The Hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations1-4. We are curious as to what structural solution has the viroporin adopted to afford selective cation conduction because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The p7 activity can be inhibited by amantadine and rimantadine2,5, which also happen to be potent blockers of the influenza M2 channel6 and licensed drugs against influenza infections7. The adamantane derivatives were subjects of HCV clinical trials8, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here, we determined the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbors, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also alludes to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter while an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording showed that these residues are indeed critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

BACKGROUND & AIMS: Activation of TGFB (transforming growth factor b) promotes liver fibrosis by activating hepatic stellate cells (HSCs), but the mechanisms of TGFB activation are not clear. We investigated the role of ECM1 (extracellular matrix protein 1), which interacts with extracellular and structural proteins, in TGFB activation in mouse livers. METHODS:We performed studies with C57BL/6J mice (controls), ECM1-knockout (ECM1-KO) mice, and mice with hepatocyte-specific knockout of EMC1 (ECM1 Dhep ). ECM1 or soluble TGFBR2 (TGFB receptor 2) were expressed in livers of mice after injection of an adeno-associated virus vector. Liver fibrosis was induced by carbon tetrachloride (CCl 4 ) administration. Livers were collected from mice and analyzed by histology, immunohistochemistry, in situ hybridization, and immunofluorescence analyses. Hepatocytes and HSCs were isolated from livers of mice and incubated with ECM1; production of cytokines and activation of reporter genes were quantified. Liver tissues from patients with viral or alcohol-induced hepatitis (with different stages of fibrosis) and individuals with healthy livers were analyzed by immunohistochemistry and in situ hybridization. RESULTS: ECM1-KO mice spontaneously developed liver fibrosis and died by 2 months of age without significant hepatocyte damage or inflammation. In liver tissues of mice, we found that ECM1 stabilized extracellular matrix-deposited TGFB in its inactive form by interacting with av integrins Gastroenterology 2019;157:1352-1367 BASIC AND TRANSLATIONAL LIVER * Authors share co-first authorship.Abbreviations used in this paper: AAV, adeno-associated virus; CLD, chronic liver disease; CCl 4 , carbon tetrachloride; ECM1, extracellular matrix protein 1; ECM1 D;hep , hepatocyte-specific deletion of extracellular matrix protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;

TRIM22, a tripartite-motif (TRIM) protein, is upregulated upon interferon alpha (IFNa) administration to hepatitis C virus (HCV)-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRIM22's targeting of the HCV NS5A protein. NS5A is important for HCV replication and for resistance to IFNa therapy. During the first 24 h following the initiation of IFNa treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells (PBMCs) of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA (siRNA)-mediated knockdown of TRIM22 diminished IFNa-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates NS5A in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNa treatment and plays an important role in controlling HCV replication in vitro.

Inappropriate neutrophil infiltration and subsequent neutrophil extracellular trap (NET) formation have been confirmed to be involved in chronic inflammatory conditions. Fatty liver disease is an increasingly severe health problem worldwide and currently considered the most common cause of chronic liver disease. Sphingosine 1-phosphate (S1P), a product of membrane sphingolipid metabolism, regulates vital physiological and pathological actions by inducing infiltration and activation of various cell types through S1P receptors (S1PRs). Here, we seek to determine the S1PR-mediated effects on neutrophil activation during chronic liver inflammation. In this study, NETs are detected in the early stage of methionine-choline-deficient and a high-fat (MCDHF) diet-induced liver injury. NET depletion by deoxyribonuclease I intraperitoneal injection significantly protects liver from MCDHF-induced liver injury in vivo. Meanwhile, we show that levels of myeloperoxidase-DNA complex (NET marker) in the serum present positive correlation with sphingosine kinase1 (S1P rate-limiting enzyme) messenger RNA expression or S1P levels in the injured liver of MCDHF-fed mice. In vitro, S1PR2 participates in the redirection of neutrophil apoptosis to NETosis via Gαi/o, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and reactive oxygen species signaling pathways. Moreover, S1PR2 knockdown in MCDHF-fed mice by S1PR2-siRNA intravenous injection significantly inhibits NET formation in damaged liver tissue and then alleviates hepatic inflammation and fibrosis. Conclusion: In the early stage of fatty liver disease, S1PR2-mediated neutrophil activation plays an important role in the evolvement of liver injury.

Inflammatory bowel disease (IBD) comprises chronic relapsing disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Here, we uncover a function of extracellular matrix protein 1 (ECM1) in promoting the pathogenesis of human and mouse IBD. ECM1 was highly expressed in macrophages, particularly tissue-infiltrated macrophages under inflammatory conditions, and ECM1 expression was significantly induced during IBD progression. The macrophage-specific knockout of ECM1 resulted in increased arginase 1 (ARG1) expression and impaired polarization into the M1 macrophage phenotype after lipopolysaccharide (LPS) treatment. A mechanistic study showed that ECM1 can regulate M1 macrophage polarization through the granulocyte-macrophage colony-stimulating factor/STAT5 signaling pathway. Pathological changes in mice with dextran sodium sulfate-induced IBD were alleviated by the specific knockout of the ECM1 gene in macrophages. Taken together, our findings show that ECM1 has an important function in promoting M1 macrophage polarization, which is critical for controlling inflammation and tissue repair in the intestine.

NLR family pyrin domain containing 3 (NLRP3) inflammasome accompanies chronic liver injury and is a critical mediator of inflammation-driven liver fibrosis. Sphingosine 1-phosphate (S1P)/S1P Receptor (S1PR) signaling participates in liver fibrogenesis by affecting bone marrow (BM)-derived monocytes/macrophage (BMM) activation. However, the relationship between S1P/S1PR signaling and NLRP3 inflammasome in BMMs remains unclear. Here, we found significantly elevated gene expression of NLRP3 inflammasome components (NLRP3, pro-interleukin-1β, and pro-interleukin-18) and the activation of NLRP3 inflammasome significantly elevated during murine chronic liver injury induced by a bile duct ligation operation, a methionine-choline-deficient and high-fat diet, or carbon tetrachloride intraperitoneal injection. Moreover, the increased expression of sphingosine kinase 1 (SphK1), the rate-limiting synthetic enzyme of S1P, was positively correlated with NLRP3 inflammasome components in both patients and mouse model livers. Flow cytometry analysis and immunofluorescence staining showed BMMs contributed to the significant proportion of NLRP3 + cells in murine inflammatory livers, but not Kupffer cells, dendritic cells, endothelial cells, T cells, and hepatocytes. Focusing on macrophages, S1P promoted NLRP3 inflammasome priming and activation in a dose-dependent manner. Blockade of S1PR 2 by JTE-013 (antagonist of S1PR 2) or S1PR 2-siRNA inhibited S1P-induced NLRP3 inflammasome priming and inflammatory cytokine (interleukin-1β and interleukin-18) secretion, whereas blockade of S1PR 1 or S1PR 3 had no such effect. in vivo, a β1,3-d-glucan-encapsulated siRNA particle (GeRP) delivery system is capable of silencing genes in macrophages specifically. Treatment with S1PR 2 siRNA-GeRPs markedly reduced NLRP3 inflammasome priming and activation and attenuated liver inflammation and fibrosis. Together, the conclusions indicated that targeting macrophage S1PR 2 retarded liver inflammation and fibrogenesis via downregulating NLRP3 inflammasome, which may represent an effective therapeutic strategy for chronic liver injury.

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