Mitochondria are important cellular organelles involved in many different functions, from energy generation and fatty acid oxidation to cell death regulation and immune responses. Accumulating evidence indicates that mitochondrial stress acts as a key trigger of innate immune responses. Critically, the dysfunctional mitochondria can be selectively eliminated by mitophagy. The elimination of dysfunctional mitochondria may function as an effective way employed by mitophagy to keep the immune system in check. In addition, mitophagy can be utilized by pathogens for immune evasion. In this review, we summarize how mitochondrial stress triggers innate immune responses and the roles of mitophagy in innate immunity and in infection, as well as the molecular mechanisms of mitophagy. Graphical abstract

The mechanism by which microRNAs (miRNAs) modulate innate immunity and autophagy has not been fully elucidated in Mycobacterium bovis (M. bovis) infections. In this study, we identified that miR-199a inhibited key innate immune responses and autophagy in murine macrophages infected with M. bovis. Using ex vivo and in vitro approaches we show that the expression of miR-199a was significantly increased during M. bovis infection. Furthermore, miR-199a suppressed autophagy and interferon-β (IFN-β) production by directly targeting TANK-binding kinase 1 (TBK1) mRNA in both J774a.1 and BMDM cells. Upregulation of miR-199a or TBK1 silencing (siTBK1) inhibited maturation of autophagosomes and increased M. bovis survival. Our results demonstrate that, by targeting of TBK1, miR-199a modulates innate immune responses and promote the intracellular survival and growth of M. bovis.

Nilotinib, a tyrosine kinase inhibitor, has been studied extensively in various tumor models; however, no information exists about the pharmacological action of nilotinib in bacterial infections. Mycobacterium bovis (M. bovis) and Mycobacterium avium subspecies paratuberculosis (MAP) are the etiological agents of bovine tuberculosis and Johne’s disease, respectively. Although M. bovis and MAP cause distinct tissue tropism, both of them infect, reside, and replicate in mononuclear phagocytic cells of the infected host. Autophagy is an innate immune defense mechanism for the control of intracellular bacteria, regulated by diverse signaling pathways. Here we demonstrated that nilotinib significantly inhibited the intracellular survival and growth of M. bovis and MAP in macrophages by modulating host immune responses. We showed that nilotinib induced autophagic degradation of intracellular mycobacterium occurred via the inhibition of PI3k/Akt/mTOR axis mediated by abelson (c-ABL) tyrosine kinase. In addition, we observed that nilotinib promoted ubiquitin accumulation around M. bovis through activation of E3 ubiquitin ligase parkin. From in-vivo experiments, we found that nilotinib effectively controlled M. bovis growth and survival through enhanced parkin activity in infected mice. Altogether, our data showed that nilotinib regulates protective innate immune responses against intracellular mycobacterium, both in-vitro and in-vivo, and can be exploited as a novel therapeutic remedy for the control of M. bovis and MAP infections.

Mycobacterium bovis , the causative agent of tuberculosis in cattle and humans, infects host macrophages and induces endoplasmic reticulum stress (ERS), mitochondrial damage, and interleukin (IL)-1β production. The relationship between these phenotypes is yet to be elucidated. In this study, we investigated the role of ERS in mitochondrial damage and IL-1β production in macrophages during infection with a virulent M. bovis strain. We found that ERS activates the inflammasome via NOD-like receptor family, pyrin domain-containing 3 (NLRP3)-caspase-8 and that IFN-inducible protein absent in melanoma 2 (AIM2) triggered mitochondrial damage. ERS increased reactive oxygen species (ROS), which promoted translocation of the inflammasome to the mitochondria. NLRP3, but not AIM2, was involved in the ERS-induced cleavage of caspase-8 and Bid, leading to mitochondrial damage, which was required for the production of mature IL-1β. Our data suggest that ERS induces macrophages to produce mature IL-1β during infection with virulent M. bovis through a positive feedback loop between mitochondrial damage and inflammasome activation. To the best of our knowledge, this is the first evidence of the involvement of ERS and mitochondrial damage in inflammasome activation during M. bovis infection.

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1β, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-β expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.

