Although Mycobacterium tuberculosis (MTB) has existed for thousands of years, its immune escape mechanism remains obscure. Increasing evidence signifies that microRNAs (miRNAs) play pivotal roles in the progression of tuberculosis (TB). RNA sequencing was used to sequence miRNAs in human acute monocytic leukemia cells (THP-1) infected by the virulent MTB-1458 strain and the avirulent vaccine strain Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Sets of differentially expressed miRNAs (DE-miRNAs) between MTB-1458/BCG-infected groups and uninfected groups were identified, among which 18 were differentially expressed only in the MTB-1458-infected THP-1 group. Then, 13 transcription factors (TFs) and 81 target genes of these 18 DE-miRNAs were matched. Gene Ontology classification as well as Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the candidate targets were predominantly involved in apoptotic-associated and interferon-γ-mediated signaling pathways. A TF-miRNA-mRNA interaction network was constructed to analyze the relationships among these 18 DE-miRNAs and their targets and TFs, as well as display the hub miRNAs, TFs, and target genes. Considering the degrees from network analysis and the reported functions, this study focused on the BHLHE40-miR-378d-BHLHE40 regulation axis and confirmed that BHLHE40 was a target of miR-378d. This cross-talk among DE-miRNAs, mRNAs, and TFs might be an important feature in TB, and the findings merited further study and provided new insights into immune defense and evasion underlying host-pathogen interactions.

Dendritic cells (DCs) are critical for both innate and adaptive immunity. Meanwhile, nitric oxide (NO) is a member of reactive nitrogen species (RNS) generally considered to play a key role in the bactericidal process in innate immunity against Mycobacterium tuberculosis complex infection. The present study therefore investigated the mechanism of NO production in murine DCs induced by Mycobacterium bovis (M.bovis) and its attenuated strain Bacillus Calmette-Guérin (BCG) infection. The expression of genes Slc7A1, Slc7A2, iNOS, and ArgI essential to NO synthesis was up-regulated in M.bovis/BCG infected DCs. IFN-γ addition further increased, while the iNOS inhibitor L-NMMA significantly inhibited their expression. Accordingly, the end products of arginine metabolism, NO and urea, were found to be significantly increased. In addition, BCG induced significantly higher levels of apoptosis in DCs compared to M.bovis shown by higher levels of DNA fragmentation using flow cytometry and release of mitochondrial Cytochrome C, and up-regulation of the genes caspase-3, caspase-8, caspase-9 and dffa critical to apoptosis by qRT-PCR detection and western blot analysis. Furthermore, IFN-γ increased, but L-NMMA decreased apoptosis of M.bovis/BCG infected DCs. In addition, mycobacterial intracellular survival was significantly reduced by IFN-γ treatment in BCG infected DCs, while slightly increased by L-NMMA treatment. Taken altogether, our data show that NO synthesis was differentially increased and associated with apoptosis in M.bovis/BCG infected DCs. These findings may significantly contribute to elucidate the pathogenesis of M.bovis.

Mycobacterium bovis (M. bovis) is a zoonotic pathogen that causes bovine and human tuberculosis. Dendritic cells play a critical role in initiating and regulating immune responses by promoting antigen-specific T-cell activation. Prostaglandin E2 (PGE2)-COX signaling is an important mediator of inflammation and immunity and might be involved in the pathogenesis of M. bovis infection. Therefore, this study aimed to reveal the character of PGE2 in the differentiation of naïve CD4⁺ T cells induced by infected dendritic cells (DCs). Murine bone marrow-derived DCs were pre-infected with M. bovis and its attenuated strain M. bovis bacillus Calmette-Guérin (BCG). Then, the infected DCs were co-cultured with naïve CD4⁺ T cells with or without the cyclooxygenase (COX) inhibitor indomethacin. Quantitative RT-PCR analysis and protein detection showed that PGE2/COX-2 signaling was activated, shown by the upregulation of PGE2 production as well as COX-2 and microsomal PGE2 synthase (mPGES1) transcription in DCs specifically induced by M. bovis and BCG infection. The further co-culture of infected DCs with naïve CD4⁺ T cells enhanced the generation of inflammatory cytokines IL-17 and IL-23, while indomethacin suppressed their production. Following this, the differentiation of regulatory T cells (Treg) and Th17 cell subsets was significantly induced by the infected DCs rather than uninfected DCs. Meanwhile, M. bovis infection stimulated significantly higher levels of IL-17 and IL-23 and the differentiation of Treg and Th17 cell subsets, while BCG infection led to higher levels of TNF-α and IL-12, but lower proportions of Treg and Th17 cells. In mice, M. bovis infection generated more bacterial load and severe abnormalities in spleens and lungs, as well as higher levels of COX-2, mPGE2 expression, Treg and Th17 cell subsets than BCG infection. In conclusion, PGE2/COX-2 signaling was activated in DCs by M. bovis infection and regulated differentiation of Treg and Th17 cell subsets through the crosstalk between DCs and naive T cells under the cytokine atmosphere of IL-17 and IL-23, which might contribute to M. bovis pathogenesis in mice.

Mycobacterium tuberculosis (M. tb) is the causative agent of tuberculosis (TB) that leads to millions of deaths each year. Extensive evidence has explored the involvement of microRNAs (miRNAs) in M. tb infection. Limitedly, the concrete function of microRNA-100-5p (miR-100-5p) in M. tb remains unexplored and largely elusive. In this study, using Bacillus Calmette–Guérin (BCG) as the model strain, we validated that miR-100-5p was significantly decreased in BCG-infected THP-1 cells. miR-100-5p inhibition effectively facilitated the apoptosis of infected THP-1 cells and reduced BCG survival by regulating the phosphatidylinositol 3-kinase/AKT pathway. Further, SMARCA5 was the target of miR-100-5p and reduced after miR-100-5p overexpression. Since BCG infection down-regulated miR-100-5p in THP-1 cells, the SMARCA5 expression was up-regulated, which in turn increased apoptosis through caspase-3 and Bcl-2 and, thereby, reducing BCG intracellular survival. Collectively, the study uncovered a new molecular mechanism of macrophage to suppress mycobacterial infection through miR-100-5p and SMARCA5 pathway.

Mycobacterium tuberculosis is considered a successful pathogen with multiple strategies to undermine host immunity. The YrbE3A is encoded by Rv1964 within the RD15 region present in the genome of Mtb, but missing in M. bovis, M. bovis BCG (Pasteur) strain, and M. smegmatis (Ms). However, little is known about its function. In this study, the YrbE3A gene was cloned into pMV261 and expressed in Ms and BCG, while the strains with the vector served as the controls. The YrbE3A was expressed on the mycobacterial membrane, and the purified protein could stimulate RAW264.7 cells to produce IL-6. Furthermore, the effect of the recombinant strains on cytokine secretion by RAW264.7 was confirmed, which varied with the host strains. Ms_YrbE3A increased significantly higher levels of TNF-α and IL-6 than did Ms_vec, while BCG_YrbE3A enhanced higher TNF-α than BCG_vec. The pathways associated with NF-κB p65 and MAPK p38/JNK, other than Erk1/2, regulated this process. In addition, mice were infected with Ms_YrbE3A and Ms-vec and were kinetically examined. Compared to Ms-vec, Ms_YrbE3A induced more serious inflammatory damage, higher levels of TNF-α and IL-6, higher numbers of lymphocytes, neutrophils, and monocytes in a time-dependent way, but lower lung bacterial load in lung. These findings may contribute to a better understanding of Mtb pathogenesis.

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette–Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.