Clinical significance of serum IL-18 levels for the diagnosis and monitoring of disease activity

Clinical significance of serum IL-18 levels for the diagnosis and monitoring of disease activity

• 1. Clinical significance of serum IL-18 levels for the diagnosis and monitoring of disease activity in systemic juvenile idiopathic arthritis and macrophage activation syndrome Masaki Shimizu, MD, PhD Department of Pediatrics and Developmental Biology Institute of Science Tokyo NextGen Therapies for SJIA & MAS 2024. Washington DC, USA. Nov 13, 2024

• 2. Systemic juvenile idiopathic arthritis (s-JIA) a systemic inflammatory disorder of unknown etiology and is characterized by arthritis and systemic features, such as spiking fever, skin rash, generalized lymphadenopathy, hepatosplenomegaly, and serositis Textbook of Pediatric Rheumatology 8th edition, Elsevier spiking fever skin rash hepatosplenomegaly

• 3. Role of cytokines in the pathogenesis of s-JIA Mellins ED, et al. Nat Rev Rheumatol. 2011;7:416-26. IL-1β, IL-6 and IL-18 are key cytokines in the pathogenesis of systemic JIA. These cytokines initiate the inflammatory cascade through their effects on the vascular endothelial cells, hypothalamus, liver, bone marrow, and synovial joints, and then drive systemic and joint inflammation.

• 4. Macrophage activation syndrome (MAS) s-JIA Viral infections Drugs IFN-γ ↑↑ • Continuous fever • Hepatosplemegaly/lymphadenopathy↑ • Coagulopathy • Cytopenia • CNS dysfunction • Hemophagocytosis in BM Cytokine Storm a life-threatening complication of s-JIA that occurs in about 5-8% of patients. clinically characterized by continuous fever, hepatosplenomegaly, altered liver function, lymphadenopathy, intravascular coagulation, profound depression of all three blood-cell lines, central nervous system dysfunction and phagocytosis of hematopoietic cells by macrophages in bone marrow examination. NK activity ↓ NK cells macrophages ×

• 5. The hallmark of MAS is uncontrolled immune response involving continual activation and expansion of T lymphocytes and macrophages, resulting in marked hypercytokinemia, namely, cytokine storm. Shimizu M. Immunol Med 2021;13:1-9 Macrophage activation syndrome (MAS)

• 6. Fever Rash Cervical lymphadenopathy Arthritis Coronary artery dilation CRP↑ GOT↑ Complication of MAS Overlapping clinical manifestations The diagnostic dilemma, s-JIA or KD ? Kawasaki Disease Scuccimarri R. Pediatr Clin North Am. 2012;59:425-45. The diagnosis of s-JIA is based on the combination of clinical symptoms and laboratory findings. Currently, there is no reliable biomarker for the diagnosis of s-JIA. Therefore, it is sometimes challenging to differentiate s-JIA from other inflammatory diseases. Kawasaki disease (KD) is much more common in Japan. We often have the diagnostic dilemma, s-JIA or KD, because clinical manifestations of s-JIA and KD are overlapping.

• 7. Kinjo N et al J Clin Apher. 2018；33：117-120 CRP (mg/dl) Feve rRas h IVI G IF X mPS L TC Z DexPCs A PS L P E PE+LCA P Plasma Dia-Filtration Day of illness (months) 0 1 2 3 4 106 105 104 103 102 101 MAS A case of s-JIA misdiagnosed with KD WBC (/mm3) ferritin (ng/ml) The s-JIA patients misdiagnosed with KD often show severe clinical courses complicated with MAS.

• 8. CQ1.　 What is a useful biomarker for the diagnosis of s-JIA？

• 9. Serum cytokine levels in patients with s-JIA Serum IL-6 and sTNF-RII levels were significantly elevated in patients with s-JIA. However, these levels were not useful for the differentiation between s-JIA and KD. Mizuta M, et al. Rheumatology. 2021;60:231-238. Mizuta M, et al. Rheumatology. 2021;60:2421-2426 IL-1β was not detected in most patients with s-JIA. De Benedetti F, et al. Clin Exp Rheumatol. 1995;13:779-84.

• 10. Serum cytokine levels in patients with s-JIA Mizuta M, et al. Rheumatology. 2021;60:231-238. Mizuta M, et al. Rheumatology. 2021;60:2421-2426 **** * **** ** **** * **** Serum IL-18 levels were massively elevated in patients with s-JIA and was useful for the differentiation between s-JIA and other diseases.

