Secondary hemophagocytic lymphohistiocytosis in adults (sHLHa) is a multiorgan inflammatory response syndrome triggered by an abnormal inflammatory response that further results in the aggregation of activated T lymphocytes and macrophages in multiple organs and the outbreak of a cytokine stor (Valade et al., 2021; Nguyen et al., 2024). The disease is usually triggered by a variety of causative factors, such as infections, malignancies, and rheumatologic-immune disorders (Griffin et al., 2020). Patients with untreated sHLHa have a high mortality rate (20%–53%, up to 70% for specific types of patients) (La Rosée et al., 2019; Rouphael et al., 2007). The main clinical features of sHLHa include persistent fever, hepatosplenomegaly, hematopenia in more than two lineages of cells, methemoglobinemia, and phagocytic phenomena. Commonly used clinical diagnostic indicators (e.g., ferritin, lactate dehydrogenase, and the splenic index) are insufficient to achieve early diagnosis and prognostic assessment of the disease (Henter, 2025). In addition, there is no single biomarker that reliably predicts disease progression and patient prognosis (Wu et al., 2024). This current situation highlights the importance and urgency of finding novel biomarkers to improve the diagnosis and prognostic assessment of sHLHa.

In recent years, S100A8 and S100A9 have been shown to be constitutively expressed in myeloid cells as endogenous alert proteins and stored as heterodimeric complexes (S100A8/9) in granules (Leukert et al., 2006). S100A8/9 is highly correlated with monocyte macrophage activation in hyperinflammatory responsive diseases, autoimmune disorders, and a variety of tumors (Manfredi et al., 2023; Muñoz-Grajales et al., 2023; Perego et al., 2020; Wang Q. et al., 2023; Yao et al., 2022) and is able to promote the production of inflammatory factor (Wen et al., 2018). In the pathology of sHLHa, the uncontrolled inflammatory cascade, the ensuing cytokine storm and immunosuppression are the main drivers of disease progression and poor prognosis (Nguyen et al., 2024). However, it is unclear whether elevated S100A8/9 levels are associated with poor survival in sHLHa patients, especially when independent from traditional risk factors (e.g., age and underlying disease).

Based on the above research background and gaps in the existing knowledge, the aim of this study was to systematically evaluate the clinical value of serum S100A8/9 levels in the diagnosis and prognostic assessment of patients with sHLHa.

HLH is a rapidly progressive and highly lethal disease (Nguyen et al., 2024). The diagnostic guidelines for adult sHLHa mostly borrow from those for pediatric pHLH (Griffin et al., 2020). Delayed diagnosis of sHLHa syndrome is significantly associated with increased in-hospital mortality rates (La Rosée et al., 2019). In this study, we evaluated for the first time the diagnostic and prognostic value of serum S100A8/9 in the primary diagnosis of sHLHa. Patients with sHLHa were better differentiated by ROC curves, with a similar diagnostic value to the HScore (P > 0.05) and a strong predictive value in identifying different etiologies (P < 0.01). In addition, RCS analysis revealed a nonlinear positive correlation between serum S100A8/9 levels and poor prognosis in adult sHLHa patients, with an inflection point of 2.44 μg/mL, and a significantly shorter survival for patients in the high-level group (median OS: 62 vs. 520 days, P = 0.0039).

In the present study, we found that serum S100A8/9 levels were significantly higher in sHLHa patients than in healthy controls (p < 0.05), a result that is consistent with a study by Muhammad S Soyfoo et al. ¹⁷. S100A8/9, an endogenous alarm protein, plays an important role in a variety of inflammatory diseases and cancers, and its elevated expression level has been widely recognized as a marker of the inflammatory response and disease progression. For example, in systemic lupus erythematosus (SLE), S100A8/9 levels are significantly elevated and correlate with disease activity (Soyfoo et al., 2009). In addition, S100A8/9 has significant proinflammatory effects on a variety of cancers (lung, liver, colorectal, etc.) and inflammatory diseases (sepsis, rheumatoid arthritis, etc.) (Başsorgun et al., 2014; Gebhardt et al., 2015; Hermani et al., 2006; Liu et al., 2022; Petersen et al., 2000; Sy et al., 2005; Zhang et al., 2023; Di Ceglie et al., 2018; Rodriguez-Smith et al., 2021; Wei et al., 2025). Our study found similar efficacy of serum S100A8/9 levels and diagnostic value of Hscore score in terms of diagnosing patients with sHLH (p > 0.05). In the present study, S100A8/9 not only effectively distinguished sHLH patients from other healthy individuals but also exhibited differential expression in sHLH patients with different etiologic subtypes (e.g., S100A8/9 levels were higher in LHLH patients than in non-LHLH patients), suggesting that sHLH patients with different etiologies may have different pathophysiologic mechanisms and inflammatory response intensities, which is also consistent with previous studies on the differences in the survival prognoses of sHLH patients with different etiologies (Jongdee et al., 2024).

The results of the present study revealed a significant positive correlation between the S100A8/9 levels of sHLHa patients and their survival (p < 0.05) and identified a threshold of 2.44 μg/mL for predicting poor prognosis. Patients had a significantly increased risk of death when their serum S100A8/9 levels exceeded this threshold. This finding is consistent with previous findings (Hou et al., 2023; Jakobsson et al., 2023; Krenkel et al., 2020; R et al., 2021; Su et al., 2022) that elevated S100A8/9 levels are strongly associated with poor prognosis in a variety of inflammatory diseases and malignancies, such as patients with severe COVID-19 and patients with advanced extranodal NK/T-cell lymphoma (ENKL) (Lee et al., 2023; Zhou et al., 2016), where S100A8/9 levels correlate with poor survival. Further multivariate Cox regression analysis confirmed that high S100A8/9 expression was an independent risk factor for poor prognosis in patients with sHLHa syndrome, suggesting its significant predictive value in clinical practice.

