Adult-onset Still’s disease (AOSD) is a rare, polygenic, systemic autoinflammatory disease characterized by recurrent fever, skin rash (typically salmon-pink in color), arthralgia or arthritis, sore throat, leukocytosis with neutrophilia, lymphadenopathy, splenomegaly, and liver dysfunction (1, 2). The etiology of AOSD is unknown, but the following are recognized as central to the pathogenesis of AOSD: an abnormal activation of innate immune cells (e.g., neutrophils, monocytes, and macrophages) and the overproduction of cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and IL-18 (1–4). The diagnosis of AOSD is based on a combination of clinical and laboratory findings, excluding infections (especially sepsis and viral infections), malignant neoplasms (especially lymphoma), and inflammatory diseases (especially polyarthritis nodosa) (5, 6). The serum level of ferritin is frequently used as a practical tool for the diagnosis of AOSD, but its diagnostic specificity is low and of limited value in the diagnosis of AOSD (3, 5, 7). The diagnosis of AOSD is thus often difficult, and we have sought to identify specific diagnostic markers for AOSD.

Serum levels of various cytokines and chemokines have been reported to be useful in the diagnosis of AOSD (8), and IL-18 has been found to be markedly elevated in the serum of AOSD patients in several studies (1, 9). IL-18 has been reported to be associated with the disease activity of AOSD (10, 11), and it may be useful as an indicator of therapeutic efficacy (12). The question of whether IL-18 could be a target for the treatment of AOSD is being investigated (13). IL-18 may play a particularly important role in the pathophysiology of AOSD, but whether IL-18 may be useful as its diagnostic biomarker remains to be determined.

Hemophagocytic lymphohistiocytosis (HLH) and hemophagocytic syndrome (HPS) are life-threatening inflammatory systemic disorders, and their clinical features (e.g., fever and splenomegaly) and laboratory data (e.g., elevated transaminases and hyperferritinemia) are very similar to those of AOSD (14). HLH in adults is usually classified as secondary (reactive), which is subdivided into infectious, neoplastic, and autoimmune-related secondary HLH (14, 15).

Macrophage activation syndrome (MAS), an acquired form of HLH that occurs in autoimmune diseases, is a common and serious complication of AOSD, and it is especially difficult to distinguish AOSD-related MAS from HLH or MAS with other underlying diseases (16). It was recently revealed that serum IL-18 levels are increased in primary (genetic) and secondary HLH, especially in pediatric patients, and several reports have compared the serum IL-18 levels between pediatric HLH cases and patients with systemic juvenile idiopathic arthritis (sJIA), a homolog of AOSD in children (17). There have been few similar comparative studies between AOSD and adult HLH. We thus conducted the present study to investigate whether serum IL-18 levels could be a useful biomarker for the differential diagnosis of AOSD with or without MAS and other forms of secondary HLH in adults.

The results of our analyses demonstrated that serum IL-18 levels could be used as a biomarker with high accuracy for the differential diagnosis of AOSD and adult HLH. AOSD, HLH, and MAS share some similar aspects of their pathogeneses (e.g., an abnormal activation of macrophages and an excessive production of various cytokines), and it was reported that serum IL-18 levels are markedly elevated in each of these diseases (1–3, 16, 21). However, few studies have compared serum IL-18 levels between AOSD, HLH, and MAS in adults (unlike sJIA, HLH and MAS in pediatric patients), because adult HLH and MAS have a lower incidence compared to those in pediatric patients, and few studies of adult HLH or MAS have been performed (14). The effectiveness of the use of serum IL-18 levels to distinguish these diseases has thus not been fully validated. To the best of our knowledge, our present investigation is the only case-control study to directly compare serum IL-18 levels between AOSD with or without MAS and other secondary HLH or MAS in adults, unlike the research regarding sJIA and pediatric HLH or MAS.