• 11. The patterns of serum cytokine profile are characteristic in each disease and are useful for the differentiation between s-JIA and other inflammatory diseases associated with MAS. Shimizu M et al. Rheumatology 2010;49:1645-1653 EBVーHLH Neopterin IL-6 sTNF-RII IL-18 sTNF-RI 10 3 3×10 3 3×10 4 10 5 5×10 3 3×10 4 10 4 3×10 3 3×10 4 10 5 5×10 4 7500 100 50 30 10 100 50 30 10 Kawasaki disease Neopterin IL-6 sTNF-RII IL-18 sTNF-RI 10 3 3×10 3 3×10 4 10 5 5×10 3 3×10 4 10 4 3×10 3 3×10 4 10 5 5×10 4 7500 100 50 30 10 100 50 30 10 s-JIA active phase Neopterin IL-6 sTNF-RII IL-18 sTNF-RI 10 3 3×10 3 3×10 4 10 5 5×10 3 3×10 4 10 4 3×10 3 3×10 4 10 5 5×10 4 7500 100 50 30 10 100 50 30 10 S-JIA inactive phase Neopterin IL-6 sTNF-RII IL-18 sTNF-RI 10 3 3×10 3 3×10 4 10 5 5×10 3 3×10 4 10 4 3×10 3 3×10 4 10 5 5×10 4 7500 100 50 30 10 100 50 30 10 Healthy children Neopterin IL-6 sTNF-RII IL-18 sTNF-RI 10 3 3×10 3 3×10 4 10 5 5×10 3 3×10 4 10 4 3×10 3 3×10 4 10 5 5×10 4 7500 100 50 30 10 100 50 30 10 s-JIA MAS Neopterin IL-6 sTNF-RII IL-18 sTNF-RI 10 3 3×10 3 3×10 4 10 5 5×10 3 3×10 4 10 4 3×10 3 3×10 4 10 5 5×10 4 7500 100 50 30 10 100 50 30 10 Serum cytokine levels in patients with s-JIA

• 12. Serum IL-18 level (pg/ml) median / range Cutoff value AUC s-JIA 34,750 (3,000-340,000) KD 360 (60-12,800) 4560 0.993 FMF 3,050 (1,180-4,400) 4800 0.9938 TRAPS 275 (180-370) 1685 1.0 Other subtypes of JIA 130 (48-455) 1728 1.0 SLE 825 (225-1,800) 2400 1.0 JDM 438 (86-1,250) 2125 1.0 Leukemia 826 (207-1,480) 2240 1.0 HCs 133 (76-215) 1608 1.0 Cut off value of serum IL-18 levels for the differentiation between s-JIA and other diseases Mizuta M, et al. Rheumatology. 2021;60:2421-2426 The cutoff value of serum IL-18 levels >4,800 pg/mL might be useful for the differentiation of s-JIA from other diseases.

• 13. Serum IL-18 levels as a biomarker for the diagnosis of s-JIA ILAR criteria PRINTO criteria ILAR criteria + IL-18(>4800pg/mL) PRINTO criteria + IL-18(>4800pg/mL) sensitivity 0.533 0.856 1.000 0.856 specificity 0.851 0.259 0.851 1.000 Kaneko S, et al. Cytokine 2024. in press • We evaluated the performance of the PRINTO criteria compared with the ILAR criteria and determined whether serum IL-18 levels improve their diagnostic performances. • The PRINTO criteria had higher sensitivity but lower specificity than the ILAR criteria. • With the addition of serum IL-18 levels ≥ 4,800 pg/mL, the sensitivity of the ILAR criteria and specificity of the PRINTO criteria were improved to 1.000 and 1.000, respectively. • Serum IL-18 levels could improve the diagnostic performance of the PRINTO and ILAR criteria for s-JIA. The PRINTO criteria plus serum IL-18 levels ≥ 4,800 pg/mL could be the best diagnostic performance for s-JIA.

• 14. CQ2. Is serum IL-18 level useful for the monitoring of disease activity in s-JIA?

• 15. Disease courses of s-JIA Singh-Grewal D et al. Arthritis Rheum. 2006;54:1595–1601 Disease activity Monophasic Polycyclic Persistent active disease for more than 24 months' duration Fever/Systemic Symptoms Arthritis Time Time Time active disease for any period of time, followed by inactive disease for any period of time and then recurrence of active disease.  a single episode of active disease not lasting more than 24 months, followed by inactive disease and without recurrence of active disease during the follow-up period Disease activity Disease activity

• 16. Serum IL-18 levels as an indicator of disease activity of s-JIA MAS MAS MAS relapses relapse Serum IL-18 levels markedly rose with the development of the complication of MAS, but gradually reduced after this manifestations resolved. However, even a few weeks after normalization of other inflammatory markers, IL-18 levels were still well above the normal value. In this patient, MAS was frequently complicated in this phase with high levels of serum IL-18. Shimizu M et al. Rheumatology 2010;49:1645-1653

