Biomarkers in Systemic Juvenile Idiopathic Arthritis, Macrophage Activation Syndrome and Their Importance in COVID Era
Biomarkers in Systemic Juvenile Idiopathic Arthritis, Macrophage Activation Syndrome and Their Importance in COVID Era
Biomarkers in Systemic Juvenile Idiopathic Arthritis, Macrophage Activation Syndrome and Their Importance in COVID Era
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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.
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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Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).
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 IFNγ 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
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
Apositive 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].
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-1 β 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-1 β - 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 ,
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].
Asummary of the abovementioned published studies on genetic biomarkers is shown in Table 1.
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].
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 (IFNγ ) 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 IFNγ , tumor necrosis factor alpha (TNFα ), and granulocyte-macrophage colonystimulating factor (GM-CSF) [34,35].
NKcells' 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+/TCR αβ -), NK T cells (CD56+/TCR αβ +), 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+/TCRαβ +), 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 MASevent had low NK function and a lack of circulating CD56 bright 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].
Due to the connection between IL-18 and the activity of NK cells triggered by this cytokine, de Jager et al. analyzed the relationship between high IL-18 plasma concentrations and the phenotype and function of NK cells in 15 patients with sJIA, the same number of patients with polyarticular JIA, and 10 healthy controls, via in vitro staining and functional assays. Using fluorescence microscopy, they investigated the binding of the IL-18 ligand, and the phosphorylation reactions of several MAP kinases and the IL-18 β receptor (IL-18R β ) were highlighted by the Western blot technique. The results indicated that IL-18 from the plasma of patients diagnosed with sJIA stimulated the activation of NK cells from healthy controls and bound to its cognate receptor. However, after IL-18 excitation, NK cells from sJIA patients failed to upregulate killer molecules such as perforin and IFNγ , nor did they trigger the phosphorylation of receptor-activated MAP kinases. As a result of an alternative stimulation of NK cells with IL-12, induction of cytotoxic activity was found. However, the authors did not find any adjunctive results of the combination of IL-18 and IL-12 in sJIA. Immunoprecipitation of IL-18R β revealed that NK cells from sJIA patients failed to phosphorylate this receptor following IL-18 stimulation, indicating that NK cell dysfunction in sJIA is directly correlated with a defect in IL-18R β phosphorylation, which has important implications for the understanding of the pathophysiological mechanisms and future therapy of sJIA [42].
Zhou et al. used flow cytometry to investigate the NK cell count, cytotoxicity, and expression of perforin, granzyme B, interferon (IFN)γ , and tumor necrosis factor (TNF)α in peripheral blood samples from 72 children with active JIA (systemic, 25; polyarticular, 24; pauciarticular, 23) and 25 controls. The authors used specimens from 220 children with JIA (systemic, 84; polyarticular, 72; pauciarticular, 64) and 150 controls for KIR2DS2, KIR2DS4, KIR3DS1, KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 typing by polymerase chain reaction with sequence-specific oligonucleotide probes. The results of the research proved that sJIA patients had lower NK cell counts, cytotoxicity, and expression of perforin and granzyme B compared to controls, whereas those with pauci- and polyarticular JIA subtypes expressed higher levels of perforin and granzyme B. In patients with sJIA, NK cells generated lower amounts of TNFα and higher amounts of IFNγ than in the pauciand polyarticular JIA groups. No significant disparities were found in KIR gene frequencies between JIA subgroups and healthy controls, except for KIR2DS4, which was lower in the sJIA group. Finally, the authors concluded that in sJIA there was a decline in the activity of NKcells, which secreted more IFNγ and less TNFα , and the incidence of KIR2DS4 was reduced [43].
Put et al. investigated NK-cell-specific transcriptional changes via RNA sequencing of highly purified NK cells from six patients with active sJIA and six healthy controls. The stimulatory cytokines-mainly for NK cells-were analyzed from plasma ( n = 18), evaluating the phenotype and cytotoxic activity of NK cells on tumor cells ( n = 10), as well as the production of IFNγ ( n = 8). For patients with sJIA, an altered gene expression profile of NK cells was found compared to healthy controls, with increased immunoinflammatory pathways, innate gene expression (TLR4 and S100A9), and decreased expression of immunoregulatory genes (i.e., interleukin 10 receptor, alpha subunit (IL10RA), and granzyme K (GZMK)). In patients with sJIA, increased IL-18 levels and a decreased IFNγ /IL-18 ratio were found. NK cells had an imbalance between inhibitory and activating receptors, with reduced G1 receptor expression and increased expression of natural cytotoxicity triggering receptor 2 (NKp44). A reduction in granzyme K expression was observed in CD56 bright NK cells, along with defective IL-18-induced IFNγ secretion and signaling. Hard-to-detect defects in the immune pathways governed by NK-such as the expression of granzyme K and the production of IFNγ stimulated by IL-18-induce the immunoinflammatory dysfunctions that characterize sJIA [44].
