaluating it. It may
also be a useful marker to diagnose the
transition to MAS from active sJIA.

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Table 4. Cont.
Reference/Year Chemokines and Other Biomarkers Predictive Effects in sJIA and MAS
[166]. Wittkowski, H.; Frosch, M.;
Wulffraat, N.; Goldbach-Mansky, R.;
Kallinich, T.; Kuemmerle-Deschner, J.;
Frühwald, M.C.; Dassmann, S.; Pham, T.H.;
Roth, J.; et al. S100A12 is a novel molecular
marker differentiating systemic-onset
juvenile idiopathic arthritis from other
causes of fever of unknown origin. Arthritis
Rheum. 2008, 58, 3924–3931.
https://doi.org/10.1002/art.24137
S100 proteins as follows:
calgranulin A (S100A8) or MRP 8
calgranulin B (S100A9/MRP14)
calgranulin C (S100A12/MRP 6)
calprotectin (CP) or MRP-8/MRP-14,
or S100A8/A9 heterocomplex
S100A12 is a useful biomarker in
confirming the positive diagnosis of sJIA
and excludes early severe systemic
infections and several inflammatory or
neoplastic disorders.
[167]. Holzinger, D.; Frosch, M.; Kastrup,
A.; Prince, F.H.; Otten, M.H.; Van
Suijlekom-Smit, L.W.; ten Cate, R.;
Hoppenreijs, E.P.; Hansmann, S.;
Moncrieffe, H.; et al. The Toll-like receptor
4 agonist MRP8/14 protein complex is a
sensitive indicator for disease activity and
predicts relapses in systemic-onset juvenile
idiopathic arthritis. Ann. Rheum. Dis. 2012,
71, 974–980. https://doi.org/10.1136/
annrheumdis-2011-200598
MRP8/14 (calprotectin) serum
concentration was the first predictive
biomarker indicating subclinical disease
activity and stratifying patients at risk of
relapse during clinically inactive sJIA.
[168]. Shenoi, S.; Ou, J.N.; Ni, C.; Macaubas,
C.; Gersuk, V.H.; Wallace, C.A.; Mellins,
E.D.; Stevens. A.M. Comparison of
biomarkers for systemic juvenile idiopathic
arthritis. Pediatr. Res. 2015, 78, 554–559.
https://doi.org/10.1038/pr.2015.144
In a small cohort, S100 proteins were
identified as specific diagnostic biomarkers
for febrile patients with sJIA. Decreased
PD-L1 surface expression on circulating
myeloid cells in sJIA indicated a possible
mechanism for the loss of peripheral
immune regulation.
[169]. Aljaberi, N.; Tronconi, E.; Schulert,
G.; Grom, A.A.; Lovell, D.J.; Huggins, J.L.;
Henrickson, M.; Brunner, H. The use of
S100 proteins testing in juvenile idiopathic
arthritis and autoinflammatory diseases in
a pediatric clinical setting: a retrospective
analysis. Pediatr. Rheumatol. Online J. 2020,
18, 7. https:
//doi.org/10.1186/s12969-020-0398-2
S100A8/9 and S100A12 proteins were
highly elevated in sJIA compared to nsJIA
and other autoinflammatory diseases.
Therefore, the authors concluded that S100
proteins (i.e., S100A8/9 and S100A12) are
valuable biomarkers of disease activity in
sJIA, but not in other autoinflammatory
syndromes or nsJIA.
[18]. Park, C.; Miranda-Garcia, M.;
Berendes, R.; Horneff, G.;
Kuemmerle-Deschner, J.; Ganser, G.;
Huppertz, H.I.; Minden, K.; Haas, J.P.;
Jansson, A.F.; et al. MRP8/14 serum levels
as diagnostic markers for systemic juvenile
idiopathic arthritis in children with
prolonged fever. Rheumatology 2021,
keab729. https://doi.org/10.1093/
rheumatology/keab729
Compared with ferritin, IL-18, ESR, soluble
IL-2 receptor, and procalcitonin, MRP8/14
showed the best accuracy in the diagnosis
of sJIA.

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Table 4. Cont.
Reference/Year Chemokines and Other Biomarkers Predictive Effects in sJIA and MAS
[170]. La, C.; Lê, P.Q.; Ferster, A.; Goffin, L.;
Spruyt, D.; Lauwerys, B.; Durez, P.;
Boulanger, C.; Sokolova, T.; Rasschaert, J.;
Badot, V. Serum calprotectin (S100A8/A9):
a promising biomarker in diagnosis and
follow-up in different subgroups of
juvenile idiopathic arthritis. RMD Open
2021, 7, e001646. https:
//doi.org/10.1136/rmdopen-2021-001646
The study confirmed the potential uses of
sCP as a biomarker in the diagnosis and
follow-up of sJIA.
[174]. Trachtman, R.; Murray, E.; Wang,
C.M.; Szymonifka, J.; Toussi, S.S.; Walters,
H.; Nellis, M.E.; Onel, K.B.; Mandl, L.A.
Procalcitonin Differs in Children With
Infection and Children With Disease Flares
in Juvenile Idiopathic Arthritis. J. Clin.
Rheumatol. 2021, 27, 87–91. https://doi.
org/10.1097/RHU.0000000000001170
Procalcitonin
The study found that CRP, ESR, and serum
PCT levels are biomarkers that can be used
to distinguish severe bacterial infection
from active JIA at onset, but PCT was the
most accurate. PCT can be used as a
biomarker to help clinicians guide therapy.
[194]. Bobek, D.; Grˇcevi´c, D.; Kovaˇci´c, N.;
Luki´c, I.K., Jeluši´c, M. The presence of high
mobility group box-1 and soluble receptor
for advanced glycation end-products in
juvenile idiopathic arthritis and juvenile
systemic lupus erythematosus. Pediatr.
Rheumatol. Online J. 2014, 12, 50.
https://doi.org/10.1186/1546-0096-12-50
HMGB1/sRAGE
AGEs and sRAGEs
The study found positive correlations
between serum HMGB1 and ESR, CRP, and
α2 globulin—and vice versa for
sRAGE—as inflammatory markers in
children with sJIA. Elevated serum HMGB1
levels have been associated with
hepatosplenomegaly and/or serosis in
sJIA.
[195]. Xu, D.; Zhang, Y.; Zhang, Z.Y.; Tang,
X.M. Association between high mobility
group box 1 protein and juvenile idiopathic
arthritis: a prospective longitudinal study.
Pediatr. Rheumatol. 2021, 19, 112. https:
//doi.org/10.1186/s12969-021-00587-1
Serum HMGB1 could be considered to be a
sensitive inflammatory biomarker for the
assessment of clinical activity in JIA; it was
found to be specifically elevated at first
presentation in children with sJIA.
[196]. Wu, J.F.; Yang, Y.H.; Wang, L.C.; Lee,
J.H.; Shen, E.Y.; Chiang, B.L. Comparative
usefulness of C-reactive protein and
erythrocyte sedimentation rate in juvenile
rheumatoid arthritis. Clin. Exp. Rheumatol.
2007, 25, 782–785. PMID: 18078633
Routine laboratory data in
sJIA-MAS
(CRP, ESR, Fg, D-dimer, serum
ferritin, ferritin/ESR ratio, LDH,
AST)
ESR is a useful biomarker in the diagnosis
of sJIA—much more valuable than
CRP—whereas a high initial CRP value
may strongly predict treatment failure.
[197]. Bloom, B.J.; Alario, A.J.; Miller, L.C.
Persistent elevation of fibrin d-dimer
predicts long term outcome in systemic
juvenile idiopathic arthritis. J. Rheumatol.
2009, 36, 422–426.
https://doi.org/10.3899/jrheum.070600
D-dimer could more accurately reflect the
disease activity and prognosis as compared
to clinical features in patients with sJIA.
[198]. Gorelik, M.; Fall, N.; Altaye, M.;
Barnes, M.G.; Thompson, S.D.; Grom, A.A.;
Hirsch, R. Follistatin-like protein 1 and the
ferritin/erythrocyte sedimentation rate
ratio are potential biomarkers for
dysregulated gene expression and
macrophage activation syndrome in
systemic juvenile idiopathic arthritis. J.
Rheumatol. 2013, 40, 1191–1199.
https://doi.org/10.3899/jrheum.121131
In sJIA, elevated serum levels of FSTL-1
prior to treatment were related to a
disorder of gene expression that predicts
hidden MAS and progression to true MAS.
Serum ferritin/ESR ratio may be superior
to ferritin alone in discerning the obvious
MAS from sJIA at onset.

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Table 4. Cont.
Reference/Year Chemokines and Other Biomarkers Predictive Effects in sJIA and MAS
[199]. Minoia, F.; Bovis, F.; Davì, S.; Horne,
A.; Fischbach, M.; Frosch, M.; Huber, A.;
Jelusic, M.; Sawhney, S.; McCurdy, D.K.;
et al. Pediatric Rheumatology International
Trials Organization, the Childhood
Arthritis & Rheumatology Research
Alliance, the Pediatric Rheumatology
Collaborative Study Group and the
Histiocyte Society. Development and initial
validation of the MS score for diagnosis of
macrophage activation syndrome in
systemic juvenile idiopathic arthritis. Ann.
Rheum. Dis. 2019, 78, 1357–1362.
https://doi.org/10.1136/annrheumdis-20
19-215211
MS score, which comprises seven variables
(CNS dysfunction, hemorrhagic
manifestations, active arthritis, platelet
count, Fg, LDH, and ferritin), could be a
valuable practical instrument that can be
used by clinicians in the early diagnosis of
sJIA-MAS.
[200]. Eloseily, E.M.; Minoia, F.; Crayne,
C.B.; Beukelman, T.; Ravelli, A.; Cron, R.Q.
Ferritin to erythrocyte sedimentation rate
ratio: simple measure to identify
macrophage activation syndrome in
systemic juvenile idiopathic arthritis. ACR
Open Rheumatol. 2019, 1, 345–349.
https://doi.org/10.1002/acr2.11048
Serum ferritin/ESR ratio is a valuable
parameter for the diagnosis of sJIA-MAS,
and the serum ferritin level alone can be
used in screening to detect MAS among
febrile patients.
[201]. Zou, L.X.; Zhu, Y.; Sun, L.; Ma, H.H.;
Yang, S.R.; Zeng, H.S.; Xiao, J.H.; Yu, H.G.;
Guo, L.; Xu, Y.P.; Lu, M.P. Clinical and
laboratory features, treatment, and outcomes
of macrophage activation syndrome in 80
children: a multi-center study in China.
World J. Pediatr. 2020, 16, 89–98. https:
//doi.org/10.1007/s12519-019-00256-0
Increased serum ferritin, ferritin/ESR ratio,
AST, and LDH, along with decreased
serum albumin, could be predictive
parameters of the emergence of sJIA-MAS.
[202]. Ganeva, M.; Fuehner, S.; Kessel, C.;
Klotsche, J.; Niewerth, M.; Minden, K.;
Foell, D.; Hinze, C.H.; Wittkowski, H.
Trajectories of disease courses in the
inception cohort of newly diagnosed
patients with JIA (ICON-JIA): the potential
of serum biomarkers at baseline. Pediatr.
Rheumatol. Online J. 2021, 19, 64. https:
//doi.org/10.1186/s12969-021-00553-x
Elevated baseline levels of CRP,
S100A8/A9, and S100A12, as well as
increased ESR, were associated with the
necessity to escalate therapy during the
first 12 months.
[209]. Dev, S.; Singh, A. Study of role of
serum amyloid A (SAA) as a marker of
disease activity in juvenile idiopathic
arthritis. J. Fam. Med. Prim. Care 2019, 8,
2129–2133. https:
//doi.org/10.4103/jfmpc.jfmpc_339_19
SAA
SAA is a more sensitive laboratory marker
than ESR and CRP for assessing the
presence of active joints in JIA.
[218]. Shimizu, M.; Nakagishi, Y.; Inoue, N.;
Mizuta, M.; Yachie, A. Leucine-rich
α2-glycoprotein as the acute-phase reactant
to detect systemic juvenile idiopathic arthritis
disease activity during anti-interleukin-6
blockade therapy: A case series. Mod.
Rheumatol. 2017, 27, 833–837. https:
//doi.org/10.1080/14397595.2016.1270795
LRG
Serum LRG levels might be a potential
biomarker of sJIA disease activity during
IL-6 blockade treatment.

