Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4 mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4 intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ-induced genes. Assessing cellular and tissue expression, classical inflammasome components such as , and predominated in neutrophils, whereas and were distinctly epithelial. Demonstrating the importance of free IL-18, transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4 mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

The term macrophage activation syndrome (MAS) defines a severe, potentially fatal disorder characterized by overwhelming inflammation and multiorgan involvement. Interleukin-18 (IL-18) is a proinflammatory cytokine belonging to the IL-1 family, the activity of which is regulated by its endogenous inhibitor IL-18 binding protein (IL-18BP). Elevated IL-18 levels have been reported in patients with MAS. Herein, we show that on repeated toll-like receptor 9 (TLR9) stimulation with unmethylated cytosine guanine dinucleotide containing single-stranded DNA (CpG), mice display severe MAS manifestations, including increased weight loss, splenomegaly, anemia, thrombocytopenia, hyperferritinemia, and bone marrow hemophagocytosis as compared with wild-type mice. Serum-free IL-18 was detected in CpG-treated mice only. Levels of interferon-γ (IFN-γ) and of IFN-γ signature genes, such as the chemokine or the transcription factor, were significantly increased in mice. Blocking IL-18 receptor signaling attenuated the severity of MAS and IFN-γ responses in mice. Blocking IFN-γ had comparable effects to IL-18 inhibition on most MAS manifestations. Our data indicate that endogenous IL-18BP exerts a protective role in CpG-induced MAS and that IL-18, which acts upstream of IFN-γ, is involved in the severity of MAS.

Objectives Systemic juvenile idiopathic arthritis (sJIA) is a childhood arthritis with features of autoinflammation and high risk of macrophage activation syndrome (MAS). IL-18 has been shown to have key roles in sJIA and MAS. We aimed to examine IL-18 levels in sJIA in relation to disease activity and history of MAS and other disease biomarkers namely S100 proteins and CXCL9. Methods Total IL-18, CXCL9 and S100 proteins levels were determined in 40 sJIA patients, and IL-18 levels were compared between patients with regards to disease activity, history of MAS, and other biomarkers. Results Total IL-18 levels were significantly higher in patients with active sJIA (median 16 499 pg/ml; interquartile range (IQR) 4816–61 839), and remained persistently elevated even in the majority of patients with inactive disease (1164 pg/ml; IQR 587–3444). Patients with history of MAS had significantly higher IL-18 levels (13 380 pg/ml; IQR 4212–62 628) as compared with those without MAS history (956.5 pg/ml; IQR 276.3–4262.5). Total IL-18 performed well with area under the curve of 0.8145 and 0.84 in predicting disease activity and history of MAS, respectively. We observed moderate correlation between IL-18 and CXCL9 (R = 0.56), S100A8/A9 (R = 0.47) and S100A12 (R = 0.46). The correlation was stronger for ferritin (R = 0.74) and overall for those with active disease. Conclusion Total IL-18 levels were elevated in the majority of sJIA patients regardless of clinical features, but were higher in patients with active disease and history of MAS. Change in IL-18 may reflect increased disease activity or development of MAS.

inhibition. Moreover, a combination of biomarkers including WBC, CRP, ferritin and LDH may be useful for the diagnosis of flare.A lack of biomarkers during tocilizumab treatment in inflammatory diseases has been a problem for clinical practice. Especially for large vessel vasculitis and adultonset Still's disease, the evaluation of disease activity is more dependent on acute-phase reactants in blood tests than RA. Our study showed that the CRP levels reflected disease activity under treatment with an IL-6 inhibitor only slightly, as was expected. Alternatively, LDH was identified as a potential biomarker to detect flare. LDH is an enzyme that exists abundantly in reticulocytes, the heart, lungs, muscles and liver, but is distributed in almost all types of organ cells. We assume that LDH elevation in active adult-onset Still's disease not only reflects liver involvement, but also is derived from systemic inflammation of multiple organs, caused by inflammatory cytokines. Indeed, LDH elevation has been reported to be relevant to systemic inflammation [6,7] and macrophage-activated syndrome [8]. Another important suggestion of our study was that a very subtle increase in CRP levels could indicate worsening in adult-onset Still's disease under tocilizumab treatment. The CRP level at relapse in tocilizumab(+) was only 0.1 mg/dl, but the increase from 0.01 mg/dl before relapse was significant.Our data is not confirmatory, due to the nature of a retrospective study with a small sample size; however, we believe that serum LDH is a promising biomarker for flare in adult-onset Still's disease treated with an IL-6 inhibitor. Since adult-onset Still's disease is a rare disease, and a flare during anti-IL-6 treatment is much rarer [4], cooperation to accumulate cases is necessary to validate our data.

