Single-cell Analyses Highlight the Proinflammatory Contribution of Low-density Neutrophils in the Acute Phase of Severe Fever With Thrombocytopenia Syndrome.

Background: Severe fever with thrombocytopenia syndrome (SFTS), caused by Dabie bandavirus (DBV) infection, is characterized by early cytokine storm as a primary pathological feature, although the precise mechanisms remain unclear. Low-density neutrophils (LDNs) are elevated in the peripheral blood of patients with autoimmune or infectious diseases and are closely associated with inflammatory damage and disease severity. However, the pathogenic contribution of LDNs to the progression of SFTS is largely unexplored. This study employed single-cell RNA sequencing (scRNA-seq) to profile the transcriptomic characteristics of LDNs during the acute phase of SFTS, aiming to reveal their compositional and functional heterogeneity following DBV infection, explore their role in the cytokine storm, and further understand their impact on disease progression.

Methods: Cells were isolated from 13 acute-phase SFTS patients with varying disease severity and 3 healthy controls using density gradient centrifugation, followed by preparation of single-cell suspensions for 3'-end scRNA-seq. Sequencing data were processed using the Seurat pipeline, including dimensionality reduction, clustering, cell-type annotation, and visualization with Uniform Manifold Approximation and Projection (UMAP). Low-density granulocytes (LDGs) and their subclusters were identified using canonical gene markers. Functional enrichment of differentially expressed genes (DEGs) was analyzed by high-dimensional Weighted Gene Co-expression Network Analysis (hdWGCNA), Gene Ontology (GO), AddModuleScore, single-sample Gene Set Enrichment Analysis (ssGSEA), and immune-related Gene Set Enrichment Analysis (irGSEA), while cellular interactions were explored using CellCall.

Results: 1. Compositional heterogeneity: The proportion of LDNs in peripheral blood increased in SFTS patients with greater disease severity during the acute phase. 2. Functional heterogeneity: (1) LDN subclusters showed functional diversity but consistently displayed pro-inflammatory or anti-infective properties. (2) With intensification of the systemic inflammatory response, the expression of multiple cytokine genes (e.g., IL6, IL8, TNFA) and gene sets of the inflammatory pathway (e.g., TNFA-SIGNALING-VIA-NFKB, INFLAMMATORY-RESPONSE) were significantly upregulated in LDNs. Concurrently, the expression of gene sets of type I interferon response pathway (e.g., INTERFERON-ALPHA-RESPONSE, INTERFERON-GAMMA-RESPONSE) and genes of interferon-induced antiviral proteins (e.g., EIF2AK2, OAS1, MX1) were also elevated. (3) In severe cases, glucocorticoid therapy downregulated expression of these inflammatory genes, demonstrating anti-inflammatory effects but potentially increasing infection risk.

Conclusions: This study revealed an increased proportion and heightened pro-inflammatory activity of LDNs during the acute phase of SFTS, closely correlating with disease severity. These findings suggest that LDNs may serve as potential early-warning biomarkers for predicting severe progression in patients with SFTS.