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Table 4. Cont.
Reference/Year Chemokines and Other Biomarkers Predictive Effects in sJIA and MAS
[219]. Shimizu, M.; Inoue, N.; Mizuta, M.;
Nakagishi, Y; Yachie, A. Serum
Leucine-Rich α2-Glycoprotein as a
Biomarker for Monitoring Disease Activity
in Patients with Systemic Juvenile
Idiopathic Arthritis. J. Immunol. Res. 2019,
2019, 3140204.
https://doi.org/10.1155/2019/3140204
Serum LRG levels were positively
correlated with serum CRP and ferritin
levels in sJIA and reflected disease activity;
thus, they may be useful for monitoring
sJIA disease activity.
[220]. Lee, P.Y.; Schulert, G.S.; Canna, S.W.;
Huang, Y.; Sundel, J.; Li, Y.; Hoyt, K.J.;
Blaustein, R.B.; Wactor, A.; Do, T.; et al.
Adenosine deaminase 2 as a biomarker of
macrophage activation syndrome in
systemic juvenile idiopathic arthritis. Ann.
Rheum. Dis. 2020, 79, 225–231. https://doi.
org/10.1136/annrheumdis-2019-216030
ADA2
The utility of research on plasma ADA2
activity as a future diagnostic biomarker of
sJIA-MAS was demonstrated. The
monocyte/macrophage origin of ADA2
and induction by IL-18 and IFN-γ provide
further support for the key role of MAS in
this life-threatening complication of sJIA.
3. Final Remarks and Conclusions
The COVID-19 pandemic—along with the associated CS—was and still is a challenge
for 21st century medicine. Concretely, in the current crisis, solutions were sought, and
initially an approach was even tried using some drugs inspired by rheumatology, including
pediatric rheumatology. The emergence of multisystem inflammatory syndrome in children
(MIS-C) was another test for pediatricians, because children and adolescents exposed to
the SARS-CoV-2 virus—who, even if they initially only had mild forms of the disease, or
were only contacts of people who were infected— developed a fulminant progression of a
disease very similar to MAS after about 2–4 weeks, which put their lives in danger [11,221].
Being increasingly common, hyperinflammatory syndromes in pediatrics—including
sJIA-MAS—rely on new biological disease-modifying anti-rheumatic drugs (bDMARDs)
targeting various cytokines involved in the pathophysiological mechanisms of the diseases,
which pediatric rheumatologists have begun to administer successfully more and more
often. A CS, or hypercytokinemia, is initially a physiological response in which the innate
immune system triggers an exaggerated and extreme discharge of pro-inflammatory signaling molecules, which would normally represent the immune system’s response to infection
or immunotherapy, but the unexpected huge amounts of cytokines can cause multiorgan
failure and death. There is still no single universally accepted definition of CS, as it differs
depending on the corresponding inflammatory response [222].
CSS is an umbrella term for a systemic inflammatory condition [223] that results
in the killing of both healthy and diseased cells, manifested by fever, fatigue, headache,
rash, and muscle and/or joint aches, and may advance to more serious symptoms such
as hypotension, tachycardia, vasodilatation, edema, hypoxemia, seizures, and even coma.
Prompt and correct diagnosis, as well as the appropriate emergency management through
immunosuppressive drugs, can hinder the irreversible multiorgan failure and save the
patient’s life.
In pediatric pathology, within CSS, MAS can appear relatively frequently in children
with sJIA—considered a prototype for this major complication that can even threaten
their lives, just like MIS-C after infection with the SAR-CoV-2 virus during the COVID19 pandemic. The major difficulty for pediatric rheumatologists is to make the correct
differential diagnosis between active sJIA and MAS that has recently started or is already
in progress, because these two entities have common clinical characteristics such as high
fever, skin eruptions, and other systemic signs of inflammation which, if not identified in
time, will evolve into hepatosplenic dysfunction, pancytopenia, consumption coagulopathy,
hyperferritinemia and, finally, multiorgan failure [16,223,224].

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sJIA-MAS continues to be a major problem in pediatric rheumatology due to its
morbidity, mortality, and lack of standardization for early and successful diagnosis.
To be as close as possible to reality and clinical requirements, new model biomarkers
are needed for an individualized approach to ensure the accurate assessment and management of patients with sJIA. In the current clinical practice generalized to date, a certain
number of specific biomarkers for JIA have already been adopted by expert guidelines
and are currently applied, such as ESR, CRP, serum ferritin, RF, HLA-B27 molecule, ANA,
etc., which can help clinicians to better discriminate between the different subtypes and
anticipate the response to the applied therapy, as well as relapses, which ideally should
indicate the most appropriate medication for that patient and the point at which the therapy
can be safely stopped after remission [225].
Already, in many clinics around the world that have significant funding for research
and patient care—but especially those with ultrahigh-performance equipment and special
analysis kits—new biomarkers are used to diagnose sJIA-MAS as quickly and accurately as
possible and to predict the response to the established emergency treatment in this aggravating, disastrous, and potentially deadly complication of sJIA, which may be misleading
for pediatric clinicians because it is so hard to diagnose and manage.
According to clinical studies, more than one-third of patients with sJIA may suffer
from a subclinical form of MAS which, if recognized in time, could be managed long before
reaching fully developed sJIA-MAS.
In children with subclinical sJIA-MAS, some biomarkers that highlight the extent of
T-cell activation and are a measure for the escalation of phagocytic macrophages—such as
sCD25 (sIL2RA) and sCD163—should be investigated to support the recognition of MAS.
In 2007, Bleesing et al. showed that elevated serum concentrations of sCD25 and
sCD163 may be predictive biomarkers of sJIA-MAS and could help detect patients with
subclinical MAS [148].
A few years later, Reddy et al. indicated that the presence of sCD25 levels > 7500 pg/mL
can be considered to be a useful biomarker in the detection of patients with subclinical
MAS [149].
In 2018, Sakumura et al., studying 63 patients with sJIA, concluded that sCD163 is a
potential biomarker for the evaluation of disease activity and remission, faithfully reflecting
macrophage activation in sJIA—even for patients treated with TCZ; its levels increased
dramatically with the progression of MAS in sJIA [150].
In 2021, Verweyen et al. studied how distinct gene expression signatures are able to
characterize strong clinical responders versus non-responders to canakinumab therapy in
sJIA patients, and indicated that a signature including upregulated CD163 expression was
associated with the non-response to this treatment [151].
Two other important biomarkers are the chemokines CXCL9 and CXCL10, which
play an important role in the local recruitment of CD8+ T cells to tissues. Some scientific
research has shown high serum concentrations of IFN-γ and IFN-γ-induced cytokines such
as IL-18BP, CXCL9, and CXCL10 in sJIA-MAS, compared to their concentrations in active
sJIA, inactive disease, or healthy controls. Serum values of CXCL9 and CXCL10 became
smaller with remission of sJIA-MAS. Thus, these two biomarkers highlight the tissue
activity of the IFN-γ pathway, and their ability to recruit CD8+ T cells—i.e., CXCL9 and
CXCL10—correlates very well with the laboratory parameters of MAS, providing a strongly
evidenced reason to act for in the vent of an IFN-γ blockage in sJIA-MAS [16,21,128,129].
According to other recent studies, CXCL9 has been shown to display the most significant increase after the onset of sJIA-MAS, and monitoring its serum levels would allow
physicians to conduct surveillance of sJIA-MAS.
Concerning the response to canakinumab in sJIA, a differential regulation of the
IL-18–IFN-γ–CXCL9 axis has been proven in patients with sJIA, and the higher the IL18/CXCL9 and IFN-γ/CXCL9 ratios, the better the clinical response to canakinumab
treatment [146,147].

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Neopterin is released by macrophages stimulated by IFN-γ and could be a useful
biomarker of immune system activation that can indicate disease activity in sJIA-MAS,
while also allowing the diagnosis of the transition to MAS in active sJIA [143].
Neopterin could be an accurate biomarker for the evaluation of underlying inflammatory processes and the flares of sJIA, together with traditional laboratory parameters for
diagnosis, clinical follow-up, and response to therapy. Concentrations of neopterin, IFN-γ,
CXCL9, CXCL10, sCD25, and IL-18 can be used as biomarkers to determine the onset of
MAS in active sJIA—even in children who have been prescribed TCZ [67,130,137,138].
Serum concentrations of MRP8/14 have been confirmed as an important biomarker for
the diagnosis of sJIA, ensuring early discrimination from other inflammatory conditions so
that targeted therapy for the patient’s benefit can be started immediately when the “window
of opportunity” is open, stopping a possible persistent chronic outcome which, until the
discovery of bDMARDs, was inevitable in more than 50% of patients [17,18,58,153].
The role of NK cells in sJIA-MAS has been widely investigated, but is still difficult
to fully understand due to the small size or insufficient number of studies and conflicting
conclusions. However, for sJIA-MAS, no strong genetic or transcriptional impairments
in NK cells’ killing pathways were detected. Functional deficits of NK cells have been
identified in sJIA-MAS due to inflammation-induced NK cell exhaustion. The high constitutive levels of cytokines (e.g., IL-6 and IL-18) could trigger the cytokine-induced NK
cell dysfunction that fails to stop the immune response; thus, continuous inflammatory
processes are generated [226].
The dramatic decrease in the number of figurative elements of the blood, coagulation abnormalities, significant liver cytolysis, and the increase in LDH as well as serum
ferritin are important laboratory data for the identification of patients with sJIA-MAS. A
concentration above 3500 µg/L has 85% sensitivity and 97% predictability for sJIA-MAS,
and extreme hyperferritinemia detected at the time of sJIA diagnosis may be a biomarker
to foreshadow sJIA-MAS [227].
Serum ferritin is accepted as an acute-phase reactant along with CRP, ESR, Fg, etc. In
sJIA, high levels of ferritin and low levels of fibrinogen are inceptive evidence of inevitable
sJIA-MAS [228].
High levels of platelets, CRP, and serum ferritin are specific biomarkers for the diagnosis MAS. Elevated serum ferritin concentrations in sJIA patients were correlated with
active disease during progression, even when the disease activity decreased. Its status
as a biomarker of disease activity is well-known, but the serum ferritin/ESR ratio may
be a more valuable biomarker than ferritin alone in differentiating MAS from new-onset
sJIA [200,229,230].
In discriminating between sJIA with and without MAS, the importance of the ferritin/ESR ratio, as well as the early identification of significant changes in serum ferritin
and neutrophil, leukocyte, and platelet counts, is essential in the early stages of MAS, as
evidenced by other recent studies [231].
Because many proposed new biomarkers will be difficult to implement in current
clinical use due to their high cost or difficulties in implementation, an ideal candidate could
be chosen from among the conventional laboratory biomarkers used already for assessing
the evolution of clinical symptoms, such as the ferritin/ESR ratio, neutrophil/lymphocyte
ratio, etc.
A neutrophil/lymphocyte ratio greater than 5.23 predicts the need for early biological
treatment in sJIA and MAS [232].
Although sJIA-MAS was classified by expert consensus in 2016, today, after 6 years,
new research insights are essential.
Biomarkers in sJIA-MAS could be relevant to differentiate disease subtypes, to assess
disease status and activity, and to predict responses to initial treatment. They can also
be very valuable tools in the pediatric rheumatologist’s decision to stop medication in a
patient who has entered clinical remission.

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There is no updated consensus regarding the validation, standardization, and reproducibility of methods for the determination and implementation of the most predictive
biomarkers for the diagnosis of sJIA-MAS, the progress of MAS, the emergency treatment
to be applied, the follow-up of the response, and the change of treatment in the event of
emergency to save the life of the patient.
It was suggested that the limitations imposed by each method of determining a single
biomarker could be overcome by using a comprehensive picture of the pathological aspects
regarding the evolution of MAS and the very rapid use of several clinical parameters and
laboratory data to include many more biomarkers.
A clinically useful biomarker, in addition to being correctly measured, must have
diagnostic and prognostic value in order to correlate well with the disease extent. It must
also be reasonably stable, present in an easily accessible sample, and its measurement
should be cost-effective. As of more recently, a multiple factor analysis that allows for
simultaneous examination of multiple parameters—classified according to their physiological meaning—could be applied. On the other hand, in sJIA-MAS, the choice of biomarkers
to be considered in the global assessment score should dictate their clinical relevance in the
examined patients.
Machine learning analysis was efficient in differentiating between JIA patients and
healthy controls, with ~90% precision. Analysis of the immune profiles could reveal
immunological disturbances in patients with different JIA subtypes, but was much more
important in patients with sJIA and in those with active disease. Such results open new
perspectives for large-scale immunophenotyping in longitudinal studies of JIA, which will
be able to better distinguish between JIA patients and healthy subjects, while the addition
of machine learning could foretell the response to treatment [233].
The clinical significance of the biomarkers in sJIA and sJIA-MAS results from a comprehensive analysis of the biomarkers revealed in all of the abovementioned revised studies,
and is highlighted as a synthesis in the following diagram (Figure 1):
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 52 of 66
Figure 1. Biomarkers in sJIA and sJIA-MAS: diagnosis; disease activity; predictors of sJIA-MAS;
diagnosis of sJIA-MAS; response to therapy in sJIA-MAS; and relapse. (Figure 1 was conceptualized
and drawn by L.M.A. using Microsoft Paint 3D for Windows 10). Legend: [↑ = increasing/high; ↑↑ =
very high; ↓ = decreasing/low; ↓↓ = very low; “+” = present; ± = present/absent; R = response].
Since the processes in sJIA-MAS take place extremely rapidly, it is imperative that
the responsible pediatrician has adequate biomarkers to label the disease, initiate therapy,
test correctly and quickly for response, and immediately prescribe a new drug if the
patient does not respond.
sJIA and sJIA-MAS remain a call to participate in an open competition for clinicians
to solve all of the questions that do not yet have a satisfactory answer, e.g., what are the
most valuable biomarkers for an immediate and correct diagnosis? Which is the
pathogenic pathway that needs to be blocked first? What is the best management practice?
How should treatment be individualized and balanced?
Figure 1. Biomarkers in sJIA and sJIA-MAS: diagnosis; disease activity; predictors of sJIA-MAS;
diagnosis of sJIA-MAS; response to therapy in sJIA-MAS; and relapse. (Figure 1 was conceptualized
and drawn by L.M.A. using Microsoft Paint 3D for Windows 10). Legend: [↑ = increasing/high;
↑↑ = very high; ↓ = decreasing/low; ↓↓ = very low; “+” = present; ± = present/absent; R = response].

