Epigenetic profiling reveals M1 macrophages as the primary drivers of trained immunity in HSV-1 latently infected mice

Macrophages play multifaceted and critical roles in controlling diverse pathogenic infections. Building on our recently published observations of macrophages exhibiting memory responses to HSV-1 infection, our current report investigates the macrophage subtype responsible for generating trained immunity against virus-induced immunopathogenesis. Using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing), an epigenetic profiling technique, we identified chromatin accessibility changes in both M1 and M2 macrophages associated with the acquisition of IRGM1, a marker of the trained phenotype. To conduct this study, we first generated M0, M1, and M2 macrophage subtypes from bone marrow (BM) derived macrophages isolated from HSV-1 latently infected wild type (WT) mice. ATAC-seq revealed that M1-generated macrophages displayed higher IRGM1-associated chromatin accessibility peaks compared to M2 and M0 subtypes, and this response was enhanced after stimulation with UV-inactivated virus. To further dissect this response, we analyzed memory responses in bone marrow-derived macrophages, spleen macrophages, corneal macrophages, and trigeminal ganglia (TG) of latently infected M1 and M2 macrophages. Flow cytometry and ATAC-seq data showed a significantly higher proportion of IRGM1 ⁺ macrophages in infected M2 -/- mice, which are enriched in M1 macrophages. These findings indicate that M1 macrophages, but not M2 macrophages, undergo trained immunity in response to HSV-1 infection. This is also confirmed by the Luminex assay, in which M1 macrophages, after stimulation, then known as primed M1 macrophages, enhance the secretion of pro-inflammatory cytokine/chemokine response to secondary HSV-1 exposure. These results uncover a previously underappreciated role for macrophage-mediated trained immunity in antiviral defense against HSV-1 infection and offer new insights into potential therapeutic targets for modulating host immune responses during HSV-1 infection.