Inflammation in Steatotic Liver Diseases: Pathogenesis and Therapeutic Targets.

Alcohol-related liver disease (ALD) and metabolic dysfunction-associated steatotic liver disease (MASLD), two main types of steatotic liver disease (SLDs), are characterized by a wide spectrum of several different liver disorders, including simple steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. Multiple immune cell-mediated inflammatory responses not only orchestrate the killing and removal of infected/damaged cells but also exacerbate the development of SLDs when excessive or persistent inflammation occurs. In recent years, single-cell and spatial transcriptome analyses have revealed the heterogeneity of liver-infiltrated immune cells in ALD and MASLD, revealing a new immunopathological picture of SLDs. In this review, we will emphasize the roles of several key immune cells in the pathogenesis of ALD and MASLD and discuss inflammation-based approaches for effective SLD intervention. In conclusion, the study of immunological mechanisms, especially highly specific immune cell population functions, may provide novel therapeutic opportunities for this life-threatening disease.

Liver tissue metabolically transformed by alcohol induces immune recognition of liver self-proteins but not in vivo inflammation.

Precision-cut liver slices (PCLSs) provide a novel model for studies of alcoholic liver disease (ALD). This is relevant, as in vivo ethanol exposure does not appear to generate significant liver damage in ethanol-fed mice, except in the National Institute on Alcohol Abuse and Alcoholism binge model of ALD. Previous studies have shown that the two metabolites of ethanol consumption, malondialdhyde (MDA) and acetaldehyde (AA), combine to form MDA-AA (MAA) adducts, which have been correlated with the development and progression of ALD. In this study, murine PCLSs were incubated with ethanol and examined for the production of MAA adducts. PCLSs were homogenized, and homogenates were injected into C57BL/6 mice. PCLSs from control-, pair-, and ethanol-fed animals served as targets in in situ cytotoxic assays using primed T cells from mice hyperimmunized with control or ethanol-exposed PCLS homogenates. A CD45.1/CD45.2 passive-transfer model was used to determine whether T cells from the spleens of mice hyperimmunized with PCLS ethanol-exposed homogenates trafficked to the liver. PCLSs incubated with ethanol generated MAA-modified proteins in situ. Cytotoxic (CD8+) T cells from immunized mice killed naïve PCLSs from control- and pair-fed mice in vitro, a response that was blunted in PCLSs from ethanol-fed mice. Furthermore, CD45.1 CD8+ T cells from hyperimmunized mice trafficked to the liver but did not initiate liver damage. This study demonstrates that exposure to liver tissue damaged by ethanol mediates robust immune responses to well-characterized alcohol metabolites and native liver proteins in vitro. Moreover, although these proinflammatory T cells traffic to the liver, these responses appear to be dampened in vivo by locally acting pathways. NEW & NOTEWORTHY This study shows that the metabolites of ethanol and lipid breakdown produce malondialdehyde-acetaldehyde adducts in the precision-cut liver slice model system. Additionally, precision-cut liver slices exposed to ethanol and harboring malondialdehyde-acetaldehyde adducts generate liver-specific antibody and T cell responses in the spleens of naïve mice that could traffic to the liver.

Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice.

UNLABELLED: Alcoholic hepatitis (AH) is a distinct spectrum of alcoholic liver disease (ALD) with intense neutrophilic (polymorphonuclear; PMN) inflammation and high mortality. Although a recent study implicates osteopontin (SPP1) in AH, SPP1 is also shown to have protective effects on experimental ALD. To address this unsettled question, we examined the effects of SPP1 deficiency in male mice given 40% calories derived from ad libitum consumption of the Western diet high in cholesterol and saturated fat and the rest from intragastric feeding of alcohol diet without or with weekly alcohol binge. Weekly binge in this new hybrid feeding model shifts chronic ASH with macrophage inflammation and perisinusoidal and pericellular fibrosis to AH in 57% (15 of 26) of mice, accompanied by inductions of chemokines (Spp1, Cxcl1, and interleukin [Il]-17a), progenitor genes (Cd133, Cd24, Nanog, and epithelial cell adhesion molecule), PMN infiltration, and clinical features of AH, such as hypoalbuminemia, bilirubinemia, and splenomegaly. SPP1 deficiency does not reduce AH incidence and inductions of progenitor and fibrogenic genes, but rather enhances the Il-17a induction and PMN infiltration in some mice. Furthermore, in the absence of SPP1, chronic ASH mice without weekly binge begin to develop AH. CONCLUSION: These results suggest that SPP1 has a protective, rather than causal, role for experimental AH reproduced in our model.

Invariant NKT cells promote alcohol-induced steatohepatitis through interleukin-1ß in mice.