• 17. 106 105 104 103 102 Disease activity polycyclic (days) 106 105 104 103 102 Systemic symptoms IL-18(pg/ml) Disease activity arthritis monophasic 106 105 104 103 102 Disease activity persistent Mizuta M, et al. Rheumatology. 2021;60:2421-26 Singh-Grewal D et al. Arthritis Rheum. 2006;54:1595–1601（一部改変） Serum IL-18 levels as an indicator of disease activity of s-JIA Serum IL-18 levels change in agreement with the disease course and reflect disease activity of s-JIA.

• 18. 0 0. 2 0. 4 0. 6 0. 8 1 0 5 1 0 1 5 persistent（n=18） Monophasic and polycyclic （n=47） Disease duration（years） Prognosis of s-JIA Takei S. Japanese J of Clin Med 2014;72:1854-60 Cumulative cure rate It seems that there are different subtypes in s-JIA, chronic arthritis type and systemic type.

• 19. CQ3. Are there different subtypes of s-JIA based on cytokine expression pattern?

• 20. ● MAS Distinct clinical features of the 2 s-JIA subsets based on their serum IL-6 and IL-18 levels Shimizu M et al. Clin Immunol 2015;160:277-81 There were 2 subsets of patients with s-JIA based on their serum IL-6 and IL-18 levels: IL-6–dominant and IL-18–dominant subset. IL-6–dominant subset had more severe joint disease, whereas IL-18–dominant subset had a more severe systemic disease and developed MAS. It seemed that high serum IL18 levels might be closely associated with the development of MAS IL-6 dominant IL-18 dominant P value Rash 19 34 P<0.05 Arthritis (number of affected joints) 2.7±5.2 0.8±1.5 P<0.001 complication of MAS 0 15 P<0.0001 Laboratory findings CRP (mg/dl) 13.3±6.5 8.2±5.7 P<0.01 AST (IU/l) 36.8±27.8 64.1±67.6 P<0.01 LDH (IU/l) 317.1±105.8 531.8±412.9 P<0.01 MMP-3 (ng/ml) (n=47) 286.3±373.8 50.5±45.1 P<0.01 IL-6 (pg/ml) 167.2±223.9 20.6±30.4 P<0.0001 IL-18 (pg/ml) 33872±59142 101809±90128 P<0.0001

• 21. CQ4. Are Serum IL-18 levels useful for the prediction and diagnosis of MAS?

• 22. Interleukin-18 for predicting the development of MAS Cut off value 47750 pg/ml Sensitivity 86.7% Specificity 70.5% Shimizu M et al. Clin Immunol. 2015；160：277-81 P<0.0001 P<0.0001 MAS - MAS + MAS phase active phase To determine clinical significance of serum IL-18 levels as a parameter to predict MAS development, we compared serum IL-18 levels in s-JIA patients with active disease who later developed MAS (MAS+) and those who did not (MAS-). Serum IL-18 levels in MAS+ patients were significantly higher than in MAS- patients. The receiver operating characteristic curve analysis indicated that the cutoff for serum IL-18 levels to predict MAS development was 47,750 pg/mL with a sensitivity of 86.7% and a specificity of 70.5% .

• 23. Interleukin-18 for predicting the development of MAS P<0.0001 P<0.0001 MAS - MAS + MAS phase active phase Shimizu M et al. Clin Immunol. 2015；160：277-81 In contrast, there was no significant difference in serum IL-18 levels in MAS+ patients whether measured before or during MAS. Serum IL-18 levels might not be useful for the diagnosis of MAS.

• 24. CQ5. What is a useful biomarker for the diagnosis of MAS？

• 25. Low middle high MAS Active TCZ(+) MAS Active(MAS+) Active(MAS-) TCZ(-) Extensive cytokine expression analysis in MAS patients revealed CXCL9 might be a useful for the diagnosis of MAS Mizuta M et al. Cytokine 2019;119：182-187 To identify the serum biomarkers for the diagnosis of MAS, we employed an antibody array that simultaneously detects 174 cytokines. CXCL9 showed the most significant increase following the development of MAS.

• 26. Biomarkers Cut off values Area under the ROC curve values Neopterin 19.5 0.9465 CXCL9 3130 0.9333 sTNFR-II/I 3.796 0.9395 Ferritin 2560 0.8671 IL-18 69250 0.8895 Takakuwa M, et al. Clin Immunol 2019:208:108252. Comparison of serum biomarkers for the diagnosis of MAS We compared the accuracy of serum biomarkers for the diagnosis of MAS. Receiver operating characteristic curve analysis revealed serum neopterin as well as CXCL9 levels is a useful marker to diagnose the transition to MAS from active-phase of s-JIA.

