Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly ...expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS.
Cellular homeostais, that is normally maintained through autophagy, is disrupted in alcoholic liver disease (ALD). Because autophagy and exosome biogenesis share common elements, we hypothesized that ...increased exosome production in ALD may be linked to disruption of autophagic function. We found impaired autophagy both in ALD and alcoholic hepatitis (AH) mouse models and human livers with ALD as indicated by increased hepatic p62 and LC3‐II levels. Alcohol reduced autophagy flux in vivo in chloroquine‐treated mice as well as in vitro in hepatocytes and macrophages treated with bafilomycin A. Our results revealed that alcohol targets multiple steps in the autophagy pathway. Alcohol‐related decrease in mechanistic target of rapamycin (mTOR) and Ras homolog enriched in brain (Rheb), that initiate autophagy, correlated with increased Beclin1 and autophagy‐related protein 7 (Atg7), proteins involved in phagophore‐autophagosome formation, in ALD. We found that alcohol disrupted autophagy function at the lysosomal level through decreased lysosomal‐associated membrane protein 1 (LAMP1) and lysosomal‐associated membrane protein 2 (LAMP2) in livers with ALD. We identified that micro‐RNA 155 (miR‐155), that is increased by alcohol, targets mTOR, Rheb, LAMP1, and LAMP2 in the authophagy pathway. Consistent with this, miR‐155‐deficient mice were protected from alcohol‐induced disruption of autophagy and showed attenuated exosome production. Mechanistically, down‐regulation of LAMP1 or LAMP2 increased exosome release in hepatocytes and macrophages in the presence and absence of alcohol. These results suggested that the alcohol‐induced increase in exosome production was linked to disruption of autophagy and impaired autophagosome and lysosome function. Conclusion: Alcohol affects multiple genes in the autophagy pathway and impairs autophagic flux at the lysosome level in ALD. Inhibition of LAMP1 and LAMP2 promotes exosome release in ALD. We identified miR‐155 as a mediator of alcohol‐related regulation of autophagy and exosome production in hepatocytes and macrophages.
Abstract Background & Aims Alcoholic liver disease (ALD) ranges from fatty liver to inflammation and cirrhosis. miRNA-155 is an important regulator of inflammation. In this study, we describe the in ...vivo role of miR-155 in ALD. Methods Wild type, WT (C57/BL6J) or miR-155 KO and TLR4 KO mice received Lieber-DeCarli diet for 5 weeks. Some mice received corn oil or CCl4 for 2 or 9 weeks. Results We found that miR-155 KO mice are protected from alcohol-induced steatosis and inflammation. The reduction in alcohol-induced fat accumulation in miR-155 KO mice was associated with increased PPRE and PPARα (miR-155 target) binding and decreased MCP1 production. Treatment with a miR-155 inhibitor increased PPAR γ expression in naive and alcohol treated RAW macrophages. Alcohol increased lipid metabolism genes (FABP4, LXRα, ACC1 and LDLR) in WT mice and this was prevented in KO mice. Alcohol diet induced increase in the number of CD163+ CD206+ infiltrating macrophages and neutrophils in WT was prevented in miR-155 KO mice. Kupffer cells isolated from miR-155 KO mice exhibited predominance of M2 phenotype when exposed to M1 polarized signals and this was due to increased C/EBPβ. Profibrotic genes were attenuated in miR-155 KO mice after alcohol diet or CCl4 treatment. Compared to WT attenuation in CCl4 induced hydroxyproline and α SMA was observed in KO mice. Finally, we show TLR4 signaling regulates miR-155 as TLR4 KO mice showed no induction of miR-155 after alcohol diet. Conclusions Collectively our results demonstrated the role of miR-155 in alcohol-induced steatohepatitis and fibrosis in vivo.
