Molecular signature of neutrophils in antiphospholipid syndrome based on epigenomic and transcriptome analysis

Background: Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and pregnancy loss. Recent studies indicate that neutrophils, particularly neutrophil extracellular traps (NETs), contribute to the development and progression of APS. However, the precise underlying mechanisms remain unclear. Methods: To address this, we analyzed epigenome and transcriptome data to identify key differentially expressed genes (DEGs) of neutrophils in APS using weighted gene co-expression network analysis (WGCNA). Two datasets (GSE102215 and GSE124565) were obtained from the GEO database. The Limma R package was used to identify DEGs, while ChAMP R was applied to analyze differentially methylated genes (DMGs) between APS patients and healthy controls. Enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed using ClusterProfiler, and TISIDB was used to examine associations with immunomodulators, chemokines, and receptors. GeneCards and Connectivity Map databases were further used for disease-related gene analysis and drug prediction. Results: GO enrichment analysis revealed that DEGs were primarily enriched in leukocyte cell-cell adhesion, regulation of leukocyte cell-cell adhesion, and cytokine-mediated signaling pathways. Correspondingly, KEGG enrichment analysis demonstrated that DEGs were mainly enriched in the ribosome, NF-kB signaling pathway, NOD-like receptor signaling pathway, and other related pathways. Through WGCNA analysis, we identified two key intersection genes, CCL5 and ITK, which were positively correlated with CD8+ T cells and eosinophils, while being negatively correlated with neutrophils and follicular helper T cells. Gene set enrichment analysis (GSEA) indicated that CCL5 was enriched in hematopoietic cell lineage, ribosome, and ribosome biogenesis in eukaryotes, and ITK was enriched in ribosome, ribosome biogenesis in eukaryotes, and T-cell receptor signaling. Similarly, gene set variation analysis (GSVA) showed that CCL5 and ITK were associated with IL-2-STAT5 signaling and the P53 pathway as well as MTORC1 signaling. Furthermore, gene regulatory network analysis suggested that CCL5 and ITK are modulated by common mechanisms involving multiple transcription factors. By examining APS-related genes in the GeneCards database, we observed a significant negative correlation between CCL5 and phosphatase and tensin homolog (PTEN) (R = −0.624), and a strong positive correlation between ITK and CD40LG (R = 0.933). Finally, drug perturbation expression profiles revealed that RS-56812, acebutolol, emetine, and proscillaridin exhibited the most significant negative correlation with disease-associated expression profiles. Conclusion: These data nominate CCL5 and ITK as APS-associated genes in neutrophils and indicate associations with multiple signaling pathways. Moreover, drugs targeting these genes may represent potential therapeutic strategies for APS.