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Since the processes in sJIA-MAS take place extremely rapidly, it is imperative that the
responsible pediatrician has adequate biomarkers to label the disease, initiate therapy, test
correctly and quickly for response, and immediately prescribe a new drug if the patient
does not respond.
sJIA and sJIA-MAS remain a call to participate in an open competition for clinicians
to solve all of the questions that do not yet have a satisfactory answer, e.g., what are the
most valuable biomarkers for an immediate and correct diagnosis? Which is the pathogenic
pathway that needs to be blocked first? What is the best management practice? How
should treatment be individualized and balanced?
The clinical significance of biomarkers in sJIA-MAS should be delineated by the latest
scientific research, reanalyzed by a group of experts to draw new guidelines in clinical
practice through an objective analysis of all of the relevant predictive biomarkers with great
sensitivity and specificity, and to offer real support to clinicians to make the best decisions
and choices under certain conditions—especially in the COVID-19 era.
Author Contributions: Conceptualization, L.M.A. and C.A.; writing—original draft preparation,
L.M.A.; review and editing, G.L. and L.M.A. All authors have read and agreed to the published
version of the manuscript.
Funding: There was no special funding for this review article.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data supporting reported results can be found by the first author L.M.A.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ALL Acute lymphoblastic leukemia
ADA2 Adenosine deaminase 2
AOSD Adult-onset Still’s disease
AGEs Advanced glycation end products
AHSP Alpha hemoglobin-stabilizing protein
ACR American College of Rheumatology
ACR 50 American College of Rheumatology score = at least 50%
improvement in disease activity
ANK Anakinra
ANAs Antinuclear antibodies
Arg-1 Arginase-1
AST Aspartate aminotransferase
ADs Autoimmune diseases
AID Autoinflammatory disease
bDMARDs Biological disease-modifying anti-rheumatic drugs
S100A12 Calgranulin C
CP, or S100A8/A9, or MRP8/14 Calprotectin
CAN Canakinumab
CASP8 Caspase-8
CCL23 C-C motif chemokine ligand 23
CDC Centers for Disease Control and Prevention
CNS Central nervous system
CXCL1 . . . 11 Chemokine (C-X-C motif) ligand 1 . . . 11
CCL2 Chemokine (C-C motif) ligand 2
CXCL9 Chemokine (C-X-C motif) ligand 9
CXCL10 Chemokine (C-X-C motif) ligand 10
CX3CR1 Chemokine (C-X3-C motif) receptor 1
CID Clinically inactive disease

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CD Cluster of differentiation
CD163 Cluster of differentiation 163
CLPs Common lymphoid progenitors
CBC Complete blood count
CTD Connective tissue disease
cNK Conventional NK
csDMARDs Conventional synthetic disease-modifying antirheumatic drugs
CS Corticosteroid
CRP C-reactive protein
CSS Cytokine storm syndrome
DAMP Damage-associated molecular pattern
DNA Deoxyribonucleic acid
DEGs Differentially expressed genes
DMARDs Disease-modifying anti-rheumatic drugs
EHEC Enterohemorrhagic Escherichia coli
ELISA Enzyme-linked immunosorbent assay
EBV Epstein–Barr virus
EBV-HLH Epstein–Barr-virus-induced hemophagocytic
lymphohistiocytosis
ESR Erythrocyte sedimentation rate
fHLH Familial hemophagocytic lymphohistiocytosis
FMF Familial Mediterranean fever
FasL Fas ligand
FUO Fever of unknown origin
Fg Fibrinogen
FSTL-1 Follistatin-like protein 1
FDA Food and Drug Administration
CFA Freund’s complete adjuvant
GEO Gene Expression Omnibus
G-CSF Granulocyte colony-stimulating factor
GM-CSF Granulocyte–macrophage colony-stimulating factor
GZMK Granzyme K
HCs Healthy controls
HSC Hematopoietic stem cell
HLH Hemophagocytic lymphohistiocytosis
HMGB1 High-mobility group box-1
hsCRP High-sensitivity C-reactive protein
HLA Human leukocyte antigen
Igs Immunoglobulins
ILCs Innate lymphoid cells
ILC1 “Unconventional” NK cells
ICAM-5 Intercellular adhesion molecule 5
IFN-α Interferon-alpha
IFN-γ Interferon-gamma
IRF5 Interferon regulatory factor 5
IL-1, IL-6, IL-18 Interleukin 1, 6, 18
IL-18Rβ IL-18β receptor
IL-1 alpha, IL-1α; IL-1 beta, IL-1β Interleukin 1 alpha, beta
IL-1Ra Interleukin-1 receptor antagonist
IL10RA Interleukin-10 receptor, alpha subunit
IL6ST Interleukin-6 signal transducer
IL-18BP IL-18-binding protein
ICAM-1 Intracellular adhesion molecule 1

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IV Intravenous
JDM Juvenile dermatomyositis
JIA Juvenile idiopathic arthritis
JADAS Juvenile Arthritis Disease Activity Score
KD Kawasaki disease
LDH Lactate dehydrogenase
LRG Leucine-rich α2-glycoprotein
LRG1 Leucine-rich α-2 glycoprotein 1
LPSs Lipopolysaccharides
CD62L L-selectin
LCLs Lymphoblastoid cell lines
M Macrophage
M1 M1 macrophages (encourage inflammation)
M2 M2 macrophages (decrease inflammation and
encourage tissue repair)
MAS Macrophage activation syndrome
sJIA-MAS Macrophage activation syndrome in sJIA
MIF Macrophage migration inhibitory factor
MHC Major histocompatibility complex
MMP1 Matrix metalloproteinase 1
MMP3 Matrix metalloproteinase 3
MEFV Mediterranean fever
MTX Methotrexate
MAPKs Mitogen-activated protein kinases
MCP1 Monocyte chemoattractant protein 1
MIS-C Multisystem inflammatory syndrome in children
MPO–DNA complexes Myeloperoxidase–DNA complexes
MRP Myeloid-related protein
MRP8 Myeloid-related protein 8
MRP14 Myeloid-related protein 14
NCR2 or NKp44 Natural cytotoxicity-triggering receptor 2
NK Natural killer
NKT Natural killer T
NCAM or CD56 Neural cell adhesion molecule, also called CD56
NETs Neutrophil extracellular traps
NETosis NET activation and release
NLR NOD-like receptor
NSAIDs Non-steroidal anti-inflammatory drugs
nsJIA Non-systemic forms of JIA
NFIL3 Nuclear factor, interleukin 3-regulated gene
NF-κB or NF-kappaB Nuclear factor kappa-light-chain-enhancer of
activated B cells
NOD Nucleotide-binding oligomerization
NLRP Nucleotide-binding oligomerization domain,
leucine-rich repeat and Pyrin domain
NLRP3 Nucleotide-binding oligomerization domain-like
receptor pyrin domain-containing protein 3
NLRs Nucleotide-binding oligomerization domain-like
receptors = NOD-like receptors
No. Number; Latin “numero”, the ablative case of
“numerus”
PAMP Pathogen-associated molecular pattern
PRRs Pattern-recognition receptors
PRINTO Pediatric Rheumatology International Trials
Organization

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PRF1 Perforin 1
PBMC Peripheral blood mononuclear cell
PMLs or PMNLs Polymorphonuclear leukocytes
PMN Polymorphonuclear neutrophil
+ Present
± Present/absent
pHLH Primary hemophagocytic lymphohistiocytosis
PCT Procalcitonin
PD-L1 Programmed death ligand-1
PKC Protein kinase C
qRT-PCR Quantitative reverse-transcription polymerase chain
reaction
ROS Reactive oxygen species
TNFSF11 Receptor activator of nuclear factor-κB ligand, RANKL
RAGE Receptor for advanced glycation end products
rIL-1Ra Recombinant IL-1 receptor antagonist
rr Reference range
R Response
RMD Rheumatic and musculoskeletal disease
RF Rheumatoid factor
S100A9 S100 calcium-binding protein A9
MS score Score for sJIA-MAS
sec-HLH Secondary hemophagocytic lymphohistiocytosis
SAA Serum amyloid A
sCP Serum calprotectin
sPCT Serum procalcitonin
SIF Severe infections
SNP Single-nucleotide polymorphism
sCD163 Soluble cluster of differentiation 163
sCD25 or sIL2RA Soluble IL-2 receptor alpha chain, or soluble IL2RA
sRAGE Soluble receptor for advanced glycation end products
sTNFR-I Soluble tumor necrosis factor receptor I
sTNFR-II Soluble tumor necrosis factor receptor II
sTNF-R II/I Soluble TNF receptor II/I
SF Synovial fluid
sJIA Systemic juvenile idiopathic arthritis
SLE Systemic lupus erythematosus
TCR T-cell receptor
Th cells, CD4+ or CD4-positive T-helper cells, or CD4+ cells, or CD4-positive cells
trNK Tissue-resident (tr)NK
TWEAK TNF-like weak inducer of apoptosis
TCZ Tocilizumab
TLR Toll-like receptor
TGF-β Transforming growth factor beta
TAMs Tumor-associated macrophages
TNF-α Tumor necrosis factor alpha
TNFR1 Tumor necrosis factor receptor 1
FcγRIII Type III Fcγ receptor
WGCNA Weighted gene co-expression network analysis
WT Wild type
Increasing/high ↑
Very high ↑↑
Decreasing/low ↓
Very low ↓↓

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References
1. Hinze, C.; Gohar, F.; Foell, D. Management of juvenile idiopathic arthritis: Hitting the target. Nat. Rev. Rheumatol. 2015, 11,
290–300. [CrossRef] [PubMed]
2. Ravelli, A.; Consolaro, A.; Horneff, G.; Laxer, R.M.; Lovell, D.J.; Wulffraat, N.M.; Akikusa, J.D.; Al-Mayouf, S.M.; Antón, J.; Avcin,
T.; et al. Treating juvenile idiopathic arthritis to target: Recommendations of an international task force. Ann. Rheum. Dis. 2018,
77, 819–828. [CrossRef] [PubMed]
3. Ailioaie, L.M.; Litscher, G. Molecular and Cellular Mechanisms of Arthritis in Children and Adults: New Perspectives on Applied
Photobiomodulation. Int. J. Mol. Sci. 2020, 21, 6565. [CrossRef] [PubMed]
4. Guzman, J.; Oen, K.; Huber, A.M.; Watanabe Duffy, K.; Boire, G.; Shiff, N.; Berard, R.A.; Levy, D.M.; Stringer, E.; Scuccimarri, R.;
et al. The risk and nature of flares in juvenile idiopathic arthritis: Results from the ReACCh-Out cohort. Ann. Rheum. Dis. 2016,
75, 1092–1098. [CrossRef] [PubMed]
5. Horton, D.B.; Salas, J.; Wec, A.; Kohlheim, M.; Kapadia, P.; Beukelman, T.; Boneparth, A.; Haverkamp, K.; Mannion, M.L.; Moorthy,
L.N.; et al. Making Decisions About Stopping Medicines for Well-Controlled Juvenile Idiopathic Arthritis: A Mixed-Methods
Study of Patients and Caregivers. Arthritis Care Res. 2021, 73, 374–385. [CrossRef]
6. Gerss, J.; Tedy, M.; Klein, A.; Horneff, G.; Miranda-Garcia, M.; Kessel, C.; Holzinger, D.; Stanevica, V.; Swart, J.F.; Cabral, D.A.;
et al. Prevention of disease flares by risk-adapted stratification of therapy withdrawal in juvenile idiopathic arthritis: Results
from the PREVENT-JIA trial. Ann. Rheum. Dis. 2022, 81, 990–997. [CrossRef]
7. Lee, J.; Schneider, R. Systemic juvenile idiopathic arthritis. Pediatr Clin. N. Am. 2018, 65, 691–709. [CrossRef]
8. Barut, K.; Adrovic, A.; Sahin, S.; Tarcin, G.; Tahaoglu, G.; Koker, O.; Yildiz, M.; Kasapcopur, O. Prognosis, complications and
treatment response in systemic juvenile idiopathic arthritis patients: A single-center experience. Int. J. Rheum. Dis. 2019, 22,
1661–1669. [CrossRef]
9. Ter Haar, N.M.; van Dijkhuizen, E.H.P.; Swart, J.F.; van Royen-Kerkhof, A.; El Idrissi, A.; Leek, A.P.; de Jager, W.; de Groot, M.C.H.;
Haitjema, S.; Holzinger, D. Treatment to Target Using Recombinant Interleukin-1 Receptor Antagonist as First-Line Monotherapy
in New-Onset Systemic Juvenile Idiopathic Arthritis: Results From a Five-Year Follow-Up Study. Arthritis Rheumatol. 2019, 71,
1163–1173. [CrossRef]
10. Newswise. The American College of Rheumatology Releases Two Updated Guidelines for Treatment of Juvenile Idiopathic
Arthritis. Available online: https://www.newswise.com/articles/the-american-college-of-rheumatology-releases-two-updatedguidelines-for-treatment-of-juvenile-idiopathic-arthritis (accessed on 10 July 2022).
11. Ailioaie, L.M.; Ailioaie, C.; Litscher, G. Implications of SARS-CoV-2 Infection in Systemic Juvenile Idiopathic Arthritis. Int. J. Mol.
Sci. 2022, 23, 4268. [CrossRef]
12. Moradinejad, M.H.; Ziaee, V. The incidence of macrophage activation syndrome in children with rheumatic disorders. Minerva
Pediatr. 2011, 63, 459–466. [PubMed]
13. Ravelli, A.; Grom, A.A.; Behrens, E.M.; Cron, R.Q. Macrophage activation syndrome as part of systemic juvenile idiopathic
arthritis: Diagnosis, genetics, pathophysiology and treatment. Genes Immun. 2012, 13, 289–298. [CrossRef] [PubMed]
14. Schulert, G.S.; Grom, A.A. Pathogenesis of macrophage activation syndrome and potential for cytokine-directed therapies. Annu.
Rev. Med. 2015, 66, 145–159. [CrossRef] [PubMed]
15. Ravelli, A.; Minoia, F.; Davì, S.; Horne, A.; Bovis, F.; Pistorio, A.; Aricò, M.; Avcin, T.; Behrens, E.M.; De Benedetti, F.; et al. 2016
Classification Criteria for Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis: A European
League Against Rheumatism/American College of Rheumatology/Paediatric Rheumatology International Trials Organisation
Collaborative Initiative. Arthritis Rheumatol. 2016, 68, 566–576. [CrossRef]
16. Schneider, R.; Canny, S.P.; Mellins, E.D. Cytokine Storm Syndrome Associated with Systemic Juvenile Idiopathic Arthritis. In
Cytokine Storm Syndrome, 1st ed.; Cron, R.Q., Behrens, E.M., Eds.; Springer Nature Switzerland AG: Cham, Switzerland, 2019;
pp. 349–379. [CrossRef]
17. Pardeo, M.; Vastert, S.J.; De Benedetti, F. It is about time: The first validated biomarker for early diagnosis of systemic juvenile
idiopathic arthritis. Rheumatology 2021, 61, 2724–2725. [CrossRef]
18. Park, C.; Miranda-Garcia, M.; Berendes, R.; Horneff, G.; Kuemmerle-Deschner, J.; Ganser, G.; Huppertz, H.I.; Minden, K.; Haas,
J.P.; Jansson, A.F.; et al. MRP8/14 serum levels as diagnostic markers for systemic juvenile idiopathic arthritis in children with
prolonged fever. Rheumatology 2022, 61, 3082–3092. [CrossRef]
19. Ruperto, N.; Brunner, H.I.; Quartier, P.; Constantin, T.; Wulffraat, N.; Horneff, G.; Brik, R.; McCann, L.; Kasapcopur, O.;
Rutkowska-Sak, L.; et al. Two randomized trials of canakinumab in systemic juvenile idiopathic arthritis. N. Engl. J. Med. 2012,
367, 2396–2406. [CrossRef]
20. De Benedetti, F.; Meazza, C.; Vivarelli, M.; Rossi, F.; Pistorio, A.; Lamb, R.; Lunt, M.; Thomson, W.; Ravelli, A.; Donn, R.;
et al. Functional and prognostic relevance of the -173 polymorphism of the macrophage migration inhibitory factor gene in
systemic-onset juvenile idiopathic arthritis. Arthritis Rheum. 2003, 48, 1398–1407. [CrossRef]
21. Yanagimachi, M.; Naruto, T.; Miyamae, T.; Hara, T.; Kikuchi, M.; Hara, R.; Imagawa, T.; Mori, M.; Sato, H.; Goto, H.; et al.
Association of IRF5 polymorphisms with susceptibility to macrophage activation syndrome in patients with juvenile idiopathic
arthritis. J. Rheumatol. 2011, 38, 769–774. [CrossRef]

