Arvin Iracheta-Vellve

STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease

Significance This paper provides previously undescribed evidence that STING, an endoplasmic reticulum (ER)-resident protein involved in DNA sensing, couples ER stress with apoptotic signaling in alcoholic liver disease. The proapoptotic role of STING is mediated by the interferon regulatory factor 3 (IRF3), and this is independent of inflammation or Type-I interferons. Activation of STING and IRF3, originally reported in the context of antiviral response, determines survival of hepatocytes in early alcoholic liver disease suggesting that innate immunity regulates hepatocyte pathophysiology independent of inflammation. 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.

Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease.

Inflammation defines the progression of ALD from reversible to advanced stages. Translocation of bacterial LPS to the liver from the gut is necessary for alcohol‐induced liver inflammation. However, it is not known whether endogenous, metabolic danger signals are required for inflammation in ALD. Uric acid and ATP, 2 major proinflammatory danger signals, were evaluated in the serum of human volunteers exposed to a single dose of ethanol or in supernatants of primary human hepatocytes exposed to ethanol. In vitro studies were used to evaluate the role of uric acid and ATP in inflammatory cross‐talk between hepatocytes and immune cells. The significance of signaling downstream of uric acid and ATP in the liver was evaluated in NLRP3‐deficient mice fed a Lieber‐DeCarli ethanol diet. Exposure of healthy human volunteers to a single dose of ethanol resulted in increased serum levels of uric acid and ATP. In vitro, we identified hepatocytes as a significant source of these endogenous inflammatory signals. Uric acid and ATP mediated a paracrine inflammatory cross‐talk between damaged hepatocytes and immune cells and significantly increased the expression of LPS‐inducible cytokines, IL‐1β and TNF‐α, by immune cells. Deficiency of NLRP3, a ligand‐sensing component of the inflammasome recognizing uric acid and ATP, prevented the development of alcohol‐induced liver inflammation in mice and significantly ameliorated liver damage and steatosis. Endogenous metabolic danger signals, uric acid, and ATP are involved in inflammatory cross‐talk between hepatocytes and immune cells and play a crucial role in alcohol‐induced liver inflammation.

Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice

BACKGROUND & AIMS The inflammasome is a well-characterized inducer of inflammation in alcoholic steatohepatitis (ASH). Inflammasome activation requires two signals for mature interleukin (IL)-1β production. Here we asked whether metabolic danger signals trigger inflammasome activation in ASH. METHODS Wild-type mice, ATP receptor 2x7 (P2rx7)-KO mice, or mice overexpressing uricase were fed Lieber-DeCarli ethanol or control diet. We also implemented a pharmacological approach in which mice were treated with probenecid or allopurinol. RESULTS The sterile danger signals, ATP and uric acid, were increased in the serum and liver of alcohol-fed mice. Depletion of uric acid or ATP, or lack of ATP signaling attenuated ASH and prevented inflammasome activation and its major downstream cytokine, IL-1β. Pharmacological depletion of uric acid with allopurinol provided significant protection from alcohol-induced inflammatory response, steatosis and liver damage, and additional protection was achieved in mice treated with probenecid, which depletes uric acid and blocks ATP-induced P2rx7 signaling. We found that alcohol-damaged hepatocytes released uric acid and ATP in vivo and in vitro and that these sterile danger signals activated the inflammasome in LPS-exposed liver mononuclear cells. CONCLUSIONS Our data indicate that the second signal in inflammasome activation and IL-1β production in ASH results from the endogenous danger signals, uric acid and ATP. Inhibition of signaling triggered by uric acid and ATP may have therapeutic implications in ASH.

MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

Background & Aim MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis. Methods Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed. Results MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor κ beta (NF-κB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and α smooth muscle actin (αSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-β (TGFβ) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein β (C/EBPβ) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice. Conclusions Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis.

Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, inflammation and steatosis in early alcoholic hepatitis in mice

Background Alcohol-induced intestinal dysbiosis disrupts homeostatic gut-liver axis function and is essential in the development of alcoholic liver disease. Here, we investigate changes in enteric microbiome composition in a model of early alcoholic steatohepatitis and dissect the pathogenic role of intestinal microbes in alcohol-induced liver pathology. Materials and methods Wild type mice received a 10-day diet that was either 5% alcohol-containing or an isocaloric control diet plus a single binge. 16S rDNA sequencing defined the bacterial communities in the cecum of alcohol- and pair-fed animals. Some mice were treated with an antibiotic cocktail prior to and throughout alcohol feeding. Liver neutrophils, cytokines and steatosis were evaluated. Results Acute-on-chronic alcohol administration induced shifts in various bacterial phyla in the cecum, including increased Actinobacteria and a reduction in Verrucomicrobia driven entirely by a reduction in the genus Akkermansia. Antibiotic treatment reduced the gut bacterial load and circulating bacterial wall component lipopolysaccharide (LPS). We found that bacterial load suppression prevented alcohol-related increases in the number of myeloperoxidase- (MPO) positive infiltrating neutrophils in the liver. Expression of liver mRNA tumor necrosis factor alpha (Tnfα), C-X-C motif chemokine ligand 1 (Cxcl1) and circulating protein monocyte chemoattractant protein-1 (MCP-1) were also reduced in antibiotic-treated alcohol-fed mice. Alcohol-induced hepatic steatosis measured by Oil-Red O staining was significantly reduced in antibiotic treated mice. Genes regulating lipid production and storage were also altered by alcohol and antibiotic treatment. Interestingly, antibiotic treatment did not protect from alcohol-induced increases in serum aminotransferases (ALT/AST). Conclusions Our data indicate that acute-on-chronic alcohol feeding alters the microflora at multiple taxonomic levels and identifies loss of Akkermansia as an early marker of alcohol-induced gut dysbiosis. We conclude that gut microbes influence liver inflammation, neutrophil infiltration and liver steatosis following alcohol consumption and these data further emphasize the role of the gut-liver axis in early alcoholic liver disease.

