Serum Cytokine Profiling Differentiates Underlying Diseases in Cytokine Storm Syndrome

Abstract

Objective: Cytokine storm syndrome (CSS), commonly associated with hemophagocytic lymphohistiocytosis (HLH), is a fatal hyperinflammatory syndrome. Differentiating the underlying diseases responsible for CSS is essential for timely therapeutic decisions. This study explored the clinical usefulness of serum cytokine profiling in distinguishing underlying diseases in patients with CSS.

Methods: Serum samples were collected from 143 adult and pediatric patients with CSS and 22 healthy controls. The cohort included patients with various diagnoses of primary and secondary HLH and Kawasaki disease (KD)-like hyperinflammatory syndromes. Serum levels of 48 cytokines were analyzed in 97 patients using a bead-based multiplex immunoassay (Luminex assay). Serum levels of interferon alpha (IFN-α), interleukin-18 (IL-18), IL-6, CXCL9, and soluble tumor necrosis factor receptor II (sTNF-RII) were measured in 165 participants using enzyme-linked immunosorbent assay (ELISA).

Results: Luminex assay categorized patients with CSS into five clusters based on serum cytokine patterns. ELISA revealed distinct cytokine patterns, wherein patients with histiocytic necrotizing lymphadenitis-associated HLH and systemic lupus erythematosus-associated macrophage activation syndrome (MAS) showed elevated IFN-α; systemic juvenile idiopathic arthritis-associated and adult-onset Still's disease-associated MAS, X-linked inhibitor of apoptosis protein deficiency with HLH, and NLRC4-associated autoinflammatory disorder exhibited higher IL-18 levels. Additionally, KD shock syndrome had higher IL-6 levels than the other groups. CXCL9 was significantly elevated in patients with virus-associated HLH, familial HLH, malignant lymphoma-associated HLH, and KD-MAS. Multisystem inflammatory syndrome in children and toxic shock syndrome also showed moderate elevations of CXCL9 and IL-6 levels.

Conclusion: Serum cytokine profiling effectively differentiates CSS subtypes, facilitating better diagnosis and personalized treatment strategies based on specific disease backgrounds.

Figure 1

Hierarchical cluster analysis with serum levels of 48 cytokines and chemokines in patients with CSS. Serum biomarker profiles of 97 patients with CSS. Data comprising 48 analytes are presented as a heat map after unsupervised hierarchical clustering of biomarker expression profiles according to correlation distance. Colors indicate column Z score. AOSD, Adult onset Still's disease; FHL, familial hemophagocytic lymphohistiocytosis; GS2, Griscelli syndrome type 2; HC, healthy controls; HLH, hemophagocytic lymphohistiocytosis; HNL, histiocytic necrotizing lymphadenitis; KDSS, Kawasaki disease shock syndrome; LAHS, lymphoma associated HLH; MAS, macrophage activation syndrome; MIS‐C, multisystem inflammatory syndrome in children; MKD, Mevalonate kinase deficiency; NLRC4‐AID, NLR‐family CARD domain‐containing protein 4 associated autoinflammatory disorder; SLE, systemic lupus erythematosus; TSS, toxic shock syndrome; VAHS, virus‐associated hemophagocytic syndrome; XIAP, X‐linked inhibitor of apoptosis.

Figure 2

Differences and correlations of serum cytokine levels quantified by ELISA and Luminex assay. (A) Comparison of serum IFN‐α2 levels determined by the Luminex assay and serum IFN‐α levels determined by ELISA. (B, C) Correlations of serum cytokine and chemokine levels. The X‐axis shows cytokine levels in serum quantified by the Luminex assay, and the Y‐axis shows cytokine levels in serum quantified by ELISA. The green dotted line indicates that the value of the ELISA/Luminex assay ratio is 10, and the blue line indicates that the ratio is 1. Statistically significant differences among each patient group are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. CSS, cytokine storm syndrome; ELISA, enzyme‐linked immunosorbent assay; HC, healthy control; HNL‐HLH, histiocytic necrotizing lymphadenitis complicating hemophagocytic lymphohistiocytosis; IFN‐α, interferon alpha; IL, interleukin; MIG, monokine induced by IFN‐γ; SLE‐MAS, systemic lupus erythematosus–macrophage activation syndrome; sTNF‐RII, soluble tumor necrosis factor receptor II; TNF, tumor necrosis factor.

Figure 3

Comparison of serum cytokine levels in patients with CSS. (A) Serum cytokine levels of patients with CSS. Bars represent median levels and interquartile ranges. Statistically significant differences among each patient group are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. All units of cytokines are pg/mL. (B) Serum cytokine profiles in each patient group. The blue hexagons show median levels of serum cytokines in patients with each background disease, and the white hexagons show those in HCs. All units of cytokines are pg/mL. GS2, Griscelli syndrome type 2; HNL, histiocytic necrotizing lymphadenitis; KDSS, Kawasaki disease shock syndrome; LAHS, lymphoma associated HLH; MKD, Mevalonate kinase deficiency; NLRC4‐AID, NLR‐family CARD domain‐containing protein 4 associated autoinflammatory disorder; TSS, toxic shock syndrome; VAHS, virus‐associated hemophagocytic syndrome; XIAP, X‐linked inhibitor of apoptosis.

Figure 4

Stepwise ROC curve analysis and classification algorithm for patients with CSS. For the 165 cases, the high group was defined as “IFN‐α–dominant CSS” with a cutoff of 18.0 pg/mL for IFN‐α, and for the remaining groups with an IFN‐α level ≤18.0 pg/mL, the high group was defined as “IL‐18–dominant CSS” with a cutoff of 13,420 pg/mL for IL‐18. For the remaining groups with an IL‐18 level ≤13,420 pg/mL, the group with an IL‐6 level >185.5 pg/mL was defined as “IL‐6–dominant CSS,” and the remaining groups were defined as “IFN‐γ–dominant CSS” with a CXCL9 level of 10,337 pg/mL as the cutoff. The group with levels <10,337 pg/mL was classified as “IL‐6– and IFN‐γ–intermediate CSS.” HNL, histiocytic necrotizing lymphadenitis; KDSS, Kawasaki disease shock syndrome; LAHS, lymphoma associated HLH; NLRC4‐AID, NLR‐family CARD domain‐containing protein 4 associated autoinflammatory disorder; TSS, toxic shock syndrome; VAHS, virus‐associated hemophagocytic syndrome; XIAP, X‐linked inhibitor of apoptosis.

Figure 5

Classification algorithm for CSS using multiple serum cytokine levels. A stepwise classification algorithm for the background diseases of CSS using five cytokines. CSS, cytokine storm syndrome; IFN, interferon; IL, interleukin.