52/61

53/61

Int. J. Mol. Sci. 2022, 23, 12757 54 of 61
46. Fukui, S.; Iwamoto, N.; Takatani, A.; Igawa, T.; Shimizu, T.; Umeda, M.; Nishino, A.; Horai, Y.; Hirai, Y.; Koga, T.; et al. M1 and
M2 monocytes in rheumatoid arthritis: A contribution of imbalance of M1/M2 monocytes to osteoclastogenesis. Front. Immunol.
2017, 8, 1958. [CrossRef]
47. Feng, D.; Huang, W.Y.; Niu, X.L.; Hao, S.; Zhang, L.N.; Hu, Y.J. Significance of Macrophage Subtypes in the Peripheral Blood of
Children with Systemic Juvenile Idiopathic Arthritis. Rheumatol. Ther. 2021, 8, 1859–1870. [CrossRef]
48. Sridharan, R.; Cavanagh, B.; Cameron, A.R.; Kelly, D.J.; O’Brien, F.J. Material stiffness influences the polarization state, function
and migration mode of macrophages. Acta Biomater. 2019, 89, 47–59. [CrossRef]
49. Wang, Q.; Ni, H.; Lan, L.; Wei, X.; Xiang, R.; Wang, Y. Fra-1 protooncogene regulates IL-6 expression in macrophages and
promotes the generation of M2d macrophages. Cell Res. 2010, 20, 701–712. [CrossRef]
50. Funes, S.C.; Rios, M.; Escobar-Vera, J.; Kalergis, A.M. Implications of macrophage polarization in autoimmunity. Immunology
2018, 154, 186–195. [CrossRef]
51. Wang, L.X.; Zhang, S.X.; Wu, H.J.; Rong, X.L.; Guo, J. M2b macrophage polarization and its roles in diseases. J. Leukoc. Biol. 2019,
106, 345–358. [CrossRef]
52. Fischer-Riepe, L.; Daber, N.; Schulte-Schrepping, J.; Véras De Carvalho, B.C.; Russo, A.; Pohlen, M.; Fischer, J.; Chasan, A.I.; Wolf,
M.; Ulas, T.; et al. CD163 expression defines specific, IRF8-dependent, immune-modulatory macrophages in the bone marrow. J.
Allergy Clin. Immunol. 2020, 146, 1137–1151. [CrossRef]
53. Thornton, S.; Tan, R.; Sproles, A.; Do, T.; Schick, J.; Grom, A.A.; DeLay, M.; Schulert, G.S. A Multiparameter Flow Cytometry
Analysis Panel to Assess CD163 mRNA and Protein in Monocyte and Macrophage Populations in Hyperinflammatory Diseases.
J. Immunol. 2019, 202, 1635–1643. [CrossRef] [PubMed]
54. Silvestre-Roig, C.; Fridlender, Z.G.; Glogauer, M.; Scapini, P. Neutrophil diversity in health and disease. Trends Immunol. 2019, 40,
565–583. [CrossRef] [PubMed]
55. Fousert, E.; Toes, R.; Desai, J. Neutrophil extracellular traps (NETs) take the central stage in driving autoimmune responses. Cells
2020, 9, 915. [CrossRef] [PubMed]
56. Ng, L.G.; Ostuni, R.; Hidalgo, A. Heterogeneity of neutrophils. Nat. Rev. Immunol. 2019, 19, 255–265. [CrossRef]
57. Gohar, F.; Kessel, C.; Lavric, M.; Holzinger, D.; Foel, D. Review of biomarkers in systemic juvenile idiopathic arthritis: Helpful
tools or just playing tricks? Arthritis Res. Ther. 2016, 18, 163. [CrossRef]
58. Nigrovic, P.A. Review: Is there a window of opportunity for treatment of systemic juvenile idiopathic arthritis? Arthritis Rheumatol.
2014, 66, 1405–1413. [CrossRef]
59. Ter Haar, N.M.; Tak, T.; Mokry, M.; Scholman, R.C.; Meerding, J.M.; de Jagerz, W.; Verwoerd, A.; Foell, D.; Vogl, T.; Roth, J.; et al.
Reversal of Sepsis-Like Features of Neutrophils by Interleukin-1 Blockade in Patients with Systemic-Onset Juvenile Idiopathic
Arthritis. Arthritis Rheumatol. 2018, 70, 943–956. [CrossRef]
60. Brown, R.A.; Henderlight, M.; Do, T.; Yasin, S.; Grom, A.A.; DeLay, M.; Thornton, S.; Schulert, G.S. Neutrophils from Children
with Systemic Juvenile Idiopathic Arthritis Exhibit Persistent Proinflammatory Activation Despite Long-Standing Clinically
Inactive Disease. Front. Immunol. 2018, 9, 2995. [CrossRef]
61. Malengier-Devlies, B.; Metzemaekers, M.; Wouters, C.; Proost, P.; Matthys, P. Neutrophil Homeostasis and Emergency Granulopoiesis: The Example of Systemic Juvenile Idiopathic Arthritis. Front. Immunol. 2021, 12, 766620. [CrossRef]
62. Torres-Ruiz, J.; Carrillo-Vázquez, D.A.; Tapia-Rodríguezc, M.; Garcia-Galicia, J.A.; Alcocer-Varela, J.; Gómez-Martín, D. The role
of low-density granulocytes and NETosis in the pathogenesis of adult-onset Still’s Disease. Clin. Exp. Rheumatol. 2019, 37, 74–82.
[PubMed]
63. Jung, J.Y.; Kim, J.W.; Suh, C.H.; Kim, H.A. Roles of Interactions Between Toll-Like Receptors and Their Endogenous Ligands
in the Pathogenesis of Systemic Juvenile Idiopathic Arthritis and Adult-Onset Still’s Disease. Front. Immunol. 2020, 11, 583513.
[CrossRef] [PubMed]
64. Kim, J.-W.; Ahn, M.H.; Jung, J.Y.; Suh, C.H.; Kim, H.A. An Update on the Pathogenic Role of Neutrophils in Systemic Juvenile
Idiopathic Arthritis and Adult-Onset Still’s Disease. Int. J. Mol. Sci. 2021, 22, 13038. [CrossRef] [PubMed]
65. Ravelli, A.; Minoia, F.; Davì, S.; Horne, A.; Bovis, F.; Pistorio, A.; Aricò, M.; Avcin, T.; Behrens, E.M.; De Benedetti, F.; et al. Expert
consensus on dynamics of laboratory tests for diagnosis of macrophage activation syndrome complicating systemic juvenile
idiopathic arthritis. RMD Open 2016, 2, e000161. [CrossRef] [PubMed]
66. Crayne, C.B.; Albeituni, S.; Nichols, K.E.; Cron, R.Q. The Immunology of Macrophage Activation Syndrome. Front. Immunol.
2019, 10, 119. [CrossRef] [PubMed]
67. De Matteis, A.; Pires Marafon, D.; Caiello, I.; Pardeo, M.; Marucci, G.; Sacco, E.; Prencipe, G.; De Benedetti, F.;
Bracaglia, C. Traditional Laboratory Parameters and New Biomarkers in Macrophage Activation Syndrome and Secondary Hemophagocytic Lymphohistiocytosis [abstract]. Arthritis Rheumatol. 2020, 72 (Suppl. S4). Available online:
https://acrabstracts.org/abstract/traditional-laboratory-parameters-and-new-biomarkers-in-macrophage-activationsyndrome-and-secondary-hemophagocytic-lymphohistiocytosis/ (accessed on 21 August 2022).
68. Jamilloux, Y.; Georgin-Lavialle, S.; Sève, P.; Belot, A.; Fautrel, B. Le temps est venu de réconcilier l’arthrite juvénile idiopathique
systémique et la maladie de Still de l’adulte [It is time to reconcile systemic juvenile idiopathic arthritis and adult-onset Still’s
disease]. Rev. Med. Interne 2019, 40, 635–636. [CrossRef]

