Epigenetic Crosstalk Between BCG-Infected Macrophages and Naïve Monocytes Potentiates Antimycobacterial Activity

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death from infectious disease. Infected cells secrete extracellular vesicles (EVs), nanosized membrane-bound particles containing bioactive molecules known to mediate intercellular communication and influence immune regulation during infection. We hypothesized that EVs secreted from Mycobacterium bovis Bacillus Calmette Guérin (BCG)-infected macrophages could epigenetically reprogram naïve monocytes and enhance their mycobactericidal activity against Mtb. A transwell co-culture system was used to enable communication via EVs and soluble factors, between BCG-infected macrophages and naïve monocytes (recipient macrophages). To assess the impact of these factors, we examined epigenetic reprogramming and the ability to control Mtb growth. BCG-infected macrophages released EVs with a distinct proteomic profile mapping to multiple tuberculosis-related pathways. Recipient macrophages exhibited altered DNA methylation patterns compared to those co-cultured with untreated, Staphylococcus aureus -infected or hydrogen peroxide-exposed macrophages. The proteomic cargo of EVs and differentially methylated genes in recipient cells showed significant interactions with TNF as a central hub enriched in both the phagosome and tuberculosis pathway. The epigenetic reprogramming was accompanied by a trend towards improved control of Mtb in vitro . BCG infection induces release of EVs that, together with soluble factors, induce targeted epigenetic remodeling in recipient macrophages. In this study, we demonstrate that extracellular vesicles (EVs) released during Mycobacterium bovis -infection carry a directed proteomic cargo mapping to several tuberculosis-related pathways. Furthermore, we show that these proteins interact with differentially methylated genes in EV-recipient macrophages. Our findings indicate that infected macrophages transmit epigenetic information via EVs. This work provides new insights into intercellular and epigenetic mechanisms linked to innate immune memory.