• 27. Schulert GS et al. Arthritis Care Res 2018; 70: 409-419より引用一部改変 Biologics modify clinical and laboratory findings of MAS Canakinumab Tocilizumab Historic cohort Definite/probable Possible Definite/probable Possible No. with Sufficient data 25 10 30 5 270 MAS 84 (21/25) 60 (6/10) 56.7 (17/30) 40(2/5) 7835 (212/270) Reasons missing ↓Ferritin (4) ↓Ferritin (4) Afebrile (7)↓Ferritin (6) Afebrile (1) ↓Ferritin (2) Treatment with biologics targeting IL-1 and IL-6 show a dramatic clinical effects for s-JIA. However, these drugs can modify and mask the clinical symptoms of MAS.

• 28. Shimizu M et al. Cytokine 2012; 58: 287-294 The diagnostic significance of IL-18 as a biomarker of s-JIA and MAS during TCZ therapy. p=0.0016 p=0.0002 Without TCZ With TCZ Increase of serum IL-18 levels in s-JIA and MAS were not suppressed by TCZ. Increase of serum IL-18 levels in s-JIA and MAS were not suppressed by CAN too. Serum IL-18 may be a useful biomarker of s-JIA and MAS even during the treatment with IL-1/IL-6 inhibitors.

• 29. Is this episode MAS? Patient 1: 8 years old boy with s-JIA receiving canakinumab and in remission Laboratory findings Normal values Jan 18 Feb 22 Feb24 WBC (/mm3) 3300-8600 7,700 2,900 2,300 Hb (g/dL) 13.7-16.8 13.1 13.3 14.1 Platelets (/mm3) 158,000-248,000 292,000 185,000 161,000 Fibrinogen (mg/dL) 200-400 ND 219 202 FDP (μg/mL) <4.9 ND 5.8 7.0 GOT/AST (IU/L) 13-30 26 97 98 LDH (IU/L) 124-222 216 435 554 CRP (mg/dL) <0.15 0.04 0.04 0.10 Ferritin (ng/mL) 14-304 20 362 687 He received canakinumab on Jan 18. He visited us with fever persisting 2 days and arthralgia on Feb 22.

• 30. Is this episode MAS? Patient 2: 5 years old girl with s-JIA receiving tocilizumab and in remission Laboratory findings Normal values Oct 3 Oct 23 Oct 24 WBC (/mm3) 3300-8600 4,900 2,300 2,300 Hb (g/dL) 13.7-16.8 11.8 11.5 11.9 Platelets (/mm3) 158,000-248,000 260,000 100,000 114,000 Fibrinogen (mg/dL) 200-400 ND 121 119 FDP D dimer (μg/mL) <0.9 ND 15.7 17.5 GOT/AST (IU/L) 13-30 23 48 42 LDH (IU/L) 124-222 235 551 589 CRP (mg/dL) <0.15 <0.03 <0.03 <0.03 Ferritin (ng/mL) 14-304 15 218 213 She received tocilizumab on Oct 3. She visited us with fever persisting 4 days on Oct 23.

• 31. Is this episode MAS? Patient 1 Patient 2 IL6 IL-1 8 103 3x103 3x104 105 50 30 10 100 5000 1x104 3x10 2 sTNF-R II CXCL 9 3x10 3 10 4 3x104 2x10 4 15x103 IL6 IL-1 8 103 3x103 3x104 105 50 30 10 100 5000 1x104 3x10 2 sTNF-R II CXCL 9 3x10 3 10 4 3x104 2x10 4 15x103 MA s-JIA active S IL6 IL-1 8 103 3x103 3x104 105 50 30 10 100 5000 1x104 3x10 2 sTNF-R II CXCL 9 3x10 3 10 4 3x104 2x10 4 15x103 IL6 IL-1 8 103 3x103 3x104 105 50 30 10 100 5000 1x104 3x10 2 sTNF-R II CXCL 9 3x10 3 10 4 3x104 2x10 4 15x103 MA S s-JIA flare Serum cytokine profile is useful for understanding what happens in the patient.

• 32. CQ5. What is the role of IL-18 in the pathogenesis of MAS?