54/61

Int. J. Mol. Sci. 2022, 23, 12757 55 of 61
69. Minoia, F.; Davì, S.; Horne, A.; Demirkaya, E.; Bovis, F.; Li, C.; Lehmberg, K.; Weitzman, S.; Insalaco, A.; Wouters, C.; et al. Clinical
features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: A
multinational, multicenter study of 362 patients. Arthritis Rheumatol. 2014, 66, 3160–3169. [CrossRef]
70. Vastert, S.; Prakken, B. Update on research and clinical translation on specific clinical areas: From bench to bedside: How insight
in immune pathogenesis can lead to precision medicine of severe juvenile idiopathic arthritis. Best practice & research. Clin.
Rheumatol. 2014, 28, 229–246. [CrossRef]
71. Pascual, V.; Allantaz, F.; Arce, E.; Punaro, M.; Banchereau, J. Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset
juvenile idiopathic arthritis and clinical response to IL-1 blockade. J. Exp. Med. 2005, 201, 1479–1486. [CrossRef]
72. Ogilvie, E.M.; Khan, A.; Hubank, M.; Kellam, P.; Woo, P. Specific gene expression profiles in systemic juvenile idiopathic arthritis.
Arthritis Rheum. 2007, 56, 1954–1965. [CrossRef]
73. Fall, N.; Barnes, M.; Thornton, S.; Luyrink, L.; Olson, J.; Ilowite, N.T.; Gottlieb, B.S.; Griffin, T.; Sherry, D.D.; Thompson, S.; et al.
Gene expression profiling of peripheral blood from patients with untreated new-onset systemic juvenile idiopathic arthritis reveals
molecular heterogeneity that may predict macrophage activation syndrome. Arthritis Rheum. 2007, 56, 3793–3804. [CrossRef]
[PubMed]
74. Vastert, S.J.; Jamilloux, Y.; Quartier, P.; Ohlman, S.; Osterling Koskinen, L.; Kullenberg, T.; Franck-Larsson, K.; Fautrel, B.; de
Benedetti, F. Anakinra in children and adults with Still’s disease. Rheumatology 2019, 58, vi9–vi22. [CrossRef]
75. Ruperto, N.; Brunner, H.I.; Quartier, P.; Constantin, T.; Wulffraat, N.M.; Horneff, G.; Kasapcopur, O.; Schneider, R.; Anton, J.;
Barash, J.; et al. Canakinumab in patients with systemic juvenile idiopathic arthritis and active systemic features: Results from
the 5-year long-term extension of the phase III pivotal trials. Ann. Rheum. Dis. 2018, 77, 1710–1719. [CrossRef] [PubMed]
76. Ling, X.B.; Park, J.L.; Carroll, T.; Nguyen, K.D.; Lau, K.; Macaubas, C.; Chen, E.; Lee, T.; Sandborg, C.; Milojevic, D.; et al. Plasma
profiles in active systemic juvenile idiopathic arthritis: Biomarkers and biological implications. Proteomics 2010, 10, 4415–4430.
[CrossRef] [PubMed]
77. Palomo, J.; Dietrich, D.; Martin, P.; Palmer, G.; Gabay, C. The interleukin (IL)-1 cytokine family—Balance between agonists and
antagonists in inflammatory diseases. Cytokine 2015, 66, 25–37. [CrossRef]
78. Kaneko, N.; Kurata, M.; Yamamoto, T.; Morikawa, S.; Masumoto, J. The role of interleukin-1 in general pathology. Inflamm. Regen.
2019, 39, 12. [CrossRef]
79. Quartier, P. Systemic Juvenile Idiopathic Arthritis/Pediatric Still’s Disease, a Syndrome but Several Clinical Forms: Recent
Therapeutic Approaches. J. Clin. Med. 2022, 11, 1357. [CrossRef]
80. Saccomanno, B.; Tibaldi, J.; Minoia, F.; Bagnasco, F.; Pistorio, A.; Guariento, A.; Caorsi, R.; Consolaro, A.; Gattorno, M.; Ravelli, A.
Predictors of Effectiveness of Anakinra in Systemic Juvenile Idiopathic Arthritis. J. Rheumatol. 2019, 46, 416–421. [CrossRef]
81. Lainka, E.; Baehr, M.; Raszka, B.; Haas, J.P.; Hügle, B.; Fischer, N.; Foell, D.; Hinze, C.; Weissbarth-Riedel, E.; Kallinich, T.; et al.
Experiences with IL-1 blockade in systemic juvenile idiopathic arthritis—Data from the German AID-registry. Pediatr. Rheumatol.
Online J. 2021, 19, 38. [CrossRef]
82. Wilson, D.C.; Marinov, A.D.; Blair, H.C.; Bushnell, D.S.; Thompson, S.D.; Chaly, Y.; Hirsch, R. Follistatin-like protein 1 is a
mesenchyme-derived inflammatory protein and may represent a biomarker for systemic-onset juvenile rheumatoid arthritis.
Arthritis Rheum. 2010, 62, 2510–2516. [CrossRef]
83. Mellins, E.D.; Macaubas, C.; Grom, A.A. Pathogenesis of systemic juvenile idiopathic arthritis: Some answers, more questions.
Nat. Rev. Rheumatol. 2011, 7, 416–426. [CrossRef] [PubMed]
84. Dinarello, C.A. Blocking IL-1 in systemic inflammation. J. Exp. Med. 2005, 201, 1355–1359. [CrossRef] [PubMed]
85. Monteagudo, L.A.; Boothby, A.; Gertner, E. Continuous Intravenous Anakinra Infusion to Calm the Cytokine Storm in Macrophage
Activation Syndrome. ACR Open Rheumatol. 2020, 2, 276–282. [CrossRef] [PubMed]
86. Wu, C.-Y.; Yang, H.-Y.; Huang, J.-L.; Lai, J.-H. Signals and Mechanisms Regulating Monocyte and Macrophage Activation in the
Pathogenesis of Juvenile Idiopathic Arthritis. Int. J. Mol. Sci. 2021, 22, 7960. [CrossRef] [PubMed]
87. Hinze, C.H.; Holzinger, D.; Lainka, E.; Haas, J.P.; Speth, F.; Kallinich, T.; Rieber, N.; Hufnagel, M.; Jansson, A.F.; Hedrich, C.; et al.
Practice and consensus-based strategies in diagnosing and managing systemic juvenile idiopathic arthritis in Germany. Pediatr.
Rheumatol. 2018, 16, 7. [CrossRef]
88. Toplak, N.; Blazina, Š.; Avˇcin, T. The role of IL-1 inhibition in systemic juvenile idiopathic arthritis: Current status and future
perspectives. Drug Des. Dev. Ther. 2018, 12, 1633–1643. [CrossRef]
89. Kelley, N.; Jeltema, D.; Duan, Y.; He, Y. The NLRP3 Inflammasome: An Overview of Mechanisms of Activation and Regulation.
Int. J. Mol. Sci. 2019, 20, 3328. [CrossRef]
90. Zhao, C.; Zhao, W. NLRP3 Inflammasome-A Key Player in Antiviral Responses. Front. Immunol. 2020, 11, 211. [CrossRef]
91. Kessel, C.; Lippitz, K.; Weinhage, T.; Hinze, C.; Wittkowski, H.; Holzinger, D.; Fall, N.; Grom, A.A.; Gruen, N.; Foell, D.
Proinflammatory Cytokine Environments Can Drive Interleukin-17 Overexpression by γ/δ T Cells in Systemic Juvenile Idiopathic
Arthritis. Arthritis Rheumatol. 2017, 69, 1480–1494. [CrossRef]
92. Brunner, H.I.; Quartier, P.; Alexeeva, E.; Constantin, T.; Kone-Paut, I.; Marzan, K.; Schneider, R.; Wulffraat, N.M.; Chasnyk, V.;
Tirosh, I.; et al. Efficacy and Safety of Canakinumab in Patients with Systemic Juvenile Idiopathic Arthritis With and Without
Fever at Baseline: Results From an Open-Label, Active-Treatment Extension Study. Arthritis Rheumatol. 2020, 72, 2147–2158.
[CrossRef]

55/61

Int. J. Mol. Sci. 2022, 23, 12757 56 of 61
93. Kostik, M.M.; Isupova, E.A.; Belozerov, K.; Likhacheva, T.S.; Suspitsin, E.N.; Raupov, R.; Masalova, V.V.; Chikova, I.A.; Dubko,
M.F.; Kalashnikova, O.V.; et al. Standard and increased canakinumab dosing to quiet macrophage activation syndrome in children
with systemic juvenile idiopathic arthritis. Front. Pediatr. 2022, 10, 894846. [CrossRef]
94. Vilaiyuk, S.; Lerkvaleekul, B.; Soponkanaporn, S.; Setthaudom, C.; Buranapraditkun, S. Correlations between serum interleukin 6,
serum soluble interleukin 6 receptor, and disease activity in systemic juvenile idiopathic arthritis patients treated with or without
tocilizumab. Cent. Eur. J. Immunol. 2019, 44, 150–158. [CrossRef]
95. de Benedetti, F.; Massa, M.; Robbioni, P.; Ravelli, A.; Burgio, G.R.; Martini, A. Correlation of serum interleukin-6 levels with joint
involvement and thrombocytosis in systemic juvenile rheumatoid arthritis. Arthritis Rheum. 1991, 34, 1158–1163. [CrossRef]
[PubMed]
96. Yilmaz, M.; Kendirl, S.G.; Altintas, D.; Bingol, G.; Antmen, B. Cytokine levels in serum of patients with juvenile rheumatoid
arthritis. Clin. Rheumatol. 2001, 20, 30–35. [CrossRef]
97. de Jager, W.; Hoppenreijs, E.P.; Wulffraat, N.M.; Wedderburn, L.R.; Kuis, W.; Prakken, B.J. Blood and synovial fluid cytokine
signatures in patients with juvenile idiopathic arthritis: A cross-sectional study. Ann. Rheum. Dis. 2007, 66, 589–598. [CrossRef]
[PubMed]
98. Brachat, A.H.; Grom, A.A.; Wulffraat, N.; Brunner, H.I.; Quartier, P.; Brik, R.; McCann, L.; Ozdogan, H.; Rutkowska-Sak, L.;
Schneider, R.; et al. Early changes in gene expression and inflammatory proteins in systemic juvenile idiopathic arthritis patients
on canakinumab therapy. Arthritis Res. Ther. 2017, 19, 13. [CrossRef] [PubMed]
99. Akioka, S. Interleukin-6 in juvenile idiopathic arthritis. Mod. Rheumatol. 2019, 29, 275–286. [CrossRef]
100. Schulert, G.S.; Minoia, F.; Bohnsack, J.; Cron, R.Q.; Hashad, S.; KonÉ-Paut, I.; Kostik, M.; Lovell, D.; Maritsi, D.; Nigrovic, P.A.;
et al. Effect of biologic therapy on clinical and laboratory features of macrophage activation syndrome associated with systemic
juvenile idiopathic arthritis. Arthritis Care Res. 2018, 70, 409–419. [CrossRef] [PubMed]
101. Chiran, D.A.; Weber, M.; Ailioaie, L.M.; Moraru, E.; Ailioaie, C.; Litscher, D.; Litscher, G. Intravenous laser blood irradiation and
tocilizumab in a patient with juvenile arthritis. Case Rep. Med. 2014, 2014, 923496. [CrossRef]
102. Bielak, M.; Husmann, E.; Weyandt, N.; Haas, J.P.; Hügle, B.; Horneff, G.; Neudorfc, U.; Lutz, T.; Lilienthal, E.; Kallinich, T.; et al.
IL-6 blockade in systemic juvenile idiopathic arthritis—Achievement of inactive disease and remission (data from the German
AID-registry). Pediatr. Rheumatol. 2018, 16, 22. [CrossRef]
103. Shimizu, M.; Mizuta, M.; Okamoto, N.; Yasumi, T.; Iwata, N.; Umebayashi, H.; Okura, Y.; Kinjo, N.; Kubota, T.; Nakagishi, Y.;
et al. Tocilizumab modifies clinical and laboratory features of macrophage activation syndrome complicating systemic juvenile
idiopathic arthritis. Pediatr. Rheumatol 2020, 18, 2. [CrossRef] [PubMed]
104. Qu, H.; Sundberg, E.; Aulin, C.; Neog, M.; Palmblad, K.; Horne, A.C.; Granath, F.; Ek, A.; Melén, E.; Olsson, M.; et al.
Immunoprofiling of active and inactive systemic juvenile idiopathic arthritis reveals distinct biomarkers: A single-center study.
Pediatr. Rheumatol. 2021, 19, 173. [CrossRef] [PubMed]
105. O’Garra, A.; Barrat, F.J.; Castro, A.G.; Vicari, A.; Hawrylowicz, C. Strategies for use of IL-10 or its antagonists in human disease.
Immunol. Rev. 2008, 223, 114–131. [CrossRef] [PubMed]
106. Steen, E.H.; Wang, X.; Balaji, S.; Butte, M.J.; Bollyky, P.L.; Keswani, S.G. The Role of the Anti-Inflammatory Cytokine Interleukin-10
in Tissue Fibrosis. Adv. Wound Care 2020, 9, 184–198. [CrossRef]
107. Imbrechts, M.; Avau, A.; Vandenhaute, J.; Malengier-Devlies, B.; Put, K.; Mitera, T.; Berghmans, N.; Burton, O.; Junius, S.; Liston,
A.; et al. Insufficient IL-10 Production as a Mechanism Underlying the Pathogenesis of Systemic Juvenile Idiopathic Arthritis. J.
Immunol. 2018, 201, 2654–2663. [CrossRef]
108. Peng, Y.; Liu, X.; Duan, Z.; Duan, J.; Zhou, Y. The Association of Serum IL-10 Levels with the Disease Activity in Systemic-Onset
Juvenile Idiopathic Arthritis Patients. Mediat. Inflamm. 2021, 2021, 6650928. [CrossRef]
109. Novick, D.; Kim, S.; Kaplanski, G.; Dinarello, C.A. Interleukin-18, more than a Th1 cytokine. Semin. Immunol. 2013, 25, 439–448.
[CrossRef]
110. Lotito, A.P.; Campa, A.; Silva, C.A.; Kiss, M.H.; Mello, S.B. Interleukin 18 as a marker of disease activity and severity in patients
with juvenile idiopathic arthritis. J. Rheumatol. 2007, 34, 823–830. [PubMed]
111. Shimizu, M.; Yokoyama, T.; Yamada, K.; Kaneda, H.; Wada, H.; Wada, T.; Toma, T.; Ohta, K.; Kasahara, Y.; Yachie, A. Distinct
cytokine profiles of systemic-onset juvenile idiopathic arthritis-associated macrophage activation syndrome with particular
emphasis on the role of interleukin-18 in its pathogenesis. Rheumatology 2010, 49, 1645–1653. [CrossRef]
112. Shimizu, M.; Nakagishi, Y.; Inoue, N.; Mizuta, M.; Ko, G.; Saikawa, Y.; Kubota, T.; Yamasaki, Y.; Takei, S.; Yachie, A. Interleukin-18
for predicting the development of macrophage activation syndrome in systemic juvenile idiopathic arthritis. Clin. Immunol. 2015,
160, 277–281. [CrossRef]
113. Xia, Y.; Cui, P.; Li, Q.; Liang, F.; Li, C.; Yang, J. Extremely elevated IL-18 levels may help distinguish systemic-onset juvenile
idiopathic arthritis from other febrile diseases. Braz. J. Med. Biol. Res. 2017, 50, e5958. [CrossRef] [PubMed]
114. Kaplanski, G. Interleukin-18: Biological properties and role in disease pathogenesis. Immunol. Rev. 2018, 281, 138–153. [CrossRef]
[PubMed]
115. Kudela, H.; Drynda, S.; Lux, A.; Horneff, G.; Kekow, J. Comparative study of Interleukin-18 (IL-18) serum levels in adult-onset
Still’s disease (AOSD) and systemic onset juvenile idiopathic arthritis (sJIA) and its use as a biomarker for diagnosis and
evaluation of disease activity. BMC Rheumatol. 2019, 3, 4. [CrossRef] [PubMed]