To assess the diagnostic ability of IL-18 in AOSD, it is necessary to conduct comparative studies with control diseases that have pathologies and clinical findings similar to those of AOSD. Between 2001, when Kawashima et al. were the first to report that serum IL-18 was markedly elevated in patients with AOSD, and the present investigation’s writing, several studies have compared serum IL-18 levels in AOSD and other inflammatory diseases in adults (8, 10, 12, 22–32). The most common control group in those reports was rheumatic diseases, mainly such as rheumatoid arthritis, SLE, and Sjogren’s syndrome. Serum IL-18 levels in AOSD patients were statistically higher than those in any of the rheumatic diseases in those studies. The control disease in the remaining studies was infection, especially sepsis in two studies (30, 31), which is categorized as ‘hyperferritinemic syndrome,’ similar to MAS or Still’s disease (33). These studies also showed that the serum IL-18 levels in patients with sepsis were approximately 100 pg/mL, which is significantly lower than the levels in AOSD patients. Interestingly, Chen et al. recently reported that serum IL-18 levels at the cut-off value 190.5 pg/ml had high discriminative ability for AOSD and coronavirus disease 2019 (COVID-19), with ROC-AUC 0.948, sensitivity 91.3% and specificity 95.8% (34).

To the best of our knowledge, no comparative study of AOSD, HLH, and MAS has been ever performed in adults, whereas there are numerous studies comparing sJIA, HLH, and MAS in pediatric populations. Krei et al. recently conducted a systematic review of 14 studies regarding the potential role of IL-18 in the diagnosis of HLH or MAS mainly in children (17). In that review, the serum IL-18 levels of the patients with active sJIA were approx. 10⁴–10⁵ pg/mL, whereas those of the HLH patients remained in the range of 10³–10⁴ pg/mL. Among the14 studies in the review, Weiss et al. reported that a total IL-18 (i.e., IL-18 bound to IL-18 binding protein [BP]) cut-off value at >11,600 pg/mL distinguished sJIA (or AOSD) or MAS complicating sJIA (or AOSD) from familial or infection-associated HLH, with 88% sensitivity and 93% specificity (35). Considering that AOSD shares pathology with sJIA, these findings seem to support the validity of our present results and further reinforce that sJIA and AOSD are roughly the same disease.

We subdivided the AOSD group and the adult HLH group into four subgroups as shown in Table 2 and Figure 1 and observed that the serum IL-18 levels in the AOSD group were significantly higher those in each subdivided adult HLH group, regardless of whether MAS was complicating with AOSD or not. There was no significant difference in serum IL-18 levels between the AOSD MAS- and MAS+ groups. In contrast to these findings, it has been reported that IL-18 or ferritin was significantly increased in AOSD patients with MAS compared to those without MAS (36, 37). These different results might be due to the small sample size of the patients (n=9) and high rate of steroid use at the time of diagnosis (n=3, 33%) in the AOSD MAS+ group.

Regarding useful laboratory data for differentiating AOSD from adult HLH other than IL-18, the present multivariate analysis identified sIL-2R, which is a 45-kDa molecule released from activated T cells (38, 39). SIL-2R is then considered a surrogate biomarker for T cell activation (40). On the other hand, it is also produced by dendritic cells, activated B cells and lymphoma cells (39), and is commonly used as a tumor marker for malignant lymphomas in clinical practice. Although serum sIL-2R levels have been also reported to be elevated not only in malignant lymphomas but also in AOSD or HLH other than LAHS (18, 38), sIL-2R in the LAHS group was markably higher compared to other three groups (espacially the AOSD MAS- group) in this study. Tsuji et al. reported that a sIL-2R/ferritin ratio of patients in LAHS was significantly higher than that of patients in benign disease-associated HLH (8.56 vs. 0.66, p=0.0004) (41). Therefore, elevated serum levels of sIL-2R in LAHS might be due in part to its excessive and abnormal secretion from malignant cells.

On the other hand, sIL-2R in the diagnosis of adult HLH has been reported to have high sensitivity but low specificity (42, 43). The same statistical trend was observed in this study (sIL-2R AOSD vs. adult HLH; sensitivity: 77.4%, specificity: 69.6%). The results of previous reports and the present study suggest that the ability of sIL-2R to differentiate between AOSD with or without MAS and adult HLH is limited.

Among our patients’ clinical features, the multivariate analysis demonstrated that the presence of arthralgia or arthritis, but not skin rash or sore throat, was an independent differential predictor in the AOSD group. After fever, arthralgia is the second most common symptom in patients with AOSD (44), occurring in more than two-thirds of patients with AOSD and in 35 of the present 46 patients (76.1%). Higher expression levels of IL-18 mRNA were observed in synovial biopsies from patients with active AOSD compared to healthy controls (23), suggesting that IL-18 may be associated with joint pain or arthritis in individuals with AOSD. The combination of these clinical findings with serum IL-18 levels may further improve the diagnostic accuracy for AOSD.

