Background Hemophagocytic lymphohistiocytosis (HLH) is an excessive systemic inflammatory reaction and organ dysfunction caused by many reasons. High-dose dexamethasone is often used to inhibit inflammation, and serum soluble VSIG4 (sVSIG4) can be used as a marker for evaluating macrophage activation to diagnose HLH.
Objective To explore the changes of phagocytosis and gene transcription expression after the high expression of VSIG4 in human primary monocytes stimulated by dexamethasone, and screen the key expression genes and pathways.
Methods Human peripheral blood mononuclear cells were isolated in vitro. CD14+ monocytes were positively selected by magnetic beads sorting and stimulated by dexamethasone. The expression of VSIG4 was detected at mRNA and protein levels respectively by qPCR, flow cytometry and Western Blot. Western Blot was used to detect the expression of sVSIG4 in cell culture supernatant, and the phagocytosis of monocytes was detected while dexamethasone stimulated the expression of VSIG4. RNA-seq was used to sequence the differential genes of human primary monocytes before and after dexamethasone stimulation, KEGG pathway enrichment and protein-to-protein interaction analysis were performed, and hub genes were found through node analysis.
Results Dexamethasone could stimulate monocytes to promote the up-regulation of VSIG4 expression at mRNA and protein levels in a dose-dependent and time-dependent manner in vitro (P＜0.01). Overexpression of VSIG4 in monocytes stimulated by dexamethasone led to extracellular domain shearing to generate sVSIG4. VSIG4+ monocytes stimulated by dexamethasone showed enhanced phagocytosis (P＜0.01). There were 491 differentially expressed genes in the difference analysis of RNA-seq, of which 159 were up-regulated and 332 were down-regulated. KEGG pathway enrichment showed that the differential genes were mainly concentrated in EB virus infection, COVID-19 infection, complement and coagulation cascades, NOD-like receptor signaling pathway, pyrimidine metabolism, C-type lectin receptor signaling pathway, phagosome, staphylococcus aureus infection, type I diabetes mellitus, RIG-I-like receptor signaling pathway, toll-like receptor signaling pathway, etc (P＜0.05). Twenty hub genes were identified from PPI network.
Conclusion Stimulation of human primary monocytes by dexamethasone in vitro can promote the up-regulation of VSIG4 expression and generate sVSIG4 at the same time. VSIG4+ monocytes may show enhanced phagocytosis. RNA-seq analysis suggests that some inflammation-related genes and signal pathways may be related to the expression regulation of VSIG4 and the pathogenesis of HLH.
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