56/61

Int. J. Mol. Sci. 2022, 23, 12757 57 of 61
116. Yasin, S.; Fall, N.; Brown, R.A.; Henderlight, M.; Canna, S.W.; Girard-Guyonvarc’h, C.; Gabay, C.; Grom, A.A.; Schulert, G.S.
IL-18 as a biomarker linking systemic juvenile idiopathic arthritis and macrophage activation syndrome. Rheumatology 2020, 59,
361–366. [CrossRef]
117. Krei, J.M.; Møller, H.J.; Larsen, J.B. The role of interleukin-18 in the diagnosis and monitoring of hemophagocytic lymphohistiocytosis/macrophage activation syndrome—A systematic review. Clin. Exp. Immunol. 2021, 203, 174–182. [CrossRef]
118. Mizuta, M.; Shimizu, M.; Inoue, N.; Ikawa, Y.; Nakagishi, Y.; Yasuoka, R.; Iwata, N.; Yachie, A. Clinical significance of interleukin18 for the diagnosis and prediction of disease course in systemic juvenile idiopathic arthritis. Rheumatology 2021, 60, 2421–2426.
[CrossRef]
119. Girard-Guyonvarc’h, C.; Palomo, J.; Martin, P.; Rodriguez, E.; Troccaz, S.; Palmer, G.; Gabay, C. Unopposed IL-18 signaling leads
to severe TLR9-induced macrophage activation syndrome in mice. Blood 2018, 131, 1430–1441. [CrossRef]
120. Girard-Guyonvarc’h, C.; Harel, M.; Gabay, C. The Role of Interleukin 18/Interleukin 18-Binding Protein in Adult-Onset Still’s
Disease and Systemic Juvenile Idiopathic Arthritis. J. Clin. Med. 2022, 11, 430. [CrossRef]
121. Schulert, G.S. The IL-18/IFN-γ axis in systemic JIA and MAS—New answers, more questions. Rheumatology 2021, 60, 3045–3047.
[CrossRef]
122. Burke, J.D.; Young, H.A. IFN-γ: A cytokine at the right time, is in the right place. Semin. Immunol. 2019, 43, 101280. [CrossRef]
123. Merli, P.; Quintarelli, C.; Strocchio, L.; Locatelli, F. The role of interferon-gamma and its signaling pathway in pediatric
hematological disorders. Pediatr. Blood Cancer 2021, 68, e28900. [CrossRef]
124. Morales-Mantilla, D.E.; King, K.Y. The Role of Interferon-Gamma in Hematopoietic Stem Cell Development, Homeostasis, and
Disease. Curr. Stem Cell Rep. 2018, 4, 264–271. [CrossRef] [PubMed]
125. Locatelli, F.; Jordan, M.B.; Allen, C.; Cesaro, S.; Rizzari, C.; Rao, A.; Degar, B.; Garrington, T.P.; Sevilla, J.; Putti, M.C.; et al.
Emapalumab in Children with Primary Hemophagocytic Lymphohistiocytosis. N. Engl. J. Med. 2020, 382, 1811–1822. [CrossRef]
[PubMed]
126. Merli, P.; Algeri, M.; Gaspari, S.; Locatelli, F. Novel Therapeutic Approaches to Familial HLH (Emapalumab in FHL). Front.
Immunol. 2020, 11, 608492. [CrossRef]
127. Garonzi, C.; Chinello, M.; Cesaro, S. Emapalumab for adult and pediatric patients with hemophagocytic lymphohistiocytosis.
Expert Rev. Clin. Pharmacol. 2021, 14, 527–534. [CrossRef]
128. Put, K.; Avau, A.; Brisse, E.; Mitera, T.; Put, S.; Proost, P.; Bader-Meunier, B.; Westhovens, R.; Eynde, B.J.V.D.; Orabona, C.; et al.
Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: Tipping the balance between
interleukin-18 and interferon-γ. Rheumatology 2015, 54, 1507–1517. [CrossRef] [PubMed]
129. Bracaglia, C.; De Graaf, K.; Marafon, D.P.; Guilhot, F.; Ferlin, W.; Prencipe, G.; Caiello, I.; Davì, S.; Schulert, G.; Ravelli, A.; et al.
Elevated circulating levels of interferon-γ and interferon-γ-induced chemokines characterise patients with macrophage activation
syndrome complicating systemic juvenile idiopathic arthritis. Ann. Rheum. Dis. 2017, 76, 166–172. [CrossRef]
130. Bracaglia, C.; Pires Marafon, D.; Caiello, I.; de Graaf, K.; Ballabio, M.; Ferlin, W.; Davì, S.; Schulert, G.; Ravelli, A.; Grom,
A.A.; et al. Biomarkers for the Diagnosis and the Identification of Risk of Macrophage Activation Syndrome (MAS) in
Systemic Juvenile Idiopathic Arthritis (sJIA) [abstract]. Arthritis Rheumatol. 2017, 69 (Suppl. S10). Available online:
https://acrabstracts.org/abstract/biomarkers-for-the-diagnosis-and-the-identification-of-risk-of-macrophage-activationsyndrome-mas-in-systemic-juvenile-idiopathic-arthritis-sjia/ (accessed on 21 August 2022).
131. Guo, L.; Xu, Y.; Qian, X.; Zou, L.; Zheng, R.; Teng, L.; Zheng, Q.; Leung Jung, L.K.; Lu, M. Sudden Hypotension and Increased
Serum Interferon-γ and Interleukin-10 Predict Early Macrophage Activation Syndrome in Patients with Systemic Juvenile
Idiopathic Arthritis. J. Pediatr. 2021, 235, 203–211.e3. [CrossRef]
132. Sethi, J.K.; Hotamisligil, G.S. Metabolic Messengers: Tumour necrosis factor. Nat. Metab. 2021, 3, 1302–1312. [CrossRef]
133. Crayne, C.; Cron, R.Q. Pediatric macrophage activation syndrome, recognizing the tip of the Iceberg. Eur. J. Rheumatol. 2019, 7,
1–8. [CrossRef] [PubMed]
134. Jang, D.I.; Lee, A.H.; Shin, H.Y.; Song, H.R.; Park, J.H.; Kang, T.B.; Lee, S.R.; Yang, S.H. The Role of Tumor Necrosis Factor Alpha
(TNF-α) in Autoimmune Disease and Current TNF-α Inhibitors in Therapeutics. Int. J. Mol. Sci. 2021, 22, 2719. [CrossRef]
[PubMed]
135. Hügle, B.; Hinze, C.; Lainka, E.; Fischer, N.; Haas, J.P. Development of positive antinuclear antibodies and rheumatoid factor in
systemic juvenile idiopathic arthritis points toward an autoimmune phenotype later in the disease course. Pediatr. Rheumatol.
2014, 12, 28. [CrossRef]
136. Shimizu, M.; Inoue, N.; Mizuta, M.; Nakagishi, Y.; Yachie, A. Characteristic elevation of soluble TNF receptor II: I ratio in
macrophage activation syndrome with systemic juvenile idiopathic arthritis. Clin. Exp. Immunol. 2018, 191, 349–355. [CrossRef]
137. Irabu, H.; Shimizu, M.; Kaneko, S.; Inoue, N.; Mizuta, M.; Nakagishi, Y.; Yachie, A. Comparison of serum biomarkers for the
diagnosis of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis during tocilizumab therapy.
Pediatr. Res. 2020, 88, 934–939. [CrossRef]
138. Mizuta, M.; Shimizu, M.; Irabu, H.; Usami, M.; Inoue, N.; Nakagishi, Y.; Wada, T.; Yachie, A. Comparison of serum cytokine
profiles in macrophage activation syndrome complicating different background rheumatic diseases in children. Rheumatology
2021, 60, 231–238. [CrossRef]
139. Silva, T.A.; Garlet, G.P.; Lara, V.S.; Martins, W., Jr.; Silva, J.S.; Cunha, F.Q. Differential expression of chemokines and chemokine
receptors in inflammatory periapical diseases. Oral Microbiol. Immunol. 2005, 20, 310–316. [CrossRef] [PubMed]

57/61

Int. J. Mol. Sci. 2022, 23, 12757 58 of 61
140. Kwak, H.B.; Lee, S.W.; Jin, H.M.; Ha, H.; Lee, S.H.; Takeshita, S.; Tanaka, S.; Kim, H.M.; Kim, H.H.; Lee, Z.H. Monokine induced
by interferon-gamma is induced by receptor activator of nuclear factor kappa B ligand and is involved in osteoclast adhesion and
migration. Blood 2005, 105, 2963–2969. [CrossRef]
141. Raman, D.; Sobolik-Delmaire, T.; Richmond, A. Chemokines in health and disease. Exp. Cell Res. 2011, 317, 575–589. [CrossRef]
142. Ruytinx, P.; Proost, P.; Van Damme, J.; Struyf, S. Chemokine-Induced Macrophage Polarization in Inflammatory Conditions. Front.
Immunol. 2018, 9, 1930. [CrossRef]
143. Takakura, M.; Shimizu, M.; Irabu, H.; Sakumura, N.; Inoue, N.; Mizuta, M.; Nakagishi, Y.; Yachie, A. Comparison of serum
biomarkers for the diagnosis of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis. Clin.
Immunol. 2019, 208, 108252. [CrossRef]
144. House, I.G.; Savas, P.; Lai, J.; Chen, A.X.Y.; Oliver, A.J.; Teo, Z.L.; Todd, K.L.; Henderson, M.A.; Giuffrida, L.; Petley, E.V.; et al.
Macrophage-Derived CXCL9 and CXCL10 Are Required for Antitumor Immune Responses Following Immune Checkpoint
Blockade. Clin. Cancer Res. 2020, 26, 487–504. [CrossRef] [PubMed]
145. Hasegawa, T.; Venkata Suresh, V.; Yahata, Y.; Nakano, M.; Suzuki, S.; Suzuki, S.; Yamada, S.; Kitaura, H.; Mizoguchi, I.; Noiri,
Y.; et al. Inhibition of the CXCL9-CXCR3 axis suppresses the progression of experimental apical periodontitis by blocking
macrophage migration and activation. Sci. Rep. 2021, 11, 2613. [CrossRef] [PubMed]
146. Mizuta, M.; Shimizu, M.; Inoue, N.; Nakagishi, Y.; Yachie, A. Clinical Significance of Serum CXCL9 Levels as a Biomarker for
Systemic Juvenile Idiopathic Arthritis Associated Macrophage Activation Syndrome. Cytokine 2019, 119, 182–187. [CrossRef]
[PubMed]
147. Hinze, T.; Kessel, C.; Hinze, C.H.; Seibert, J.; Gram, H.; Foell, D. A dysregulated interleukin-18-interferon-γ-CXCL9 axis impacts
treatment response to canakinumab in systemic juvenile idiopathic arthritis. Rheumatology 2021, 60, 5165–5174. [CrossRef]
148. Bleesing, J.; Prada, A.; Siegel, D.M.; Villanueva, J.; Olson, J.; Ilowite, N.T.; Brunner, H.I.; Griffin, T.; Graham, T.B.; Sherry, D.D.;
et al. The diagnostic significance of soluble CD163 and soluble interleukin-2 receptor alpha-chain in macrophage activation
syndrome and untreated new-onset systemic juvenile idiopathic arthritis. Arthritis Rheum. 2007, 56, 965–971. [CrossRef]
149. Reddy, V.V.; Myles, A.; Cheekatla, S.S.; Singh, S.; Aggarwal, A. Soluble CD25 in serum: A potential marker for subclinical
macrophage activation syndrome in patients with active systemic onset juvenile idiopathic arthritis. Int. J. Rheum. Dis. 2014, 17,
261–267. [CrossRef]
150. Sakumura, N.; Shimizu, M.; Mizuta, M.; Inoue, N.; Nakagishi, Y.; Yachie, A. Soluble CD163, a unique biomarker to evaluate the
disease activity, exhibits macrophage activation in systemic juvenile idiopathic arthritis. Cytokine 2018, 110, 459–465. [CrossRef]
151. Verweyen, E.L.; Pickering, A.; Grom, A.A.; Schulert, G.S. Distinct Gene Expression Signatures Characterize Strong Clinical
Responders Versus Nonresponders to Canakinumab in Children with Systemic Juvenile Idiopathic Arthritis. Arthritis Rheumatol.
2021, 73, 1334–1340. [CrossRef]
152. Dik, W.A.; Heron, M. Clinical significance of soluble interleukin-2 receptor measurement in immune-mediated diseases. Neth. J.
Med. 2020, 78, 220–231. [PubMed]
153. Frosch, M.; Ahlmann, M.; Vogl, T.; Wittkowski, H.; Wulffraat, N.; Foell, D.; Roth, J. The myeloid-related proteins 8 and 14
complex, a novel ligand of toll-like receptor 4, and interleukin-1 β form a positive feedback mechanism in systemic-onset juvenile
idiopathic arthritis. Arthritis Rheum. 2009, 60, 883–891. [CrossRef] [PubMed]
154. Nirmala, N.; Grom, A.; Gram, H. Biomarkers in systemic juvenile idiopathic arthritis: A comparison with biomarkers in
cryopyrin-associated periodic syndromes. Curr. Opin. Rheumatol. 2014, 26, 543–552. [CrossRef] [PubMed]
155. Ahn, J.G. Role of Biomarkers in Juvenile Idiopathic Arthritis. J. Rheum. Dis. 2020, 27, 233–240. [CrossRef]
156. Miller, Y.I.; Choi, S.-H.; Wiesner, P.; Fang, L.; Harkewicz, R.; Hartvigsen, K.; Boullier, A.; Gonen, A.; Diehl, C.J.; Que, X.; et al.
Oxidation-specific epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate
immunity. Circ. Res. 2011, 108, 235–248. [CrossRef]
157. Wang, S.; Song, R.; Wang, Z.; Jing, Z.; Wang, S.; Ma, J. S100A8/A9 in Inflammation. Front. Immunol. 2018, 9, 1298. [CrossRef]
158. Marongiu, L.; Gornati, L.; Artuso, I.; Zanoni, I.; Granucci, F. Below the surface: The inner lives of TLR4 and TLR9. J. Leukoc. Biol.
2019, 106, 147–160. [CrossRef]
159. Jukic, A.; Bakiri, L.; Wagner, E.F.; Tilg, H.; Adolph, T.E. Calprotectin: From biomarker to biological function. Gut 2021, 70,
1978–1988. [CrossRef]
160. Degraeuwe, P.L.; Beld, M.P.; Ashorn, M.; Canani, R.B.; Day, A.S.; Diamanti, A.; Fagerberg, U.L.; Henderson, P.; Kolho, K.L.; Van
de Vijver, E.; et al. Faecal calprotectin in suspected paediatric inflammatory bowel disease. J. Pediatr. Gastroenterol. Nutr. 2015, 60,
339–346. [CrossRef]
161. Austermann, J.; Spiekermann, C.; Roth, J. S100 Proteins in Rheumatic Diseases. Nat. Rev. Rheumatol. 2018, 14, 528–541. [CrossRef]
162. Mylemans, M.; Nevejan, L.; Van Den Bremt, S.; Stubbe, M.; Cruyssen, B.V.; Moulakakis, C.; Berthold, H.; Konrad, C.; Bossuyt, X.;
Van Hoovels, L. Circulating calprotectin as a biomarker in neutrophil-associated inflammation: Pre-analytical recommendations
and reference values depending on the type of sample. Clin. Chim. Acta 2021, 517, 149–155. [CrossRef]
163. Ometto, F.; Friso, L.; Astorri, D.; Botsios, C.; Raffeiner, B.; Punzi, L.; Doria, A. Calprotectin in rheumatic diseases. Exp. Biol. Med.
2017, 242, 859–873. [CrossRef] [PubMed]
164. Romand, X.; Bernardy, C.; Nguyen, M.V.C.; Courtier, A.; Trocme, C.; Clapasson, M.; Paclet, M.H.; Toussaint, B.; Gaudin, P.; Baillet,
A. Systemic calprotectin and chronic inflammatory rheumatic diseases. Jt. Bone Spine 2019, 86, 691–698. [CrossRef] [PubMed]

