Pranoti Mandrekar

A Recent Perspective on Alcohol, Immunity, and Host Defense

BACKGROUND Multiple line of clinical and experimental evidence demonstrates that both acute, moderate, and chronic, excessive alcohol use result in various abnormalities in the functions of the immune system. METHODS Medline and PubMed databases were used to identify published reports with particular interest in the period of 2000-2008 in the subject of alcohol use, infection, inflammation, innate, and adaptive immunity. RESULTS This review article summarizes recent findings relevant to acute or chronic alcohol use-induced immunomodulation and its consequences on host defense against microbial pathogens and tissue injury. Studies with in vivo and in vitro alcohol administration are both discussed. The effects of alcohol on lung infections, trauma and burn injury, liver, pancreas, and cardiovascular diseases are evaluated with respect to the role of immune cells. Specific changes in innate immune response and abnormalities in adaptive immunity caused by alcohol intake are detailed. CONCLUSION Altered inflammatory cell and adaptive immune responses after alcohol consumption result in increased incidence and poor outcome of infections and other organ-specific immune-mediated effects.

The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88

The Toll‐like receptor 4 (TLR4) that recognizes endotoxin, a trigger of inflammation in alcoholic liver disease (ALD), activates two signaling pathways utilizing different adapter molecules: the common TLR adapter, myeloid differentiation factor 88 (MyD88), or Toll/interleukin immune‐response–domain‐containing adaptor inducing interferon (IFN)‐β. The MyD88 pathway induces proinflammatory cytokine activation, a critical mediator of ALD. Here we evaluated the role of MyD88 in alcohol‐induced liver injury in wild‐type, TLR2‐deficient, TLR4‐deficient, or MyD88‐deficient (knockout [KO]) mice after administration of the Lieber‐De‐Carli diet (4.5% volume/volume ethanol) or an isocaloric liquid control diet for 5 weeks. Alcohol feeding resulted in a significant increase in serum alanine aminotransferase levels, liver steatosis and triglyceride levels suggesting liver damage in WT, TLR2‐KO, and MyD88‐KO but not in TLR4‐KO mice. Expression of inflammatory mediators (tumor necrosis factor–α and interleukin‐6) and TLR4 coreceptors (CD14 and MD2) was significantly higher in livers of alcohol‐fed WT, TLR2‐KO, or MyD88‐KO, but not in TLR4‐KO mice, compared to controls. Reactive oxygen radicals produced by cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complexes contribute to alcoholic liver damage. Alcohol feeding–induced expression and activation of cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complex were prevented by TLR4‐deficiency but not by MyD88‐deficiency. Liver expression of interferon regulatory factor 3 (IRF3), a MyD88‐independent signaling molecule, was not affected by chronic alcohol treatment in whole livers of WT mice or in any of the KO mice. However, the induction of IRF7, an IRF3‐inducible gene, was found in Kupffer cells of alcohol‐fed WT mice. Alcohol feeding also induced nuclear factor–κB activation in a TLR4‐dependent MyD88‐independent manner. Conclusion: While TLR4 deficiency was protective, MyD88 deficiency failed to prevent alcohol‐induced liver damage and inflammation. These results suggest that the common TLR adapter, MyD88, is dispensable in TLR4‐mediated liver injury in ALD. (HEPATOLOGY 2008.)

Signalling pathways in alcohol-induced liver inflammation☆

The pathogenesis of alcoholic liver injury involves interactions of several intracellular signalling pathways in different cell types of the liver. Alcohol-induced sensitization of liver macrophages to portal endotoxin/lipopolysaccharide (LPS) is considered a hallmark of alcoholic liver disease (ALD). Intracellular mechanisms associated with LPS-induced signalling play a crucial role in the initiation and progression of alcoholic liver injury, and are being extensively explored. LPS recognition by Toll-like receptor 4 (TLR4) on macrophages and other cell types in the liver, activation of downstream signalling pathways culminating in activation of transcription factors such as NFkappaB, AP-1 leads to increased inflammatory cytokine production in ALD. In addition, LPS-induced MAPK such as ERK and p38 also contribute to liver injury. The importance of alcohol-induced reactive oxygen species and interactions with TLR pathways in macrophages leading to inflammation is becoming increasingly evident. Collectively, these signalling pathways induce pro- and anti-inflammatory cytokines that play an important role in ALD. In this review we describe the key signalling intermediates leading to alcohol-induced inflammation in alcoholic liver disease.