BACKGROUND & AIMS: It was reported that alcohol consumption activated the NLRP3 inflammasome in Kupffer cells, leading to mature interleukin (IL)-1ß release in alcoholic liver injury; however, how IL-1ß promotes liver injury remains unclear. METHODS: We investigated the role of IL-1ß in alcoholic steatohepatitis by using a chronic plus single-binge ethanol consumption mouse model. RESULTS: Here, liver steatosis was accompanied by notably increased invariant natural killer T (iNKT) cell numbers and activation, and iNKT-deficient Jα18(-/-) mice developed less alcohol-induced steatosis, with reduced liver inflammation and neutrophil infiltration. Kupffer cells and IL-1ß were required for the hepatic iNKT accumulation, as either blocking IL-1ß signaling with a recombinant IL-1 receptor antagonist (IL-1Ra), depleting Kupffer cells by clodronate liposomes, or specifically silencing IL-1ß in Kupffer cells by nanoparticle-encapsulated siRNA, resulted in inhibited hepatic iNKT cell accumulation and activation, as well as amelioration of alcoholic fatty liver. In addition, IL-1ß overexpression in hepatocytes was sufficient to compensate for Kupffer cell depletion. Increased gene and protein expression of mature IL-1ß correlated with elevated expression of the NLRP3 inflammasome components NLRP3, ASC, and cleaved caspase-1 in Kupffer cells from ethanol-exposed wild-type mice. NLRP3 deficiency led to the attenuation of alcoholic steatosis, similarly as Kupffer cell depletion, almost without hepatic NKT cells. CONCLUSIONS: After alcohol-exposure Kupffer cell-derived IL-1ß triggered by NLRP3 activation, recruits and activates hepatic iNKT cells, subsequently promoting liver inflammation and neutrophil infiltration, and inducing alcoholic liver injury.

Differences in innate immune signaling between alcoholic and non-alcoholic steatohepatitis.

The similar histopathological characteristics of alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH), and the crucial role of the innate immune response in both conditions may lead to the assumption that ASH and NASH represent the same pathophysiological entities caused by different risk factors. In this review paper, we elaborate on the pathophysiological differences between these two entities and highlight the disease-specific involvement of signaling molecules downstream of the Toll-like receptor 4, and the differential mechanism by which the inflammasome contributes to ASH versus NASH. Our findings emphasize that ASH and NASH have disease-specific mechanisms and therefore represent distinct biological entities. Further studies are needed to dissect the emerging differences in pathogenesis of these two conditions.

Successful transplantation of reduced-sized rat alcoholic fatty livers made possible by mobilization of host stem cells.

Livers from Lewis rats fed with 7% alcohol for 5 weeks were used for transplantation. Reduced sized (50%) livers or whole livers were transplanted into normal DA recipients, which, in this strain combination, survive indefinitely when the donor has not been fed alcohol. However, none of the rats survived a whole fatty liver transplant while six of seven recipients of reduced sized alcoholic liver grafts survived long term. SDF-1 and HGF were significantly increased in reduced size liver grafts compared to whole liver grafts. Lineage-negative Thy-1+CXCR4+CD133+ stem cells were significantly increased in the peripheral blood and in allografts after reduced size fatty liver transplantation. In contrast, there were meager increases in cells reactive with anti Thy-1, CXCR4 and CD133 in peripheral blood and allografts in whole alcoholic liver recipients. The provision of plerixafor, a stem cell mobilizer, salvaged 5 of 10 whole fatty liver grafts. Conversely, blocking SDF-1 activity with neutralizing antibodies diminished stem cell recruitment and four of five reduced sized fatty liver recipients died. Thus chemokine insufficiency was associated with transplant failure of whole grafts, which was overcome by the increased regenerative requirements promoted by the small grafts and mediated by SDF-1 resulting in stem cell influx.

Hepatic expression of CCL2 in alcoholic liver disease is associated with disease severity and neutrophil infiltrates.

Serum levels and liver expression of CCL2 are increased in patients with alcoholic hepatitis (AH). In an experimental model of alcoholic liver disease (ALD), CCL2 was implicated in proinflammatory cytokines activation and hepatic lipid metabolism, but its role in human disease is currently unknown. In a large cohort of ALD patients, we analysed plasma levels and liver expression of CCL2 and their association with liver disease severity and histological lesions. We also studied the relationship between -2518 A > G CCL2 and CCR2 190 A/G polymorphisms and severity of ALD. We show that CCL2 plasma levels are increased in ALD patients compared with healthy subjects. AH patients had significantly higher plasma levels and hepatic expression of CCL2 than patients without AH. Plasma levels and hepatic expression of CCL2 were associated with disease severity. CCL2 liver expression was correlated with neutrophil infiltrate and interleukin (IL)-8 expression, but not with steatosis. Moreover, there were more G-allele carriers of -2518 A > G CCL2 polymorphism in severe AH patients than in other ALD patients. Our results demonstrate that CCL2 is increased in ALD, particularly in severe forms, and suggest a role for CCL2 in the pathogenesis of ALD via neutrophil recruitment.

Role of cannabinoid receptors in alcoholic hepatic injury: steatosis and fibrogenesis are increased in CB2 receptor-deficient mice and decreased in CB1 receptor knockouts.