Objective Infection with the novel coronavirus SARS–CoV‐2 triggers severe illness with high mortality in a subgroup of patients. Such a critical course of COVID‐19 is thought to be associated with the development of cytokine storm, a condition seen in macrophage activation syndrome (MAS) and secondary hemophagocytic lymphohistiocytosis (HLH). However, specific data demonstrating a clear association of cytokine storm with severe COVID‐19 are still lacking. The aim of this study was to directly address whether immune activation in COVID‐19 does indeed mimic the conditions found in these classic cytokine storm syndromes. Methods Levels of 22 biomarkers were quantified in serum samples from patients with COVID‐19 (n = 30 patients, n = 83 longitudinal samples in total), patients with secondary HLH/MAS (n = 50), and healthy controls (n = 9). Measurements were performed using bead array assays and single‐marker enzyme‐linked immunosorbent assay. Serum biomarker levels were assessed for correlations with disease outcome. Results In patients with secondary HLH/MAS, we observed pronounced activation of the interleukin‐18 (IL‐18)–interferon‐γ axis, increased serum levels of IL‐1 receptor antagonist, intercellular adhesion molecule 1, and IL‐8, and strongly reduced levels of soluble Fas ligand in the course of SARS–CoV‐2 infection. These observations appeared to discriminate immune dysregulation in critical COVID‐19 from the well‐recognized characteristics of other cytokine storm syndromes. Conclusion Serum biomarker profiles clearly separate COVID‐19 from MAS or secondary HLH in terms of distinguishing the severe systemic hyperinflammation that occurs following SARS–CoV‐2 infection. These findings could be useful in determining the efficacy of drugs targeting key molecules and pathways specifically associated with systemic cytokine storm conditions in the treatment of COVID‐19.

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Objective Dominantly inherited PSTPIP1 mutations cause a spectrum of autoinflammatory manifestations epitomized by PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome.). The connections between PSTPIP1 and PAPA syndrome are poorly understood, although evidence suggests involvement of pyrin inflammasome activation. Interleukin‐18 (IL‐18) is an inflammasome‐activated cytokine associated with susceptibility to macrophage activation syndrome (MAS). This study was undertaken to investigate an association of IL‐18 with PAPA syndrome. Methods Clinical and genetic data and serum samples were obtained from patients referred to institutions due to symptoms indicative of PAPA syndrome. Serum IL‐18, IL‐18 binding protein (IL‐18BP), and CXCL9 levels were assessed by bead‐based assay, and free IL‐18 levels were assessed by enzyme‐linked immunosorbent assay. Results The symptoms of PSTPIP1‐positive patients with PAPA syndrome overlapped with those of mutation‐negative patients with PAPA‐like conditions, but mutation‐positive patients had earlier onset and a greater proportion had a history of arthritis. We found uniform elevation of total serum IL‐18 in treated PAPA syndrome patients at levels nearly as high as those seen in NLRC4‐associated autoinflammation with infantile enterocolitis patients, and well above levels found in most familial Mediterranean fever patients. Serum IL‐18 elevation in PAPA syndrome patients persisted despite fluctuations in disease activity. Levels of the soluble IL‐18 antagonist IL‐18BP were modestly elevated, and PAPA syndrome patients had detectable free IL‐18. PAPA syndrome was rarely associated with elevation of CXCL9, an indicator of interferon‐γ activity, but no PAPA syndrome patients had a history of MAS. Conclusion PAPA syndrome is a refractory and often disabling monogenic autoinflammatory disease associated with chronic and unopposed elevation of serum IL‐18 levels but not with risk of MAS. These findings affect our understanding of the diseases in which IL‐18 is overproduced and suggest a link between pyrin inflammasome activation, IL‐18, and autoinflammation, without susceptibility to MAS.

IL-18 binding protein; Il-18bp 2/2 , Il-18bp-deficient; KO, knockout; KO→KO, Il-18bp 2/2 BM transplanted into Il-18bp 2/2 recipient mice; KO→WT, Il-18bp 2/2 BM transplanted into WT recipient mice; MAS, macrophage activation syndrome; pt-Il18bp, primary Il18bp transcript; sJIA, systemic juvenile idiopathic arthritis; WT, wild-type; WT→KO, WT BM transplanted into Il-18bp 2/2 recipient mice; WT→WT, WT BM transplanted into WT recipient mice.