The pathophysiology of sJIA and MAS still has many unclear elements, although it is known that there are high levels of IL-18 and reduced activity of NK cells in patients with active disease. Ohya et al. explored the phosphorylation of mitogen-activated protein kinase (MAPK) p38 and the enhancer of nuclear factor κ light chain of activated B cells
(NFκ B) 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 NFκ B 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 NFκ B 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., TNFα , 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-1 β , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17, INFα , INFγ , and TNFα . 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 (TGFβ ), participating in tissue regeneration and incorporation of pro-inflammatory molecules, thereby preventing the inflammatory
response. M2b macrophages secrete IL-1, IL-6, IL-10, and TNFα under the influence of immune complexes or lipopolysaccharides (LPSs), resulting in the activation of Th2 cells and the reducing inflammation. M2c macrophages are activated by IL-10, TGFβ , and glucocorticoids; they secrete IL-10 and TGFβ , which suppress the inflammatory response. The M2a subtype, in tandem with Th2 cells, participates in allergic reactions, while M2b is involved in immune regulation and M2c can secrete anti-inflammatory cytokines with a role in reducing inflammation, reshaping the matrix and tissues. M2d macrophages are polarized by IL-6 and adenosine, and are known as tumor-associated macrophages (TAMs). In sJIA, the cluster of differentiation 163 (CD163), arginase-1 (Arg-1), and chemokine (C-C motif) ligand 2 (CCL2) or monocyte chemoattractant protein 1 (MCP1) genes are closely related to M2 macrophages and are expressed on the surface of monocytes [49-51].
CD163 is a potent receptor specific to monocytes and macrophages for haptoglobinhemoglobin complexes. CD163 is strongly induced by the anti-inflammatory cytokine IL-10, as well as by glucocorticoids and IL-6. CD163 easily induces TNF-like weak inducer of apoptosis (TWEAK) and is a receptor for a variety of bacteria and viruses. CD163 is widely used as a marker for alternately activated macrophages. CD163 +-activated macrophages have an immunomodulatory profile and are valued as anti-inflammatory markers [52].
CD163 helps regulate and extinguish inflammation, eliminates free hemoglobin, and is strongly expressed in myeloid cells in patients with sJIA and MAS [53].
2.2.3. Neutrophils and Neutrophil Extracellular Traps
Neutrophils are cells that are part of the innate immune system, with an important role in the phagocytosis of pathogens; they originate from hematopoietic stem cells, have a short lifespan, and represent more than 50% of circulating white blood cells. The nuclear morphology of these cells gives them a phenotypic and functional heterogeneity. In addition to their ability to migrate, as well as their phagocytic and immunomodulatory functions, they can form neutrophil extracellular traps (NETs) in the form of a network of fibers consisting of cytoskeletal proteins, proteases, antimicrobial peptides, histones, and chromatin deoxyribonucleic acid [54-56].
It has been observed that in the early stages of sJIA's onset, the innate immune cells with a defensive role-such as neutrophils and macrophages-are much more abundant in peripheral blood and play an essential role in the development of systemic inflammation. These cells produce several mediators involved in the pathogenesis of sJIA, of which IL-1 appears to play a pivotal role [57,58].
Ter Haar et al. investigated the role of neutrophils in 50 patients with sJIA receiving therapy with a recombinant IL-1 receptor antagonist (rIL-1Ra; anakinra). At the onset of the disease, the number of neutrophils increased significantly and was correlated directly with the pro-inflammatory factors, and after a few days of treatment with anakinra the neutrophil count returned to normal. RNA sequencing research has revealed an important adjustment of the inflammatory processes triggered by neutrophils in active disease patients, identical to the blood transcriptome analysis of patients with sepsis. Neutrophils from patients with active sJIA had a charged phenotype defined by increased reactive oxygen species (ROS) production, decreased L-selectin (CD62L) levels, and degranulation of secretory vesicles, which was completely changed after anakinra therapy in clinical remission patients. Those with a short period from the onset of sJIA had many neutrophils-especially immature ones-and a complete resolution of the disease under anti-rIL-1Ra, in contrast to those with a disease duration of more than one month, who had a normal number of neutrophils, but with an unsatisfactory response to anakinra treatment. These findings firmly suggest that neutrophils play an important role in sJIA's onset and in the early inflammation stage, and that their counts and pro-inflammatory activity are influenced by IL-1 blockade [59].