In our present patient population, the serum IL-18 levels in the AOSD group showed a strong correlation not only with ferritin but also with sIL-2R, LDH, and liver deviation enzymes, consistent with previous reports (45), and all of these are considered disease activity markers of AOSD (17, 46). In patients with active AOSD, an increase in serum levels of sIL-2R correlated with IL-18 is presumed to be a mechanism by which IL-18 induces interferon-gamma production by activating T cells with IL-18 receptors, especially IL-17-positive γδ cells (47). In the present study, LDH showed the strongest correlation with IL-18 (r= 0.85, p<0.0001), and the correlation between AST and IL-18 was stronger than that between AST and IL-18. These results might indicate that IL-18 expression is widely distributed not only in the liver but also in organs throughout the body.

The prognosis of AOSD with MAS is worse than that of AOSD without MAS (48), and the mortality rate of AOSD with MAS has been high, ranging from 10% to 41% (49, 50). Therefore, early prediction of progression to MAS is also important in the diagnosis of AOSD (16). IL-18 may also be useful for early diagnosis of AOSD-related MAS, but we could not show any difference in serum IL-18 levels between the AOSD MAS- and AOSD MAS+ group in this study. On the other hand, the HScore is an indicator developed for the diagnosis of secondary HLH, mainly in adults (51), and has recently been reported to be useful for the diagnosis of rheumatic disease-associated HLH, including AOSD (20). Although there were some missing data, the HScore was significantly higher in the AOSD MAS+ group compared to the AOSD MAS- group in the four-group comparison of the current study. The result suggests that the HScore may be useful as a predictor of MAS in the diagnosis of AOSD.

There are several study limitations to address. This was a retrospective and single-center study, and the possibility of unintentional selection bias in the enrollment of patients thus cannot be fully excluded. For example, 17 of the 31 patients (54.8%) in the adult HLH group had LAHS, while Ramos-Casals et al. verified the causative diseases of 2,197 cases of adult HLH or MAS, of which 798 (39.8%) were lymphoma (14). There might have been a partial bias in the underlying diseases of our present adult HLH group. In addition, free IL-18 (i.e., IL-18 not bound to IL-18 BP) in the serum is elevated in patients with AOSD and has been shown to be closely associated with disease activity, and IL-18 BP has been reported to bind to free IL-18 and neutralize its activity (8, 47). The assay that we used in this study could not accurately evaluate the activity of IL-18 because it could not distinguish between free IL-18 not bound to IL-18 BP and IL-18 complexed with IL-18 BP. Finally, eight patients in the AOSD group and eight patients in the adult HLH group were using a steroid or immunosuppressive agent at the time of blood collection. This may have affected the production of cytokines, but as shown in Supplementary Figure S1, the ROC-AUC of IL-18 in the untreated AOSD and adult HLH groups with a cutoff value of 18,550 pg/mL was 0.87 (95%CI: 0.78–0.94, 87.0% sensitivity, 92.1% specificity), and there was not a clear difference compared to the cases involving treated patients. There is an opportunity to diagnose AOSD patients and provide treatment interventions, and thus this result might be more in line with daily clinical practice.

Serum IL-18 levels were shown to be a very useful biomarker for differentiating AOSD from adult HLH. In particular, when MAS is present in AOSD patients, the measurement of serum IL-18 levels may facilitate the distinction between AOSD-associated MAS and other secondary HLH/HPS. Although the diagnosis of AOSD remains challenging, the results of this study may improve the diagnostic performance for AOSD.

The original contributions presented in the study are included in the study/Supplementary Material. Further inquiries can be directed to the corresponding author.

This study complied with the standards of the Declaration of Helsinki, and was registered and approved by the Research Ethics Committee of the Faculty of Medicine of Kindai University (approval no. R02-218). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Conception and design, TS, YN, MF, and IM. Acquisition of data, TS, DT, and KaK. Statistical analysis, TS, YN, and KoK. Manuscript preparation, TS, YN, and MF. All authors contributed to the manuscript’s revision and have read and approved the submitted version

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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