58/61

Int. J. Mol. Sci. 2022, 23, 12757 59 of 61
165. Altobelli, E.; Angeletti, P.M.; Petrocelli, R.; Lapergola, G.; Farello, G.; Cannataro, G.; Breda, L. Serum Calprotectin a Potential
Biomarker in Juvenile Idiopathic Arthritis: A Meta-Analysis. J. Clin. Med. 2021, 10, 4861. [CrossRef] [PubMed]
166. Wittkowski, H.; Frosch, M.; Wulffraat, N.; Goldbach-Mansky, R.; Kallinich, T.; Kuemmerle-Deschner, J.; Frühwald, M.C.;
Dassmann, S.; Pham, T.H.; Roth, J.; et al. S100A12 is a novel molecular marker differentiating systemic-onset juvenile idiopathic
arthritis from other causes of fever of unknown origin. Arthritis Rheum. 2008, 58, 3924–3931. [CrossRef] [PubMed]
167. Holzinger, D.; Frosch, M.; Kastrup, A.; Prince, F.H.; Otten, M.H.; Van Suijlekom-Smit, L.W.; ten Cate, R.; Hoppenreijs, E.P.;
Hansmann, S.; Moncrieffe, H.; et al. The Toll-like receptor 4 agonist MRP8/14 protein complex is a sensitive indicator for disease
activity and predicts relapses in systemic-onset juvenile idiopathic arthritis. Ann. Rheum. Dis. 2012, 71, 974–980. [CrossRef]
[PubMed]
168. Shenoi, S.; Ou, J.N.; Ni, C.; Macaubas, C.; Gersuk, V.H.; Wallace, C.A.; Mellins, E.D.; Stevens, A.M. Comparison of biomarkers for
systemic juvenile idiopathic arthritis. Pediatr. Res. 2015, 78, 554–559. [CrossRef] [PubMed]
169. Aljaberi, N.; Tronconi, E.; Schulert, G.; Grom, A.A.; Lovell, D.J.; Huggins, J.L.; Henrickson, M.; Brunner, H. The use of S100
proteins testing in juvenile idiopathic arthritis and autoinflammatory diseases in a pediatric clinical setting: A retrospective
analysis. Pediatr. Rheumatol. 2020, 18, 7. [CrossRef]
170. La, C.; Lê, P.Q.; Ferster, A.; Goffin, L.; Spruyt, D.; Lauwerys, B.; Durez, P.; Boulanger, C.; Sokolova, T.; Rasschaert, J.; et al. Serum
calprotectin (S100A8/A9): A promising biomarker in diagnosis and follow-up in different subgroups of juvenile idiopathic
arthritis. RMD Open 2021, 7, e001646. [CrossRef]
171. Deftos, L.J.; Roos, B.A.; Parthemore, J.G. Calcium and skeletal metabolism. West. J. Med. 1975, 123, 447–458.
172. Dandona, P.; Nix, D.; Wilson, M.F.; Aljada, A.; Love, J.; Assicot, M.; Bohuon, C. Procalcitonin increase after endotoxin injection in
normal subjects. J. Clin. Endocrinol. Metab. 1994, 79, 1605–1608. [CrossRef]
173. Trippella, G.; Galli, L.; De Martino, M.; Lisi, C.; Chiappini, E. Procalcitonin performance in detecting serious and invasive bacterial
infections in children with fever without apparent source: A systematic review and meta-analysis. Expert Rev. Anti-Infect. Ther.
2017, 15, 1041–1057. [CrossRef] [PubMed]
174. Trachtman, R.; Murray, E.; Wang, C.M.; Szymonifka, J.; Toussi, S.S.; Walters, H.; Nellis, M.E.; Onel, K.B.; Mandl, L.A. Procalcitonin
Differs in Children With Infection and Children With Disease Flares in Juvenile Idiopathic Arthritis. J. Clin. Rheumatol. 2021, 27,
87–91. [CrossRef] [PubMed]
175. Kim, J.K.; Park, S.; Lee, M.J.; Song, Y.R.; Han, S.H.; Kim, S.G.; Kang, S.W.; Choi, K.H.; Kim, H.J.; Yoo, T.H. Plasma levels of soluble
receptor for advanced glycation end products (sRAGE) and proinflammatory ligand for RAGE (EN-RAGE) are associated with
carotid atherosclerosis in patients with peritoneal dialysis. Atherosclerosis 2012, 220, 208–214. [CrossRef] [PubMed]
176. Xie, J.; Mendez, J.D.; Mendez-Valenzuela, V.; Aguilar-Hernandez, M.M. Cellular signalling of the receptor for advanced glycation
end products (RAGE). Cell Signal. 2013, 25, 2185–2197. [CrossRef] [PubMed]
177. Selvin, E.; Halushka, M.K.; Rawlings, A.M.; Hoogeveen, R.C.; Ballantyne, C.M.; Coresh, J.; Astor, B.C. sRAGE and risk of diabetes,
cardiovascular disease, and death. Diabetes 2013, 62, 2116–2121. [CrossRef]
178. Pratte, K.A.; Curtis, J.L.; Kechris, K.; Couper, D.; Cho, M.H.; Silverman, E.K.; DeMeo, D.L.; Sciurba, F.C.; Zhang, Y.; Ortega,
V.E.; et al. Soluble receptor for advanced glycation end products (sRAGE) as a biomarker of COPD. Respir. Res. 2021, 22, 127.
[CrossRef]
179. Geroldi, D.; Falcone, C.; Emanuele, E. Soluble receptor for advanced glycation end products: From disease marker to potential
therapeutic target. Curr. Med. Chem. 2006, 13, 1971–1978. [CrossRef]
180. Grauen Larsen, H.; Marinkovic, G.; Nilsson, P.M.; Nilsson, J.; Engström, G.; Melander, O.; Orho-Melander, M.; Schiopu, A.
High Plasma sRAGE (Soluble Receptor for Advanced Glycation End Products) Is Associated With Slower Carotid Intima-Media
Thickness Progression and Lower Risk for First-Time Coronary Events and Mortality. Arter. Thromb. Vasc. Biol. 2019, 39, 925–933.
[CrossRef]
181. Prasad, K. Is there any evidence that AGE/sRAGE is a universal biomarker/risk marker for diseases? Mol. Cell. Biochem. 2019,
451, 139–144. [CrossRef]
182. Zhang, X.; Cao, X.; Dang, M.; Wang, H.; Chen, B.; Du, F.; Li, H.; Zeng, X.; Guo, C. Soluble receptor for advanced glycation
end-products enhanced the production of IFN-γ through the NF-κB pathway in macrophages recruited by ischemia/reperfusion.
Int. J. Mol. Med. 2019, 43, 2507–2515. [CrossRef]
183. Klune, J.R.; Dhupar, R.; Cardinal, J.; Billiar, T.R.; Tsung, A. HMGB1: Endogenous danger signaling. Mol. Med. 2008, 14, 476–484.
[CrossRef] [PubMed]
184. Andersson, U.; Rauvala, H. Introduction: HMGB1 in inflammation and innate immunity. J. Intern. Med. 2011, 270, 296–300.
[CrossRef]
185. Yang, H.; Wang, H.; Andersson, U. Targeting Inflammation Driven by HMGB1. Front. Immunol. 2020, 11, 484. [CrossRef]
[PubMed]
186. Andersson, U.; Harris, H.E. The role of HMGB1 in the pathogenesis of rheumatic disease. Biochim. Biophys. Acta 2010, 1799,
141–148. [CrossRef] [PubMed]
187. Harris, H.E.; Andersson, U.; Pisetsky, D.S. HMGB1: A multifunctional alarmin driving autoimmune and inflammatory disease.
Nat. Rev. Rheumatol. 2012, 8, 195–202. [CrossRef] [PubMed]
188. Magna, M.; Pisetsky, D.S. The role of HMGB1 in the pathogenesis of inflammatory and autoimmune diseases. Mol. Med. 2014, 20,
138–146. [CrossRef] [PubMed]

