BCG-trained macrophages promote pan-anti-tumor activity through epigenetic rewiring of NOX2-ROS axis.

backgroundBacillus Calmette-Guerin (BCG)-induced trained immunity in the macrophages is characterized by exaggerated inflammatory cytokine production with favourable effects on disease controls. However, how BCG-trained macrophages exert anti-tumor effects and underlying mechanisms remain to be clarified.

methodsPan-anti-tumor activity induced by BCG training was evaluated in mouse models with grafted tumors. The proportion and function of tumor infiltrating macrophages (TAMs) and CD8+T cells, as well as the level of reactive oxygen species (ROS), were detected by flow cytometry. The secretion of IL-1β and TNF-α were detected by Enzyme-Linked Immunosorbent Assay (ELISA). The expression and activation of NOX2 complex and NF-κB were detected by qRT-PCR, immunoblotting and immunofluorescence. The methylation modification level and chromatin opening level were detected by CUT&RUN and ATAC sequencing. RNA sequencing was used to detect the transcriptome of macrophages and bladder cancer tissues.

resultsBCG-trained mice exhibited pan-anti-tumor activity where TAMs originated from newly bone marrow hematopoiesis were the predominant effectors. The anti-tumor effects of BCG-trained TAMs were mediated by ROS production in the tumor microenvironment (TME), resulting from the overactivation of NADPH oxidase 2 (NOX2) complex. Epigenetic rewiring of NOX2 complex occurred both in the myeloid progenitor of BCG-trained mice as well as in BCG-trained macrophages marked by increased deposition of H3K4me3 at the promoter regions of NOX2 complex genes, which in turn facilitated the accessibility of transcription factor such as NF-kB and enhanced transcriptional activation. Clinically, NOX2 gene signatures correlated with a favourable prognosis in bladder cancer patients receiving BCG intravesical instillation.

conclusionsOur findings reveal that BCG training reprograms TAMs to overproduce ROS through epigenetic rewiring of NOX2-ROS axis. Systemic BCG training becomes an effective and promising strategy to remodel the TME with enhanced pan-anti-tumor activity of infiltrating macrophages.