Endoplasmic reticulum stress-induced hepatocellular death pathways mediate liver injury and fibrosis via Stimulator of Interferon Genes.

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-β (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4. Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4. In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease

The spectrum of alcoholic liver disease (ALD) is a major cause of mortality with limited therapies available. Because alcohol targets numerous signaling pathways in hepatocytes and in immune cells, the identification of a master regulatory target that modulates multiple signaling processes is attractive. In this report, we assessed the role of spleen tyrosine kinase (SYK), a nonreceptor tyrosine kinase, which has a central modulatory role in multiple proinflammatory signaling pathways involved in the pathomechanism of ALD. Using mouse disease models that represent various phases in the progression of human ALD, we found that alcohol, in all of these models, induced SYK activation in the liver, both in hepatocytes and liver mononuclear cells. Furthermore, significant SYK activation also occurred in liver samples and peripheral blood mononuclear cells of patients with ALD/alcoholic hepatitis compared to controls. Functional inhibition of SYK activation in vivo abrogated alcohol‐induced hepatic neutrophil infiltration, resident immune cell activation, as well as inflammasome and extracellular signal‐regulated kinase 1 and 2‐mediated nuclear factor kappa B activation in mice. Strikingly, inhibition of SYK activation diminished alcohol‐induced hepatic steatosis and interferon regulatory factor 3‐mediated apoptosis. Conclusion: Our data demonstrate a novel, functional, and multicellular role for SYK phosphorylation in modulating immune cell‐driven liver inflammation, hepatocyte cell death, and steatosis at different stages of ALD. These novel findings highlight SYK as a potential multifunctional target in the treatment of alcoholic steatohepatitis. (Hepatology 2016;64:1057‐1071)

Modulation of cellular stress response via the erythropoietin/CD131 heteroreceptor complex in mouse mesenchymal-derived cells

Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO signaling in defined cell populations. In this study, we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO signaling strongly modulated transcriptional, translational, or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able to overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1), and activator protein 1 (AP1). We conclude that alternative EPO signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis.Key message• Erythropoietin (EPO) triggers an alternative pathway via heteroreceptor EPO/CD131.• ARA290 peptide specifically binds EPO/CD131 but not the canonical EPO/EPO receptor.• Oxidative stress and inflammation promote cell surface expression of CD131.• ARA290 prevents tumor necrosis factor-mediated inhibition of stress-related genes.• Alternative EPO signaling modulates inflammation and promotes tissue homeostasis.

Inflammasome activation in the liver: Focus on alcoholic and non-alcoholic steatohepatitis

Upregulation of the inflammatory cascade is a major element both in the progression of steatohepatitis to severe alcoholic hepatitis as well as in the progression of NASH to advanced NASH with fibrosis. The mechanisms underpinning these changes are only partially understood. Activation of the inflammatory cascade requires multiple stimuli and in this report, we discuss the role of inflammasomes that activate IL-1β as well as the sterile and pathogen-derived danger signals that results in inflammasome activation and inflammation in alcoholic and non-alcoholic steatohepatitis. The dynamics of inflammasome activation, the cell types involved and the trigger signals appear to be somewhat different between ASH and NASH. Further studies are needed to dissect the pathology-related differences between these two major forms of steatohepatitis. Clinical and therapeutic implications of inflammasome activation in steatohepatitis are also discussed.

Alcohol-induced miR-155 and HDAC11 inhibit negative regulators of the TLR4 pathway and lead to increased LPS responsiveness of Kupffer cells in alcoholic liver disease

Inflammation promotes the progression of alcoholic liver disease. Alcohol sensitizes KCs to gut‐derived endotoxin (LPS); however, signaling pathways that perpetuate inflammation in alcoholic liver disease are only partially understood. We found that chronic alcohol feeding in mice induced miR‐155, an inflammatory miRNA in isolated KCs. We hypothesized that miR‐155 might increase the responsiveness of KCs to LPS via targeting the negative regulators of LPS signaling. Our results revealed that KCs that were isolated from alcohol‐fed mice showed a decrease in IRAK‐M, SHIP1, and PU.1, and an increase in TNF‐α levels. This was specific to KCs, as no significant differences were observed in these genes in hepatocytes. We found a causal effect of miR‐155 deficiency on LPS responsiveness, as KCs that were isolated from miR‐155 KO mice showed a greater induction of IRAK‐M, SHIP1, and suppressor of cytokine signaling 1 after LPS treatment. C/EBPβ, a validated miR‐155 target, stimulates IL‐10 transcription. We found a higher induction of C/EBPβ and IL‐10 in KCs that were isolated from miR‐155 KO mice after LPS treatment. Gain‐ and loss‐of‐function studies affirmed that alcohol‐induced miR‐155 directly regulates IRAK‐M, SHIP1, suppressor of cytokine signaling 1, and C/EBPβ, as miR‐155 inhibition increased and miR‐155 overexpression decreased these genes in LPS or alcohol‐pretreated wild‐type KCs. HDAC11, a regulator of IL‐10, was significantly increased and IL‐10 was decreased in KCs that were isolated from alcohol‐fed mice. Functionally, knockdown of HDAC11 with small interfering RNA resulted in an IL‐10 increase in LPS or alcohol‐pretreated Mϕ. We found that acetaldehyde and NF‐κB pathways regulate HDAC11 levels. Collectively, our results indicate that the alcohol‐induced responsiveness of KCs to LPS, in part, is governed by miR‐155 and HDAC11.