• 33. Day1 Day8 Day10 Pump insert 　　 CpG injection C57BL/6 Normal Sarine IL-1β IL-18 IL-1β+IL-18 MAS Mizuta M, et al. J Allergy Clin Immunol. 2023;152:940-948 To clarify the role of IL-18 and IL-1β in the pathogenesis of MAS, we developed a mouse model to evaluate the role of each cytokine with TLR9 stimulation after continuous infusion with IL-18, IL-1β, and a combination of both for 7 days. New murine model of MAS

• 34. *** *** IL-18+IL-1β+CpG IL-1β+CpG Body weight was significantly decreased in the IL-1β and the combination groups. Splenomegaly was observed in all groups, whereas hepatomegaly was noted in IL-18 group only. Clinical manifestations after CpG injection Mizuta M, et al. J Allergy Clin Immunol. 2023;152:940-948 ** *** * IL-18+IL-1β+CpG IL-18+CpG IL-1β+CpG NS+CpG Untreated IL-18+IL-1β+CpG IL-18+CpG IL-1β+CpG NS+CpG Untreated

• 35. Laboratory findings after CpG injection WBC (x 10 2/μL) RBC (x 10 4/μL) * ferritin (ng/mL) NS IL-18 IL-18 +IL-1β IL-1β Day 8 10 8 10 8 10 8 10 NK cell count (x 10 2/μL) NS IL-18 IL-18 +IL-1β IL-1β Day 8 10 8 10 8 10 8 10 NS IL-18 IL-18 +IL-1β IL-1β Day 8 10 8 10 8 10 8 10 NS IL-18 IL-18 +IL-1β IL-1β Day 8 10 8 10 8 10 8 10 * ** CD8+ T cell count (x 10 2/μL) NS IL-18 IL-18 +IL-1β IL-1β Day 8 10 8 10 8 10 8 10 * Lymphocyte count (x 10 2/μL) NS IL-18 IL-18 +IL-1β IL-1β Day 8 10 8 10 8 10 8 10 * ** Plt (x 10 4/μL) NS IL-18 IL-18 +IL-1β IL-1β Day 8 10 8 10 8 10 8 10 * NS IL-18 IL-18 +IL-1β IL-1β Day 8 10 8 10 8 10 8 10 * CD4+ T cell count (x 10 2/μL) Decreased T cell numbers, anemia, and thrombocytopenia were observed in the combination group. Mizuta M, et al. J Allergy Clin Immunol. 2023;152:940-948

• 36. IL-18+IL-1β IL-18 IL-1β NS Untreated IFN-γ/GAPDH * *** * ** *** *** IL-18+IL-1β IL-18 IL-1β NS Untreated TNF-α/GAPDH * IL-18+IL-1β IL-18 IL-1β NS Untreated *** IL-18+IL-1β IL-18 IL-1β NS Untreated ** IL-18+IL-1β IL-18 IL-1β NS Untreated IFN-γ, CXCL9, and IL-12A mRNA levels were upregulated and IL-10 mRNA levels in the spleen were downregulated in the IL-18 group. Mizuta M, et al. J Allergy Clin Immunol. 2023;152:940-948 Cytokine mRNA expression levels after CpG injection IFN-γ CXCL9 TNF-α IL-12A IL-10

• 37. * *** ** ** * TNFα/GAPDH * *** IFN-γ/GAPDH TNF-α/GAPDH Cytokine mRNA levels were regulated by IL-18 in a dose-dependent manner Mizuta M, et al. J Allergy Clin Immunol. 2023;152:940-948 TNF-α, CXCL9, and IL-12A mRNA levels were upregulated, IL-10 mRNA was downregulated only in mice with extremely elevated plasma IL-18 levels. IFN-γ CXCL9 TNF-α IL-12A IL-10

• 38. Distinct roles of IL-18 and IL-1β in murine model of MAS Mizuta M, et al. J Allergy Clin Immunol. 2023;152:940-948 IL-18 and IL-1β have distinct roles in the pathogenesis of MAS. Dual blockade of IL-18 and IL-1β might be necessary to treat MAS.

• 39. Conclusions • Serum IL-18 level is a useful biomarker for the diagnosis and monitoring of disease activity of s-JIA. • There are 2 subsets of s-JIA patients with distinct clinical features based on their serum IL-6 and IL-18 levels. • Serum IL-18 level is useful for the prediction of the development of MAS. • Serum neopterin and CXCL9 levels are useful biomarkers for the diagnosis of MAS. • Serum cytokine profile including IL-18 and CXCL9 is useful for understating the pathology in patients with s-JIA, in particular, in patients receiving biologics. • IL-18 and IL-1β have distinct roles in the pathogenesis of MAS. Dual blockade of IL-18 and IL-1β might be necessary to treat MAS.

• 40. Thank you for your attentions. Pediatric Rheumatology and Nephrology Team in Kanazawa University and Institute of Science Tokyo