Hepatitis C Virus Core and Nonstructural Protein 3 Proteins Induce Pro- and Anti-inflammatory Cytokines and Inhibit Dendritic Cell Differentiation

Antiviral immunity requires recognition of viral pathogens and activation of cytotoxic and Th cells by innate immune cells. In this study, we demonstrate that hepatitis C virus (HCV) core and nonstructural protein 3 (NS3), but not envelope 2 proteins (E2), activate monocytes and myeloid dendritic cells (DCs) and partially reproduce abnormalities found in chronic HCV infection. HCV core or NS3 (not E2) triggered inflammatory cytokine mRNA and TNF-α production in monocytes. Degradation of I-κBα suggested involvement of NF-κB activation. HCV core and NS3 induced production of the anti-inflammatory cytokine, IL-10. Both monocyte TNF-α and IL-10 levels were higher upon HCV core and NS3 protein stimulation in HCV-infected patients than in normals. HCV core and NS3 (not E2) inhibited differentiation and allostimulatory capacity of immature DCs similar to defects in HCV infection. This was associated with elevated IL-10 and decreased IL-2 levels during T cell proliferation. Increased IL-10 was produced by HCV patients’ DCs and by core- or NS3-treated normal DCs, while IL-12 was decreased only in HCV DCs. Addition of anti-IL-10 Ab, not IL-12, ameliorated T cell proliferation with HCV core- or NS3-treated DCs. Reduced allostimulatory capacity in HCV core- and NS3-treated immature DCs, but not in DCs of HCV patients, was reversed by LPS maturation, suggesting more complex DC defects in vivo than those mediated by core or NS3 proteins. Our results reveal that HCV core and NS3 proteins activate monocytes and inhibit DC differentiation in the absence of the intact virus and mediate some of the immunoinhibitory effects of HCV via IL-10 induction.

Up-regulation of microRNA-155 in macrophages contributes to increased tumor necrosis factor {alpha} (TNF{alpha}) production via increased mRNA half-life in alcoholic liver disease

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability.

Hepatitis C Virus (HCV) core protein-induced, monocyte-mediated mechanisms of reduced IFN-α and plasmacytoid dendritic cell loss in chronic HCV infection

IFN-α production by plasmacytoid dendritic cells (PDCs) is critical in antiviral immunity. In the present study, we evaluated the IFN-α-producing capacity of PDCs of patients with chronic hepatitis C virus (HCV) infection in treatment-naive, sustained responder, and nonresponder patients. IFN-α production was tested in PBMCs or isolated PDCs after TLR9 stimulation. Treatment-naive patients with chronic HCV infection had reduced frequency of circulating PDCs due to increased apoptosis and showed diminished IFN-α production after stimulation with TLR9 ligands. These PDC defects correlated with the presence of HCV and were in contrast with normal PDC functions of sustained responders. HCV core protein, which was detectable in the plasma of infected patients, reduced TLR9-triggered IFN-α and increased TNF-α and IL-10 production in PBMCs but not in isolated PDCs, suggesting HCV core induced PDC defects. Indeed, addition of rTNF-α and IL-10 induced apoptosis and inhibited IFN-α production in PDCs. Neutralization of TNF-α and/or IL-10 prevented HCV core-induced inhibition of IFN-α production. We identified CD14+ monocytes as the source of TNF-α and IL-10 in the HCV core-induced inhibition of PDC IFN-α production. Anti-TLR2-, not anti-TLR4-, blocking Ab prevented the HCV core-induced inhibition of IFN-α production. In conclusion, our results suggest that HCV interferes with antiviral immunity through TLR2-mediated monocyte activation triggered by the HCV core protein to induce cytokines that in turn lead to PDC apoptosis and inhibit IFN-α production. These mechanisms are likely to contribute to HCV viral escape from immune responses.

