Integrative profiling of transcriptome and H3K27ac modification reveals changes associated with BCG-induced trained immunity in bovine immune cells

We previously demonstrated that Bacillus Calmette Guérin (BCG) vaccination induces trained immunity in bovine monocytes and gamma delta (γδ) T cells, enhancing IL-1β and IL-6 production. To define the underlying molecular mechanisms, we applied a multi-omic approach to circulating monocytes and alveolar macrophages (amac). Twenty Holstein-Angus heifers were randomly separated into two groups and immunized subcutaneously with 6 million CFUs of BCG Danish strain (n = 10), or saline (n = 10), and boosted after 2 weeks. Monocytes (4 weeks post-boost) and amac (6 weeks post-boost) were analyzed for cytokine production, transcriptional responses (QuantSeq 3' mRNA sequencing), and histone modifications (CUT&Tag H3K27ac). BCG vaccination increased IL-1β (p = 0.020 and IL-6 (p < 0.0001) production in monocytes, and increased IL-1β (p = 0.041) in amac following heterologous stimulation. Transcriptomic analysis revealed an expanded breadth of stimulation-responsive genes in BCG-trained monocytes, (1439 vs 1214 differentially expressed genes (DEGs, FDR < 0.05) with LPS; 6513 vs 5605 DEGs with Poly(I:C)/Imiquimod). Gene ontology analysis demonstrated enrichment of innate immune response pathways among BCG-enhanced genes. H3K27ac profiling identified 227 differential peaks (DP, p < 0.01, |FC|> 1.25) in monocytes, most of which are bovine-specific compared to human, and 383 DP in amac.These datasets provide evidence that BCG induces trained immunity in bovine monocytes and macrophages through enhanced cytokine capacity, transcriptional reprogramming, and epigenetic modifications.