MicroRNAs are fine tuners of diverse biological responses and are expressed in various cell types of the liver. Here we hypothesized that circulating microRNAs (miRNAs) may serve as biomarkers of ...liver damage and inflammation. We studied miRNA‐122, which is abundant in hepatocytes, and miR‐155, ‐146a, and ‐125b, which regulate inflammation in immune cells in mouse models of alcoholic liver disease (ALD), drug (acetaminophen, APAP)‐induced liver injury (DILI), and Toll‐like receptor (TLR) 9+4 ligand‐induced inflammatory cell‐mediated liver damage. We found that serum/plasma miR‐122 correlated with alanine aminotransferase (ALT) increases in the liver damage caused by alcohol, APAP, and TLR9 (CpG)+4 (LPS) ligands. MiR‐155, a regulator of inflammation, was increased in serum/plasma in alcoholic and inflammatory liver injury. Alcohol failed to increase serum miR‐122 in TLR4‐deficient and p47phox‐deficient mice that were protected from ALD. We found the most robust increase in plasma miR‐122 in DILI and it correlated with the highest ALT levels. Consistent with the massive inflammatory cell infiltration in the liver, plasma miR‐155 and miR‐146a were significantly elevated after CpG+LPS administration. We show for the first time that, depending on the type of liver injury, circulating miRNAs are associated either with the exosome‐rich or protein‐rich compartments. In ALD and in inflammatory liver injury, serum/plasma miR‐122 and miR‐155 were predominantly associated with the exosome‐rich fraction, whereas in DILI/APAP injury these miRNAs were present in the protein‐rich fraction. Conclusion: Our results suggest that circulating miRNAs may serve as biomarkers to differentiate between hepatocyte injury and inflammation and the exosome versus protein association of miRNAs may provide further specificity to mechanisms of liver pathology. (HEPATOLOGY 2012;56:1946–1957)
Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their ...role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.
Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of proinflammatory cytokines, including IL-1β. IL-1β, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) ...are all important regulators of the IL-1 signaling complex, which plays a role in inflammation. Furthermore, IL-1β maturation is dependent on caspase-1 (Casp-1). Using IL-1Ra-treated mice as well as 3 mouse models deficient in regulators of IL-1β activation (Casp-1 and ASC) or signaling (IL-1R1), we found that IL-1β signaling is required for the development of alcohol-induced liver steatosis, inflammation, and injury. Increased IL-1β was due to upregulation of Casp-1 activity and inflammasome activation. The pathogenic role of IL-1 signaling in ALD was attributable to the activation of the inflammasome in BM-derived Kupffer cells. Importantly, in vivo intervention with a recombinant IL-1Ra blocked IL-1 signaling and markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. Furthermore, physiological doses of IL-1β induced steatosis, increased the inflammatory and prosteatotic chemokine MCP-1 in hepatocytes, and augmented TLR4-dependent upregulation of inflammatory signaling in macrophages. In conclusion, we demonstrated that Casp-1-dependent upregulation of IL-1β and signaling mediated by IL-1R1 are crucial in ALD pathogenesis. Our findings suggest a potential role of IL-1R1 inhibition in the treatment of ALD.
Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is ...indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax B-cell lymphoma 2 (Bcl2)-associated X protein and contributed to hepatocyte apoptosis. Deficiency of STING prevented IRF3 phosphorylation by ethanol or ER stress, and absence of IRF3 prevented hepatocyte apoptosis. The pathogenic role of IRF3 in ALD was independent of inflammation or Type-I interferons. Thus, STING and IRF3 are key determinants of ALD, linking ER stress signaling with the mitochondrial pathway of hepatocyte apoptosis.
Background
Chronic alcohol impairs gut barrier function and induces inflammatory cytokines. The effects of acute alcohol binge on the gut are partially understood. Micro‐RNA‐155 (miR‐155), a ...modulator of cytokine and T‐cell immune response in the gut, stabilizes tumor necrosis factor‐α (TNFα) mRNA. Here, we investigated the role of the inflammation modulator miR‐155 as well as the effects of acute binge and chronic alcohol feeding in the small bowel (SB) in mice.
Methods
For the acute alcohol binge, wild‐type (WT) mice received 5 g/kg 50% alcohol/d or equal amount of water oral gavage for 3 days. WT and miR‐155‐deficient (miR‐155‐knockout KO) mice received ethanol containing Lieber‐DeCarli or isocaloric control diet for 5 weeks. MiR‐155, antimicrobial peptide, regenerating islet‐derived 3‐beta (Reg3b), inflammation markers, Src homology 2‐containing inositol phosphatase‐1 (SHIP1), TNFα, and nuclear factor‐κB (NF‐κB) were measured in proximal intestinal tissue. Endotoxin was measured in the serum.
Results
Acute alcohol binge enhanced, whereas chronic alcohol feeding decreased, Reg3b mRNA and protein levels in the SB. Both acute binge and chronic alcohol feeding increased serum endotoxin levels, intestinal NF‐κB activation and TNFα mRNA levels. However, TNFα protein and miR‐155 were increased only after chronic alcohol feeding in the SB. Furthermore, miR‐155‐KO mice were protected from chronic alcohol‐induced increase in serum endotoxin, intestinal TNFα, and NF‐κB activation. Also, alcohol‐fed miR‐155‐KO mice had no decrease of Reg3b and SHIP1 levels.