To examine activated neutrophil subsets, the release of S100 alarmin, and gene expression signatures in patients with different spectra of sJIA activity, Brown et al. analyzed samples from 23 active sJIA patients and 22 children with clinically inactive disease (CID). Control samples were obtained from children undergoing evaluation for joint pain but
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-1 β 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].
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.
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
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-1 β
Pro-inflammatory cytokines in the innate immune system-such as IL-1, IL-6, IL-18, and TNFα -are directly involved in the inflammatory process of sJIA. For example, IL1 β -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-1 β 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-
sion of the cytokine IL-1 β . 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-1 β into IL-1 β [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-1 β , 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-1 β ; 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-1 β 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,asapotentially 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-1 β -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
involvement and platelet counts-but not during remission. This study highlighted the important role of IL-6 in the pathogenesis of sJIA [95].
IL-6 levels are elevated in both serum and synovial fluid in patients with sJIA, directly related to fever, growth deficits, anemic syndrome, and thrombocytosis [96,97].
In a study on early changes in inflammatory gene and protein expression in sJIA managed with anti-IL-1 β human monoclonal antibody therapy, Brachat et al. measured gene expression in febrile sJIA patients and matched healthy controls. Transcriptional response was assessed by changes in gene expression from baseline to day 3 using the adapted sJIA-ACR 50 response. The pro-inflammatory cytokines IL-6 and IL-18 were measured up to day 197. The strongest clinical response was seen in patients with higher baseline expression of dysregulated genes and a strong transcriptional response on day 3. IL-6 decreased on day 3 ( ⥠8-fold decrease) and remained suppressed. IL-18 decreased at day 57 ( ⥠1.5-fold decrease). The authors identified an important response signature to canakinumab for the majority of sJIA patients, with the transcript levels for sJIA overexpressed genes quickly returning to normal-including those associated with the IL-1 and IL-6 signaling pathways-as well as a marked decrease in transcription of the gene encoding the target, i.e., IL-1 β , meaning that this treatment interrupts a positive feedback loop between IL-1 β signaling and IL-1 β production. Canakinumab treatment in sJIA led to downregulation of innate immune response genes and downregulation of IL-6, but also decreased the clinical symptoms, paving the way for further research [98].
IL-6 is a cytokine with a complex role in local and systemic inflammation in the pathogenesis of sJIA, as well as in complications of the disease. As it is not known exactly how it works, the choice of IL-6 inhibitors should be made in the context of genetic and biological factors, as well as the type and phase of the disease [99].
Tocilizumab (TCZ), a humanized monoclonal antibody against the human IL-6 receptor, has radically altered the course of patients with sJIA and MAS. However, MAS may occur in patients with sJIA under TCZ therapy. Thus, it could be concluded that blocking IL-6 with TCZ is not sufficient to inhibit the pathogenic mechanism of sJIA and MAS [100,101].
Bielak et al. reported the results of treatment with biological agents for 200 patients with sJIA from the German Registry of Autoinflammatory Diseases (AID), of whom 46 received TCZ therapy as an IL-6 blocker, causing the disease to become inactive and/or enter remission after one year from the initiation of the treatment [102].
In a study on the action of TCZ in sJIA-MAS, Shimizu et al. combined expert consensus with an analysis of clinical and laboratory data for 12 patients treated with TCZ, compared with 18 sJIA-MAS patients without treatment. Patients who received TCZ were less febrile and had significantly lower levels of ferritin, triglycerides, and CRP than the control group. TCZ-treated patients with MAS associated with sJIA were less likely to be classified as having MAS based on the MAS classification criteria, due to the absence of fever and lower ferritin levels compared to untreated patients, leading to the conclusion that TCZ could alter the clinical and laboratory features of MAS associated with sJIA. Thus, when patients with sJIA are evaluated during TCZ treatment, the criteria for MAS cannot be applied, and great care must be taken not to fall into the trap of underdiagnosing MAS [103].
Aiming to elucidate the immune profile of sJIA to identify clinical biomarkers correlated with new immune mechanisms, Qu et al. analyzed plasma samples from 21 patients diagnosed with sJIA, compared with 60 age- and sex-matched healthy controls, via a highly sensitive proteomic immunoassay and analysis of canonical pathways associated with cellular functions. The authors investigated well-known sJIA biomarkers such as IL-6, IL-18, and S100A12, which have been shown to be elevated during active sJIA compared to healthy controls. IL-18 was the only one of these biomarkers with elevated levels in inactive sJIA compared to healthy controls. Other novel factors-such as 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. HMGB1 levels were found to be higher in active than in inactive
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 IFNγ -deficient BALB/c mice, as the corresponding wild-type (WT) mice are known to develop only a mild and transient inflammatory reaction. Diseased IFNγ -deficient mice showed defective IL-10 production in CD4 + regulatory T cells, CD19 + B cells, and CD3-CD122 + CD49b + NKcells, 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
a form of active cytokine to induce IFNγ . IL-18 can be found as a constitutive protein in almost all human cells and in healthy animals; it is buffered by its natural inhibitoran IL-18-binding protein called IL-18BP, which has a high affinity and regulates IL-18 activity [109].