59/61

$Int. J. Mol. Sci. 2022, 23, 12757 60 of 61 189. Paudel, Y.N.; Angelopoulou, E.; Piperi, C.; Balasubramaniam, V.; Othman, I.; Shaikh, M.F. Enlightening the role of high mobility group box 1 (HMGB1) in inflammation: Updates on receptor signalling. Eur. J. Pharmacol. 2019, 858, 172487. [CrossRef] 190. Moser, B.; Hudson, B.I.; Schmidt, A.M. Soluble RAGE: A hot new biomarker for the hot joint? Arthritis Res. Ther. 2005, 7, 142. [CrossRef] 191. Yang, H.; Wang, H.; Chavan, S.S.; Andersson, U. High Mobility Group Box Protein 1 (HMGB1): The Prototypical Endogenous Danger Molecule. Mol. Med. 2015, 21, S6–S12. [CrossRef] 192. Zhong, H.; Li, X.; Zhou, S.; Jiang, P.; Liu, X.; Ouyang, M.; Nie, Y.; Chen, X.; Zhang, L.; Liu, Y.; et al. Interplay between RAGE and TLR4 Regulates HMGB1-Induced Inflammation by Promoting Cell Surface Expression of RAGE and TLR4. J. Immunol. 2020, 205, 767–775. [CrossRef] 193. Schierbeck, H.; Pullerits, R.; Pruunsild, C.; Fischer, M.; Holzingerc, D.; Laestadius, Å.; Sundberg, E.; Harris, H.E. HMGB1 levels are increased in patients with juvenile idiopathic arthritis, correlated with early onset of disease, and are independent of disease duration. J. Rheumatol. 2013, 40, 1604–1613. [CrossRef] [PubMed] 194. Bobek, D.; Grˇcevi´c, D.; Kovaˇci´c, N.; Luki´c, I.K.; Jeluši´c, M. The presence of high mobility group box-1 and soluble receptor for advanced glycation end-products in juvenile idiopathic arthritis and juvenile systemic lupus erythematosus. Pediatr. Rheumatol. 2014, 12, 50. [CrossRef] [PubMed] 195. Xu, D.; Zhang, Y.; Zhang, Z.Y.; Tang, X.M. Association between high mobility group box 1 protein and juvenile idiopathic arthritis: A prospective longitudinal study. Pediatr. Rheumatol. 2021, 19, 112. [CrossRef] [PubMed] 196. Wu, J.F.; Yang, Y.H.; Wang, L.C.; Lee, J.H.; Shen, E.Y.; Chiang, B.L. Comparative usefulness of C-reactive protein and erythrocyte sedimentation rate in juvenile rheumatoid arthritis. Clin. Exp. Rheumatol. 2007, 25, 782–785. [PubMed] 197. Bloom, B.J.; Alario, A.J.; Miller, L.C. Persistent elevation of fibrin d-dimer predicts long term outcome in systemic juvenile idiopathic arthritis. J. Rheumatol. 2009, 36, 422–426. [CrossRef] 198. Gorelik, M.; Fall, N.; Altaye, M.; Barnes, M.G.; Thompson, S.D.; Grom, A.A.; Hirsch, R. Follistatin-like protein 1 and the ferritin/erythrocyte sedimentation rate ratio are potential biomarkers for dysregulated gene expression and macrophage activation syndrome in systemic juvenile idiopathic arthritis. J. Rheumatol. 2013, 40, 1191–1199. [CrossRef] 199. Minoia, F.; Bovis, F.; Davì, S.; Horne, A.; Fischbach, M.; Frosch, M.; Huber, A.; Jelusic, M.; Sawhney, S.; McCurdy, D.K.; et al. Pediatric Rheumatology International Trials Organization, the Childhood Arthritis & Rheumatology Research Alliance, the Pediatric Rheumatology Collaborative Study Group and the Histiocyte Society. Development and initial validation of the MS score for diagnosis of macrophage activation syndrome in systemic juvenile idiopathic arthritis. Ann. Rheum. Dis. 2019, 78, 1357–1362. [CrossRef] 200. Eloseily, E.M.; Minoia, F.; Crayne, C.B.; Beukelman, T.; Ravelli, A.; Cron, R.Q. Ferritin to erythrocyte sedimentation rate ratio: Simple measure to identify macrophage activation syndrome in systemic juvenile idiopathic arthritis. ACR Open Rheumatol. 2019, 1, 345–349. [CrossRef] 201. Zou, L.X.; Zhu, Y.; Sun, L.; Ma, H.H.; Yang, S.R.; Zeng, H.S.; Xiao, J.H.; Yu, H.G.; Guo, L.; Xu, Y.P.; et al. Clinical and laboratory features, treatment, and outcomes of macrophage activation syndrome in 80 children: A multi-center study in China. World J. Pediatr. 2020, 16, 89–98. [CrossRef] 202. Ganeva, M.; Fuehner, S.; Kessel, C.; Klotsche, J.; Niewerth, M.; Minden, K.; Foell, D.; Hinze, C.H.; Wittkowski, H. Trajectories of disease courses in the inception cohort of newly diagnosed patients with JIA (ICON-JIA): The potential of serum biomarkers at baseline. Pediatr. Rheumatol. 2021, 19, 64. [CrossRef] 203. Shimojima, Y.; Matsuda, M.; Gono, T.; Ishii, W.; Ikeda, S. Serum amyloid A as a potent therapeutic marker in a refractory patient with polymyalgia rheumatica. Intern. Med. 2005, 44, 1009–1012. [CrossRef] [PubMed] 204. Cantarini, L.; Giani, T.; Fioravanti, A.; Iacoponi, F.; Simonini, G.; Pagnini, I.; Spreafico, A.; Chellini, F.; Galeazzi, M.; Cimaz, R. Serum amyloid A circulating levels and disease activity in patients with juvenile idiopathic arthritis. Yonsei Med. J. 2012, 53, 1045–1048. [CrossRef] 205. Feyzkhanova, G.U.; Voloshin, S.A.; Novikov, A.A.; Aleksandrova, E.N.; Smoldovskaya, O.V.; Rubina, A.Y. Analysis of rheumatoid factor and acute phase proteins using microarrays in patients with rheumatoid arthritis. Klin. Lab. Diagn. 2022, 67, 43–47. [CrossRef] [PubMed] 206. Sori´c Hosman, I.; Kos, I.; Lamot, L. Serum Amyloid A in Inflammatory Rheumatic Diseases: A Compendious Review of a Renowned Biomarker. Front. Immunol. 2021, 11, 631299. [CrossRef] [PubMed] 207. Li, H.; Xiang, X.; Ren, H.; Xu, L.; Zhao, L.; Chen, X.; Long, H.; Wang, Q.; Wu, Q. Serum Amyloid A is a biomarker of severe Coronavirus Disease and poor prognosis. J. Infect. 2020, 80, 646–655. [CrossRef] [PubMed] 208. Cheng, L.; Yang, J.Z.; Bai, W.H.; Li, Z.Y.; Sun, L.F.; Yan, J.J.; Zhou, C.L.; Tang, B.P. Prognostic value of serum amyloid A in patients with COVID-19. Infection 2020, 48, 715–722. [CrossRef] [PubMed] 209. Dev, S.; Singh, A. Study of role of serum amyloid A (SAA) as a marker of disease activity in juvenile idiopathic arthritis. J. Fam. Med. Prim. Care 2019, 8, 2129–2133. [CrossRef] 210. Wang, X.; Abraham, S.; McKenzie, J.A.G.; Jeffs, N.; Swire, M.; Tripathi, V.B.; Luhmann, U.F.O.; Lange, C.A.K.; Zhai, Z.; Arthur, H.M.; et al. LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling. Nature 2013, 499, 306–311. [CrossRef] [PubMed]$

60/61

Int. J. Mol. Sci. 2022, 23, 12757 61 of 61
211. Urushima, H.; Fujimoto, M.; Mishima, T.; Ohkawara, T.; Honda, H.; Lee, H.; Kawahata, H.; Serada, S.; Naka, T. Leucine-rich alpha
2 glycoprotein promotes Th17 differentiation and collagen-induced arthritis in mice through enhancement of TGF-β-Smad2
signaling in naïve helper T cells. Arthritis Res. Ther. 2017, 19, 137. [CrossRef] [PubMed]
212. Serada, S.; Fujimoto, M.; Ogata, A.; Terabe, F.; Hirano, T.; Iijima, H.; Shinzaki, S.; Nishikawa, T.; Ohkawara, T.; Iwahori, K.; et al.
iTRAQ-based proteomic identification of leucine-rich alpha-2 glycoprotein as a novel inflammatory biomarker in autoimmune
diseases. Ann. Rheum. Dis. 2010, 69, 770–774. [CrossRef]
213. Yoshimura, T.; Mitsuyama, K.; Sakemi, R.; Takedatsu, H.; Yoshioka, S.; Kuwaki, K.; Mori, A.; Fukunaga, S.; Araki, T.; Morita, M.;
et al. Evaluation of Serum Leucine-Rich Alpha-2 Glycoprotein as a New Inflammatory Biomarker of Inflammatory Bowel Disease.
Mediat. Inflamm. 2021, 2021, 8825374. [CrossRef] [PubMed]
214. Nakajima, H.; Nakajima, K.; Takaishi, M.; Ohko, K.; Serada, S.; Fujimoto, M.; Naka, T.; Sano, S. The Skin-Liver Axis Modulates the
Psoriasiform Phenotype and Involves Leucine-Rich α-2 Glycoprotein. J. Immunol. 2021, 206, 1469–1477. [CrossRef]
215. Druhan, L.J.; Lance, A.; Li, S.; Price, A.E.; Emerson, J.T.; Baxter, S.A.; Gerber, J.M.; Avalos, B.R. Leucine Rich α-2 Glycoprotein: A
Novel Neutrophil Granule Protein and Modulator of Myelopoiesis. PLoS ONE 2017, 12, e0170261. [CrossRef]
216. Camilli, C.; Hoeh, A.E.; De Rossi, G.; Moss, S.E.; Greenwood, J. LRG1: An emerging player in disease pathogenesis. J. Biomed. Sci.
2022, 29, 6. [CrossRef]
217. Zou, Y.; Xu, Y.; Chen, X.; Wu, Y.; Fu, L.; Lv, Y. Research Progress on Leucine-Rich Alpha-2 Glycoprotein 1: A Review. Front.
Pharmacol. 2022, 12, 809225. [CrossRef] [PubMed]
218. Shimizu, M.; Nakagishi, Y.; Inoue, N.; Mizuta, M.; Yachie, A. Leucine-rich α2-glycoprotein as the acute-phase reactant to detect
systemic juvenile idiopathic arthritis disease activity during anti-interleukin-6 blockade therapy: A case series. Mod. Rheumatol.
2017, 27, 833–837. [CrossRef]
219. Shimizu, M.; Inoue, N.; Mizuta, M.; Nakagishi, Y.; Yachie, A. Serum Leucine-Rich α2-Glycoprotein as a Biomarker for Monitoring
Disease Activity in Patients with Systemic Juvenile Idiopathic Arthritis. J. Immunol. Res. 2019, 2019, 3140204. [CrossRef] [PubMed]
220. Lee, P.Y.; Schulert, G.S.; Canna, S.W.; Huang, Y.; Sundel, J.; Li, Y.; Hoyt, K.J.; Blaustein, R.B.; Wactor, A.; Do, T.; et al. Adenosine
deaminase 2 as a biomarker of macrophage activation syndrome in systemic juvenile idiopathic arthritis. Ann. Rheum. Dis. 2020,
79, 225–231. [CrossRef] [PubMed]
221. Nakra, N.A.; Blumberg, D.A.; Herrera-Guerra, A.; Lakshminrusimha, S. Multi-System Inflammatory Syndrome in Children (MISC) Following SARS-CoV-2 Infection: Review of Clinical Presentation, Hypothetical Pathogenesis, and Proposed Management.
Children 2020, 7, 69. [CrossRef] [PubMed]
222. Fajgenbaum, D.C.; June, C.H. Cytokine Storm. N. Engl. J. Med. 2020, 383, 2255–2273. [CrossRef] [PubMed]
223. Henderson, L.A. Deciphering the cytokine fingerprint of macrophage activation syndrome. Lancet Rheumatol. 2021, 3, e535–e538.
[CrossRef]
224. Tang, S.; Li, S.; Zheng, S.; Ding, Y.; Zhu, D.; Sun, C.; Hu, Y.; Qiao, J.; Fang, H. Understanding of cytokines and targeted therapy in
macrophage activation syndrome. Semin. Arthritis Rheum. 2021, 51, 198–210. [CrossRef] [PubMed]
225. Consolaro, A.; Varnier, G.C.; Martini, A.; Ravelli, A. Advances in biomarkers for paediatric rheumatic diseases. Nat. Rev.
Rheumatol. 2015, 11, 265–275. [CrossRef] [PubMed]
226. Vandenhaute, J.; Wouters, C.H.; Matthys, P. Natural Killer Cells in Systemic Autoinflammatory Diseases: A Focus on Systemic
Juvenile Idiopathic Arthritis and Macrophage Activation Syndrome. Front. Immunol 2020, 10, 3089. [CrossRef]
227. Javaux, C.; El-Jammal, T.; Neau, P.-A.; Fournier, N.; Gerfaud-Valentin, M.; Perard, L.; Fouillet-Desjonqueres, M.; Le Scanff, J.;
Vignot, E.; Durupt, S.; et al. Detection and Prediction of Macrophage Activation Syndrome in Still’s Disease. J. Clin. Med. 2022, 11,
206. [CrossRef]
228. Çakan, M.; Karada ˘g, ¸S.G.; Tanatar, A.; Ayaz, N.A. The frequency of macrophage activation syndrome and disease course in
systemic juvenile idiopathic arthritis. Mod. Rheumatol. 2020, 30, 900–904. [CrossRef]
229. Zaripova, L.N.; Midgley, A.; Christmas, S.E.; Beresford, M.W.; Baildam, E.M.; Oldershaw, R.A. Juvenile idiopathic arthritis: From
aetiopathogenesis to therapeutic approaches. Pediatr. Rheumatol. 2021, 19, 135. [CrossRef] [PubMed]
230. Das, G.; Sarkar, S.; Datta, S. Evaluation of serum ferritin and its correlation with disease activity in non-systemic juvenile
idiopathic arthritis. Int. J. Contemp. Pediatr. 2021, 8, 625–630. [CrossRef]
231. Høeg, P.E.; Glerup, M.; Mahler, B.; Høst, C.; Herlin, T. Evaluation of Macrophage Activation Syndrome in Patients with Systemic
Juvenile Idiopathic Arthritis: A Single Center Experience. Int. J. Rheumatol. 2022, 2022, 1784529. [CrossRef] [PubMed]
232. Adiguzel Dundar, H.; Acari, C.; Turkucar, S.; Unsal, E. Treatment of systemic JIA: When do we need a biologic? Real world data
of a single center. Mod. Rheumatol. 2021, 31, 684–690. [CrossRef]
233. Van Nieuwenhove, E.; Lagou, V.; Van Eyck, L.; Dooley, J.; Bodenhofer, U.; Roca, C.; Vandebergh, M.; Goris, A.; Humblet-Baron, S.;
Wouters, C.; et al. Machine learning identifies an immunological pattern associated with multiple juvenile idiopathic arthritis
subtypes. Ann. Rheum. Dis. 2019, 78, 617–628. [CrossRef] [PubMed]

61/61