VSL#3 probiotic treatment attenuates fibrosis without changes in steatohepatitis in a diet‐induced nonalcoholic steatohepatitis model in mice

Nonalcoholic fatty liver disease (NAFLD) and its advanced stage, nonalcoholic steatohepatitis (NASH), are the most common causes of chronic liver disease in the United States. NASH features the metabolic syndrome, inflammation, and fibrosis. Probiotics exhibit immunoregulatory and anti‐inflammatory activity. We tested the hypothesis that probiotic VSL#3 may ameliorate the methionine‐choline‐deficient (MCD) diet–induced mouse model of NASH. MCD diet resulted in NASH in C57BL/6 mice compared to methionine‐choline‐supplemented (MCS) diet feeding evidenced by liver steatosis, increased triglycerides, inflammatory cell accumulation, increased tumor necrosis factor α levels, and fibrosis. VSL#3 failed to prevent MCD‐induced liver steatosis or inflammation. MCD diet, even in the presence of VSL#3, induced up‐regulation of serum endotoxin and expression of the Toll‐like receptor 4 signaling components, including CD14 and MD2, MyD88 adaptor, and nuclear factor κB activation. In contrast, VSL#3 treatment ameliorated MCD diet–induced liver fibrosis resulting in diminished accumulation of collagen and α‐smooth muscle actin. We identified increased expression of liver peroxisome proliferator‐activated receptors and decreased expression of procollagen and matrix metalloproteinases in mice fed MCD+VSL#3 compared to MCD diet alone. MCD diet triggered up‐regulation of transforming growth factor beta (TGFβ), a known profibrotic agent. In the presence of VSL#3, the MCD diet–induced expression of TGFβ was maintained; however, the expression of Bambi, a TGFβ pseudoreceptor with negative regulatory function, was increased. In summary, our data indicate that VSL#3 modulates liver fibrosis but does not protect from inflammation and steatosis in NASH. The mechanisms of VSL#3‐mediated protection from MCD diet–induced liver fibrosis likely include modulation of collagen expression and impaired TGFβ signaling. (HEPATOLOGY 2009.)

MicroRNA expression profile in Lieber-DeCarli diet-induced alcoholic and methionine choline deficient diet-induced nonalcoholic steatohepatitis models in mice.

BACKGROUND Alcoholic and nonalcoholic steatohepatitis are leading causes of liver diseases worldwide. While of different etiology, these share common pathophysiological mechanisms and feature abnormal fat metabolism, inflammation and fibrosis. MicroRNAs (miRNA) are highly conserved noncoding RNAs that control gene expression at the post-transcriptional level either via the degradation of target mRNAs or the inhibition of translation. Each miRNA controls the expression of multiple targets; miRNAs have been linked to regulation of lipid metabolism and inflammation. METHODS We fed Lieber-DeCarli alcohol or methionine-choline-deficient (MCD) diets to C57Bl6 and analyzed livers for histopathology, cytokines by ELISA, alanine aminotransferase (ALT) by biochemical assay, and microRNA profile by microarray. RESULTS Both Lieber-DeCarli and MCD diets lead to development of liver steatosis, liver injury, indicated by increased ALT, and elevated levels of serum TNFalpha, suggesting that animal models portray the pathophysiological features of alcoholic and nonalcoholic fatty liver, respectively. We identified that Lieber-deCarli diet up-regulated 1% and down-regulated 1% of known miRNA; MCD diet up-regulated 3% and down-regulated 1% of known miRNA, compared to controls. Of miRNAs that changed expression levels, 5 miRNAs were common in alcoholic and nonalcoholic fatty livers: the expression of both miR-705 and miR-1224 was increased after Lieber-DeCarli or MCD diet feeding. In contrast, miR-182, miR-183, and miR-199a-3p were down-regulated in Lieber-deCarli feeding, while MCD diet lead to their up-regulation, compared to corresponding controls. CONCLUSIONS Our findings indicate etiology-specific changes in miRNA expression profile during steatohepatitis models, which opens new avenues for research in the pathophysiology of alcoholic and nonalcoholic fatty liver disease.