We also found that S100A8/9 levels in sHLHa patients were significantly correlated (P < 0.05) with liver function indices (ALT, AST, LDH, α-HBDH, ADA). This result is consistent with that of the study by Chang et al. (2024), suggesting that the upregulation of S100A8/9 expression may affect the survival prognosis of patients by exacerbating liver injury and fibrosis. Calcineurin S100A8/9, binds to RAGE, scavenger receptor (CD36), CD33 or Toll-like receptor 4 (TLR4) to control protein phosphorylation, various enzyme activities, Ca2+ homeostasis, NADPH oxidase activation and intermediate filament polymerisation (Möller et al., 2023; Boucher et al., 2024), This association may reflect a pathophysiological mechanism of multiorgan dysfunction in sHLHa patients, especially in the presence of liver damage, which may be further exacerbated by S100A8/9 through the promotion of inflammatory responses and cytokine storms, this may lead to variability in serum S100A8/9 levels in different patients.

Although the exact mechanism of action of S100A8/9 in sHLHa is unclear, we propose a possible hypothesis that lymphocyte expansion and sustained activation of monocyte macrophages may lead to the upregulation of S100A8/9 expression, and this upregulation induces the secretion of proinflammatory cytokines (e.g., TNFα, IL-1β, and IL-6) and chemokines (e.g., MIP-1α and MIP-1α) by monocyte macrophages through the activation of the TLR4/myeloid differentiation Factor 88 (MyD88)/nuclear factor KB (NF-κ B) signaling pathway, which induces monocyte macrophages to secrete proinflammatory cytokines, thereby triggering a cytokine storm leading to multiorgan dysfunction (Yao et al., 2022; Hou et al., 2023; Al-Samkari and Berliner, 2018; Ramos-Casals et al., 2014; Cristaldi et al., 2023; Lu et al., 2024; Xin et al., 2024; Ye et al., 2025; Zheng et al., 2022). In addition, Haijun Wen et al. reported that the V140A mutation in the ECSIT gene activated the NF-κ B pathway by promoting the formation of S100A8/9, which in turn induced macrophage overactivation to secrete cytokines, ultimately leading to phagocytosis syndrome (Wen et al., 2018). These findings further support the critical role of S100A8/9 in the pathogenesis of sHLHa. Recent studies have also shown that the proinflammatory mechanism of S100A8/9 in vivo is highly similar to the pathogenesis of HLH characterized by a cytokine storm. For example, Giada Ingoglia et al. reported that macrophage agonistic anti-CD40 antibody treatment induced HLH by establishing a macrophage-centered mouse model (Ingoglia et al., 2020). Fan Su et al. demonstrated that a nanocomplex loaded with the S100A8/9 inhibitor ABR2527 effectively blocked neutrophil-S100A8/9-toll-like receptor (TLRS)-inflammatory vesicle signalling, thereby attenuating sepsis-induced acute lung injury in preclinical mice (Su et al., 2025). Recent BLOOD findings that neutrophil activation triggers elevated associated inflammatory factors in AOSD-MAS patients have elucidated the mechanism by which rucotinib can modulate neutrophil activation through broad inhibition of the JAK-STAT signalling pathway (Ma et al., 2025). Notably, lipid rafts can act as signalling hubs in the vascular system and in the inflammatory response (Warda et al., 2025). Larisa Dubrovsky’s study showed that inhibitors of methyl-β-cyclodextrin, statins, and the lipid raft-associated receptor IGF1R prevented lipopolysaccharide (LPS) from binding to TLR4 to activate NF-κB, which stimulated macrophages to release higher levels of proinflammatory cytokines (induced by extracellular vesicles of HIV- 1 Nef extracellular vesicles induced) (Dubrovsky et al., 2022). Sulfonamides block TLR4-lipid raft co-localisation to inhibit LPS-induced hyperinflammatory responses (Kim et al., 2020). Wang H et al. demonstrated the potential therapeutic value of biocompatible LPS-stimulated macrophage membrane-encapsulated nanoparticles (LMNPs) in the inhibition of HLH (Wang H. et al., 2023). These studies provide a theoretical basis for future interventional therapies targeting S100A8/9 or its downstream pathways.

Although this study provides new insights into the role of S100A8/9 in sHLHa, several limitations remain. First, this study was a single-center, retrospective design with a small sample size, which may have introduced selection bias. Second, the lack of longitudinal data limits the assessment of changes in S100A8/9 dynamics. Finally, the failure to analyze other proinflammatory cytokines or inflammatory markers comprehensively limits the full validation of the S100A8/9 performance profile. Future studies should aim to validate these findings in larger, multicenter prospective cohorts and explore the value of the molecular mechanisms of S100A8/9 in sHLHa and its potential therapeutic targets. In addition, combined analysis with other inflammatory markers or therapeutic targets may further increase the value of S100A8/9 for clinical applications.

This study demonstrated that S100A8/9 is not only effective in differentiating sHLHa patients from other healthy individuals but also provides important information for etiologic typing and survival prediction. S100A8/9 levels were nonlinearly and positively correlated with poor survival in sHLHa patients, with a significant threshold effect (2.44 μg/mL). Therefore, S100A8/9 is expected to be an important biomarker for the diagnosis, prognostic assessment and personalization of sHLHa.