Background and Aims Microbial dysbiosis is associated with alcohol‐related hepatitis (AH), with the mechanisms yet to be elucidated. The present study aimed to determine the effects of alcohol and zinc deficiency on Paneth cell (PC) antimicrobial peptides, α‐defensins, and to define the link between PC dysfunction and AH. Approach and Results Translocation of pathogen‐associated molecular patterns (PAMPs) was determined in patients with severe AH and in a mouse model of alcoholic steatohepatitis. Microbial composition and PC function were examined in mice. The link between α‐defensin dysfunction and AH was investigated in α‐defensin‐deficient mice. Synthetic human α‐defensin 5 (HD5) was orally given to alcohol‐fed mice to test the therapeutic potential. The role of zinc deficiency in α‐defensin was evaluated in acute and chronic mouse models of zinc deprivation. Hepatic inflammation was associated with PAMP translocation and lipocalin‐2 (LCN2) and chemokine (C‐X‐C motif) ligand 1 (CXCL1) elevation in patients with AH. Antibiotic treatment, lipopolysaccharide injection to mice, and in vitro experiments showed that PAMPs, but not alcohol, directly induced LCN2 and CXCL1. Chronic alcohol feeding caused systemic dysbiosis and PC α‐defensin reduction in mice. Knockout of functional α‐defensins synergistically affected alcohol‐perturbed bacterial composition and the gut barrier and exaggerated PAMP translocation and liver damage. Administration of HD5 effectively altered cecal microbial composition, especially increased Akkermansia muciniphila, and reversed the alcohol‐induced deleterious effects. Zinc‐regulated PC homeostasis and α‐defensins function at multiple levels, and dietary zinc deficiency exaggerated the deleterious effect of alcohol on PC bactericidal activity. Conclusions Taken together, the study suggests that alcohol‐induced PC α‐defensin dysfunction is mediated by zinc deficiency and involved in the pathogenesis of AH. HD5 administration may represent a promising therapeutic approach for treating AH.

ObjectiveMitochondrial dysfunction plays a dominant role in the pathogenesis of alcoholic liver disease (ALD); however, the underlying mechanisms remain to be fully understood. We previously found that hepatic activating transcription factor 4 (ATF4) activation was associated with mitochondrial dysfunction in ALD. This study aimed to investigate the function and mechanism of ATF4 in alcohol-induced hepatic mitochondrial dysfunction.DesignATF4 activation was detected in the livers of patients with severe alcoholic hepatitis (AH). The role of ATF4 and mitochondrial transcription factor A (TFAM) in alcohol-induced liver damage was determined in hepatocyte-specific ATF4 knockout mice and liver-specific TFAM overexpression mice, respectively.ResultsHepatic PERK-eIF2α-ATF4 ER stress signalling was upregulated in patients with AH. Hepatocyte-specific ablation of ATF4 in mice ameliorated alcohol-induced steatohepatitis. ATF4 ablation also attenuated alcohol-impaired mitochondrial biogenesis and respiratory function along with the restoration of TFAM. Cell studies confirmed that TFAM expression was negatively regulated by ATF4. TFAM silencing in hepatoma cells abrogated the protective effects of ATF4 knockdown on ethanol-mediated mitochondrial dysfunction and cell death. Moreover, hepatocyte-specific TFAM overexpression in mice attenuated alcohol-induced mitochondrial dysfunction and liver damage. Mechanistic studies revealed that ATF4 repressed the transcription activity of nuclear respiratory factor 1 (NRF1), a key regulator of TFAM, through binding to its promoter region. Clinical relevance among ATF4 activation, NRF1–TFAM pathway disruption and mitochondrial dysfunction was validated in the livers of patients with AH.ConclusionThis study demonstrates that hepatic ATF4 plays a pathological role in alcohol-induced mitochondrial dysfunction and liver injury by disrupting the NRF1–TFAM pathway.

Mycobacterium avium subspecies paratuberculosis (MAP) persistently survive and replicate in mononuclear phagocytic cells by adopting various strategies to subvert host immune response. Interleukin-10 (IL-10) upregulation via inhibition of macrophage bactericidal activity is a critical step for MAP survival and pathogenesis within the host cell. Mitogen-activated protein kinase p38 signaling cascade plays a crucial role in the elevation of IL-10 and progression of MAP pathogenesis. The contribution of microRNAs (miRNAs) and their influence on the activation of macrophages during MAP pathogenesis are still unclear. In the current study, we found that miRNA-27a-3p (miR-27a) expression is downregulated during MAP infection both in vivo and in vitro. Moreover, miR-27a is also downregulated in toll-like receptor 2 (TLR2)-stimulated murine macrophages (RAW264.7 and bone marrow-derived macrophage). ELISA and real-time qRT-PCR results confirm that overexpression of miR-27a inhibited MAP-induced IL-10 production in macrophages and upregulated pro-inflammatory cytokines, while miR-27a inhibitor counteracted these effects. Luciferase reporter assay results revealed that IL-10 and TGF-β-activated protein kinase 1 binding protein 2 (TAB 2) are potential targets of miR-27a. In addition, we demonstrated that miR-27a negatively regulates TAB 2 expression and diminishes TAB 2-dependent p38/JNK phosphorylation, ultimately downregulating IL-10 expression in MAP-infected macrophages. Furthermore, overexpression of miR-27a significantly inhibited the intracellular survival of MAP in infected macrophages. Our data show that miR-27a augments antimicrobial activities of macrophages and inhibits the expression of IL-10, demonstrating that miR-27a regulates protective innate immune responses during MAP infection and can be exploited as a novel therapeutic target in the control of intracellular pathogens, including paratuberculosis.

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.

Mycobacteria can trigger the AIM2 inflammasome, autophagy activation and type-I interferon release, which are both activated by cytosolic DNA. We have recently demonstrated that activation of the AIM2 inflammasome during M. bovis infection is the result of mycobacterial translocation into the cytosol. To elucidate the effects of inflammasome activation on autophagy, we investigated the role of the AIM2 inflammasome from macrophages infected with a virulent strain of M. bovis. The results showed that the M. bovis-induced AIM2 inflammasome activation decreases autophagy in immortalized and primary murine macrophages. This relied on the inflammasome sensor AIM2 which conjugates with cytosolic DNA to inhibit the STING-dependent pathway involved in selective autophagy and interferon-β release in Mycobacterium-infected macrophages. These results suggest that the AIM2 cytosolic DNA sensor may conjugate competitively with cytosolic M. bovis DNA to restrict M. bovis induced STING-TBK1-dependent autophagy activation and IFN-β secretion.