Comparative transcriptomic analysis of interactions between RAW264.7 macrophages and Mycobacterium tuberculosis, Mycobacterium bovis, and BCG
Tuberculosis is caused by the Mycobacterium tuberculosis complex (MTBC), posing a significant threat to human and animal health worldwide.To investigate the differences in macrophage responses to infections by various members of the MTBC complex, mice's RAW264.7 macrophages were infected with Mycobacterium bovis (M.bovis), Mycobacterium tuberculosis (M.tb) and BCG respectively. RNA sequencing was used to analyze the transcriptional changes in the macrophages after infection by each strain, and the sequencing data was compared between them.The results showed that, compared with the control group, 2120, 2801 and 2022 unique DEGs were identified in M.tb, M.bovis and BCG infection groups respectively, among which there were 756 differential genes in three infection groups.GO and KEGG enrichment analysis showed that differentially expressed genes mainly participated in biological processes such as immune response, apoptosis and cytokine activity during bacterial infection, and were enriched in signal pathways related to host defense against pathogens.The results of RT-qPCR and Western Blot experiments showed that M.bovis and M.tb successfully activated the NF-kB and JAK-STAT pathways in host cells.After inhibiting key factors of the NF-kB and JAK-STAT pathways using specific inhibitors, the results showed that upon P65 inhibition, IL6 expression in the M.tb-infected group was significantly upregulated, while TNF-a expression was unaffected; in the M.bovis-infected group, IL6 expression was significantly downregulated, while TNF-a expression was significantly upregulated. Following STAT3 inhibition, IL6 expression in the M.tb-infected group was significantly downregulated, while TNF-a expression was unaffected; no significant changes in IL6 or TNF-a expression were observed in the M.bovis-infected group. Upon JAK-STAT pathway inhibition, IL6 expression was significantly upregulated in both the M.tb-infected and M.bovis-infected groups, and TNF-a expression was also significantly upregulated in both groups.Overall, our research based on RNA sequencing transcriptomics identified the regulatory patterns of gene expression after different members of MTBC infect the same host. We also conducted preliminary investigations into the pathogenic mechanisms, with the aim of providing theoretical insights for a deeper understanding of MTBC's pathogenic processes