An essential role for monocyte chemoattractant protein‐1 in alcoholic liver injury: Regulation of proinflammatory cytokines and hepatic steatosis in mice

The importance of chemokines in alcoholic liver injury has been implicated. The role of the chemokine, monocyte chemoattractant protein‐1 (MCP‐1), elevated in patients with alcoholic liver disease is not yet understood. Here, we evaluated the pathophysiological significance of MCP‐1 and its receptor, chemokine (C‐C motif) receptor 2 (CCR2), in alcoholic liver injury. The Leiber‐DeCarli diet containing alcohol or isocaloric control diets were fed to wild‐type (WT) and MCP‐1‐deficient knockout (KO) mice for 6 weeks. In vivo and in vitro assays were performed to study the role of MCP‐1 in alcoholic liver injury. MCP‐1 was increased in Kupffer cells (KCs) as well as hepatocytes of alcohol‐fed mice. Alcohol feeding increased serum alanine aminotransferase in WT and CCR2KO, but not MCP‐1KO, mice. Alcohol‐induced liver steatosis and triglyceride were attenuated in alcohol‐fed MCP‐1KO, but high in CCR2KO mice, compared to WT, whereas serum endotoxin was high in alcohol‐fed WT and MCP‐1KO mice. Expression of liver proinflammatory cytokines tumor necrosis factor alpha, interleukin (IL)‐1β, IL‐6, KC/IL‐8, intercellular adhesion molecule 1, and cluster of differentiation 68 was induced in alcohol‐fed WT, but inhibited in MCP‐1KO, mice independent of nuclear factor kappa light‐chain enhancer of activated B cell activation in KCs. Oxidative stress, but not cytochrome P450 2E1, was prevented in chronic alcohol‐fed MCP‐1KO mice, compared to WT. Increased expression of peroxisome proliferator‐activated receptor (PPAR)α and PPARγ was accompanied by nuclear translocation, DNA binding, and induction of fatty acid metabolism genes acyl coenzyme A oxidase and carnitine palmitoyltransferase 1A in livers of alcohol‐fed MCP‐1KO mice, compared to WT controls. In vitro assays uncovered an inhibitory effect of recombinant MCP‐1 on PPARα messenger RNA and peroxisome proliferator response element binding in hepatocytes independent of CCR2. Conclusion: Deficiency of MCP‐1 protects mice against alcoholic liver injury, independent of CCR2, by inhibition of proinflammatory cytokines and induction of genes related to fatty acid oxidation, linking chemokines to hepatic lipid metabolism. (HEPATOLOGY 2011)

Pattern recognition receptors: A contemporary view on liver diseases

Pattern recognition receptors (PRRs) function as sensors of microbial danger signals enabling the vertebrate host to initiate an immune response. PRRs are present not only in immune cells but also in liver parenchymal cells and the complexity of the cell populations provide unique aspects to pathogen recognition and tissue damage in the liver. This review discusses the role of different PRRs in pathogen recognition in the liver, and focuses on the role of PRRs in hepatic inflammation, cholestasis, ischemia, repair and fibrosis. PRRs as novel therapeutic targets are evaluated. (HEPATOLOGY 2006;44:287–298.)