Conclusions
These results demonstrate that both acute binge and chronic ethanol administration result in increased serum‐endotoxin levels. Our study identifies a novel role for miR‐155 in chronic alcohol‐induced intestinal inflammation and barrier dysfunction.
Display omitted
•Alcohol treatment induces spontaneous neutrophil extracellular trap (NET) formation.•Alcohol impairs further NET release by the same neutrophils upon subsequent activation.•Alcohol ...reduces efferocytosis by macrophages, leading to less clearance of NETotic neutrophils.•Depletion of neutrophils in vivo alleviates hepatic injury after binge-drinking.
Neutrophil extracellular traps (NETs) are an important strategy utilized by neutrophils to immobilize and kill invading microorganisms. Herein, we studied NET formation and the process of neutrophil cell death (NETosis), as well as the clearance of NETs by macrophages (MΦ) (efferocytosis) in acute sepsis following binge drinking.
Healthy volunteers consumed 2 ml of vodka/kg body weight, before blood endotoxin and 16 s rDNA were measured. Peripheral neutrophils were isolated and exposed to alcohol followed by phorbol 12-myristate 13-acetate (PMA) stimulation. Mice were treated with three alcohol binges and intraperitoneal lipopolysaccharide (LPS) to assess the dynamics of NET formation and efferocytosis. In vivo, anti-Ly6G antibody (IA8) was used for neutrophil depletion.
Inducers of NETs (endotoxin and bacterial DNA) significantly increased in the circulation after binge alcohol drinking in humans. Ex vivo, alcohol alone increased NET formation, but upon PMA stimulation alcohol attenuated NET formation. Binge alcohol in mice resulted in a biphasic response to LPS. Initially, binge alcohol reduced LPS-induced NET formation and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-naïve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after LPS challenge in mice receiving binge alcohol, suggesting decreased NET formation. However, in the efferocytosis phase (15 h after LPS) citrullinated histone-H3 was increased in the liver in alcohol binge mice, suggesting decreased clearance of NETs. In vitro alcohol treatment reduced efferocytosis and phagocytosis of NETotic neutrophils and promoted expression of CD206 on MΦ. Finally, depletion of neutrophils prior to binge alcohol ameliorated LPS-induced systemic inflammation and liver injury in mice.
Dysfunctional NETosis and efferocytosis following binge drinking exacerbate liver injury associated with sepsis.
Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.
Alcohol abuse is a leading cause of liver disease characterized by liver inflammation, fatty liver, alcoholic hepatitis, or liver cirrhosis. Immunomodulatory effects of alcohol on monocytes and ...macrophages contribute to alcoholic liver disease. Alcohol use, an independent risk factor for progression of hepatitis C virus (HCV) infection-mediated liver disease, impairs host defense and alters cytokine production and monocyte/macrophage activation. We hypothesized that alcohol and HCV have synergistic effects on the phenotype and function of monocytes. Our data show that acute alcohol binge drinking in healthy volunteers results in increased frequency of CD16(+) and CD68(+) and M2-type (CD206(+), dendritic cell DC-SIGN(+)-expressing and IL-10-secreting) circulating CD14(+) monocytes. Expression of HCV-induced CD68 and M2 markers (CD206 and DC-SIGN) in normal monocytes was further enhanced in the presence of alcohol. The levels of microRNA (miR)-27a was significantly upregulated in monocytes cultured in the presence of alcohol or alcohol and HCV as compared with HCV alone. The functional role of miR-27a in macrophage polarization was demonstrated by transfecting monocytes with an miR-27a inhibitor that resulted in reduced alcohol- and HCV- mediated monocyte activation (CD14 and CD68 expression), polarization (CD206 and DC-SIGN expression), and IL-10 secretion. Overexpression of miR-27a in monocytes enhanced IL-10 secretion via activation of the ERK signaling pathway. We found that miR-27a promoted ERK phosphorylation by downregulating the expression of ERK inhibitor sprouty2 in monocytes. Thus, we identified that sprouty2 is a target of miR-27a in human monocytes. In summary, our study demonstrates the regulatory role of miR-27a in alcohol-induced monocyte activation and polarization.
PDF