Lotito et al., in a group of 50 patients with JIA (13 systemic, 13 polyarticular, 24 oligoarticular) and 25 controls, were among the first to investigate the levels of IL18 in synovial fluid (SF) and serum, in correlation with the activity and severity of the disease. The concentrations of the studied cytokines (i.e., IL-1, IL-1Ra, IL-6, and IL-18) were higher in the sera of patients with JIA than in controls. IL-18 levels were as high in serum as in SF and were positively correlated with IL-1, IL-1Ra, IL-6, and other disease activity parameters (i.e., CRP, number of active joints, and radiological score). However, the levels of IL-18 and IL-6 in SF and serum were much higher in patients with sJIA than in those with other types of the disease. The authors concluded that IL-18 is involved in the pathophysiology of JIA, reflects the severity of the disease, and could be a target for the treatment of arthritis [110].
Shimizu et al. studied serum levels of the cytokines IL-18, IL-6, neopterin, and TNFα receptor types I and II, along with ferritin concentrations, in 5 subjects with sJIA complicated by MAS (sJIA-MAS), 10 patients with Epstein-Barr-virus-induced hemophagocytic lymphohistiocytosis (EBV-HLH), 22 patients with KD, and 28 healthy controls. Biological data were compared with clinical symptoms of sJIA-MAS. Serum samples revealed significantly higher levels of IL-18 in patients with sJIA-MAS compared to patients with EBV-HLH or KD. In contrast, IL-6 levels in KD subjects were significantly higher than those in EBV-HLH or sJIA-MAS subjects, but serum neopterin levels in EBV-HLH patients were higher than those in sJIA-MAS or KD patients. A positive correlation was observed between serum levels of IL-18 and parameters of disease activity such as CRP, ferritin, and LDH. Serum IL-18 also remained elevated in patients with sJIA in the inactive stage of the disease, while clinical parameters and other cytokines normalized. The serum level of IL-18 can be considered to be a biomarker of sJIA activity, and monitoring its profile could be beneficial for distinguishing MAS/HLH and estimating sJIA disease activity [111].
In another study, Shimizu et al. measured serum IL-6/IL-18 levels in 76 patients with sJIA, including 15 with MAS, and compared them with clinical symptoms. Depending on the IL-6/IL-18 serum concentrations, two distinct subsets were detected: The IL-6-dominant subset was more likely to develop much more severe joint disease. The IL-18-dominant subset included a larger number of patients who developed MAS and in whom IL-18 concentrations during the active phase were significantly higher, compared to those without MAS. The minimum serum level found for IL-18 was 47,750 pg/mL, which predicted the development of MAS. IL-18 could be involved in the pathophysiology of MAS, and serum concentrations > 47,750 pg/mL could be useful in predicting the initiation of MAS [112].
Xia et al. investigated whether IL-18 could be a valuable biomarker in the diagnosis of sJIA, comparatively investigating 23 patients with sJIA, 20 patients with acute lymphoblastic leukemia (ALL), 18 patients with severe infections (SIFs), 26 patients with Kawasaki disease (KD), 18 patients with JIA, and 25 healthy controls. Serum levels of IL-6, IL-18, S100A8, and S100A9 were measured. The results indicated significantly higher serum IL-6 concentrations in all patient groups, without significant differences between them, compared to healthy controls. S100A8 was significantly increased in sJIA vs. ALL, JIA, and healthy controls, but was not significantly different vs. the SIF and KD groups. S100A9 in sJIA was greatly increased compared to ALL and healthy controls, but not significantly different from SIF, KD, and JIA. The levels of IL-18 in sJIA were much higher than in groups with febrile diseases, allowing the authors to conclude that serum IL-18 could be a biomarker in the differential diagnosis of sJIA compared to other febrile diseases [113].
As an IFNγ -stimulating production factor, IL-18 is a cytokine that plays an important role in Th1 and NK cell activation, but also in Th2, IL-17-producing γδ T cells, and macrophage activation in the processes of intracellular defense against bacteria and some viruses. IL-18 shares the same signaling pathway as IL-1 to initiate NFκ B and sets in
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