BackgroundMycobacterium bovis (M. bovis) is the principal causative agent of bovine tuberculosis; however, it may also cause serious infection in human being. Type I IFN is a key factor in reducing viral multiplication and modulating host immune response against viral infection. However, the regulatory pathways of Type I IFN signaling during M. bovis infection are not yet fully explored. Here, we investigate the role of Type I IFN signaling in the pathogenesis of M. bovis infection in mice.MethodsC57BL/6 mice were treated with IFNAR1-blocking antibody or Isotype control 24 h before M. bovis infection. After 21 and 84 days of infection, mice were sacrificed and the role of Type I IFN signaling in the pathogenesis of M. bovis was investigated. ELISA and qRT-PCR were performed to detect the expression of Type I IFNs and related genes. Lung lesions induced by M. bovis were assessed by histopathological examination. Viable bacterial count was determined by CFU assay.ResultsWe observed an abundant expression of Type I IFNs in the serum and lung tissues of M. bovis infected mice. In vivo blockade of Type I IFN signaling reduced the recruitment of neutrophils to the lung tissue, mediated the activation of macrophages leading to an increased pro-inflammatory profile and regulated the inflammatory cytokine production. However, no impact was observed on T cell activation and recruitment in the early acute phase of infection. Additionally, blocking of type I IFN signaling reduced bacterial burden in the infected mice as compared to untreated infected mice.ConclusionsAltogether, our results reveal that Type I IFN mediates a balance between M. bovis-mediated inflammatory reaction and host defense mechanism. Thus, modulating Type I IFN signaling could be exploited as a therapeutic strategy against a large repertoire of inflammatory disorders including tuberculosis.

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis complex imposing a high zoonotic threat to human health. The limited efficacy of BCG (Bacillus Calmette–Guérin) and upsurges of drug-resistant tuberculosis require new effective vaccination approaches and anti-TB drugs. Poly (lactic-co-glycolic acid) (PLGA) is a preferential drug delivery system candidate. In this study, we formulated PLGA nanoparticles (NPs) encapsulating the recombinant protein bovine neutrophil β-defensin-5 (B5), and investigated its role in immunomodulation and antimicrobial activity against M. bovis challenge. Using the classical water–oil–water solvent-evaporation method, B5-NPs were prepared, with encapsulation efficiency of 85.5% ± 2.5%. These spherical NPs were 206.6 ± 26.6 nm in diameter, with a negatively charged surface (ζ-potential −27.1 ± 1.5 mV). The encapsulated B5 protein from B5-NPs was released slowly under physiological conditions. B5 or B5-NPs efficiently enhanced the secretion of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-10 in J774A.1 macrophages. B5-NPs-immunized mice showed significant increases in the production of TNF-α and immunoglobulin A (IgA) in serum, and the proportion of CD4+ T cells in spleen compared with B5 alone. In immunoprotection studies, B5-NPs-immunized mice displayed significant reductions in pulmonary inflammatory area, bacterial burden in the lungs and spleen at 4-week after M. bovis challenge. In treatment studies, B5, but not B5-NPs, assisted rifampicin (RIF) with inhibition of bacterial replication in the lungs and spleen. Moreover, B5 alone also significantly reduced the bacterial load in the lungs and spleen. Altogether, our findings highlight the significance of the B5-PLGA NPs in terms of promoting the immune effect of BCG and the B5 in enhancing the therapeutic effect of RIF against M. bovis.

Mitophagy is a selective autophagy mechanism for eliminating damaged mitochondria and plays a crucial role in the immune evasion of some viruses and bacteria. Here, we report that Mycobacterium bovis ( M. bovis ) utilizes host mitophagy to suppress host xenophagy to enhance its intracellular survival. M. bovis is the causative agent of animal tuberculosis and human tuberculosis. In the current study, we show that M. bovis induces mitophagy in macrophages, and the induction of mitophagy is impaired by PINK1 knockdown, indicating the PINK1-PRKN/Parkin pathway is involved in the mitophagy induced by M. bovis . Moreover, the survival of M. bovis in macrophages and the lung bacterial burden of mice are restricted by the inhibition of mitophagy and are enhanced by the induction of mitophagy. Confocal microscopy analysis reveals that induction of mitophagy suppresses host xenophagy by competitive utilization of p-TBK1. Overall, our results suggest that induction of mitophagy enhances M. bovis growth while inhibition of mitophagy improves growth restriction. The findings provide a new insight for understanding the intracellular survival mechanism of M. bovis in the host. Abbreviations: BMDM: mouse bone marrow-derived macrophage; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; BCL2L13: BCL2-like 13 (apoptosis facilitator); CCCP: carbonyl cyanide m-cholorophenyl hydrazone; FUNDC1: FUN14 domain-containing 1; FKBP8: FKBP506 binding protein 8; HCV: hepatitis C virus; HBV: hepatitis B virus; IFN: interferon; L. monocytogenes: Listeria monocytogenes; M. bovis: Mycobacterium bovis ; Mtb: Mycobacterium tuberculosis ; Mdivi-1: mitochondrial division inhibitor 1; PINK1: PTEN-induced putative kinase 1; TBK1: TANK-binding kinase 1; TUFM: Tu translation elongation factor, mitochondrial; TEM: transmission electron microscopy