BACKGROUND: Alcohol is a common cause of hepatic liver injury with steatosis and fibrosis. Cannabinoid receptors (CB) modulate steatosis, inflammation and fibrogenesis. To investigate the differences between CB(1) and CB(2) in the hepatic response to chronic alcohol intake, we examined CB knockout mice (CB(1)(-/-), CB(2)(-/-)). METHODS: Eight- to 10-week-old CB(1)(-/-), CB(2)(-/-) and wild-type mice received 16% ethanol for 35 weeks. Animals receiving water served as controls. We analysed triglyceride and hydroxyproline contents in liver homogenates. mRNA levels of CBs, pro-inflammatory cytokines [tumour necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1ß] and profibrotic factors [α-smooth muscle actin (α-SMA), procollagen-Ia, platelet-derived growth factor ß receptor (PDGFß-R)] were analysed by reverse transcription-polymerase chain reaction (RT-PCR). Histology (hemalaun and eosin, oil-red O, CD3, CD45R, CD45, F4/80, Sirius red) characterized hepatic steatosis, inflammation and fibrosis. Activation of lipogenic pathways, activation and proliferation of hepatic stellate cell (HSC) were assessed by western blot [fatty acid synthase (FAS), sterol regulatory element binding protein 1c (SREBP-1c), α-SMA, proliferating cell nuclear antigen (PCNA), cathepsin D]. RESULTS: Hepatic mRNA levels of the respective CBs were increased in wild-type animals and in CB(1)(-/-) mice after ethanol intake. Ethanol intake in CB(2)(-/-) mice induced much higher steatosis (SREBP-1c mediated) and inflammation (B-cell predominant infiltrates) compared with wild-type animals and CB(1)(-/-) mice. HSC activation and collagen production were increased in all groups after forced ethanol intake, being most pronounced in CB(2)(-/-) mice and least pronounced in CB(1)(-/-) mice. DISCUSSION: The fact that CB(2) receptor knockout mice exhibited the most pronounced liver damage after ethanol challenge indicates a protective role of CB(2) receptor expression in chronic ethanol intake. By contrast, in CB(1) knockouts, the effect of ethanol was attenuated, suggesting aggravation of fibrogenesis and SREBP-1c-mediated steatosis via CB(1) receptor expression after ethanol intake.

Hepatic Macrophage as a Key Player in Fatty Liver Disease.

Fatty liver disease, characterized by excessive inflammation and lipid deposition, is becoming one of the most prevalent liver metabolic diseases worldwide owing to the increasing global incidence of obesity. However, the underlying mechanisms of fatty liver disease are poorly understood. Accumulating evidence suggests that hepatic macrophages, specifically Kupffer cells (KCs), act as key players in the progression of fatty liver disease. Thus, it is essential to examine the current evidence of the roles of hepatic macrophages (both KCs and monocyte-derived macrophages). In this review, we primarily address the heterogeneities and multiple patterns of hepatic macrophages participating in the pathogenesis of fatty liver disease, including Toll-like receptors (TLRs), NLRP3 inflammasome, lipotoxicity, glucotoxicity, metabolic reprogramming, interaction with surrounding cells in the liver, and iron poisoning. A better understanding of the diverse roles of hepatic macrophages in the development of fatty liver disease may provide a more specific and promising macrophage-targeting therapeutic strategy for inflammatory liver diseases.

Harmful effect of adipose tissue on liver lesions in patients with alcoholic liver disease.

BACKGROUND & AIMS: Adipose tissue is an important source of cytokines. Excess weight is an independent risk factor for steatosis, acute alcoholic hepatitis (AAH), and cirrhosis in patients with alcoholic liver disease (ALD). In this study, we investigated the role of adipose tissue in human ALD. PATIENTS AND METHODS: Fifty patients with ALD underwent liver and abdominal subcutaneous adipose tissue biopsies and supplied blood samples for the investigation of cytokine gene expression and secretion, as well as liver histology. RESULTS: The levels of TNF-alpha and IL-10 in adipose tissue were higher in patients with AAH. IL-10 level in adipose tissue was also correlated with fibrosis score. TNF-alpha gene expression in adipose tissue was correlated with Maddrey score, blood C-reactive protein (CRP) concentration and liver IL-6 concentration. IL-6 production levels in the liver were higher in patients with AAH and correlated with AAH score, liver histological lesions, liver TNF-alpha concentration, Maddrey score, and blood CRP concentration. Plasma concentrations of soluble forms of TNF-receptor were correlated with inflammatory lesions in the liver, Maddrey score and fibrosis score. CONCLUSION: In patients with ALD, inflammation occurs not only in the liver, but also in the adipose tissue. Adipose tissue inflammation is correlated with the severity of pathological features in the liver. Our findings may account for the harmful interactions between body mass index, AAH, fibrosis, and cirrhosis in alcoholic patients.

Allicin Modifies the Composition and Function of the Gut Microbiota in Alcoholic Hepatic Steatosis Mice.

The intestinal microbiome plays an important role in the pathogenesis of liver diseases. Alcohol intake induces gut microbiota dysbiosis and alters its function. This study investigated the antibiotic effect of allicin in mice with hepatic steatosis. Male C57BL/6 mice were administered an ethanol diet supplemented with allicin (5 and 20 mg/(kg bw day)) for 4 weeks. Allicin modified the gut microbiota composition. Cecal microbiota exhibited a positive correlation with alcohol and hepatic triacylglycerol, but were suppressed with allicin. Ethanol diet with 5 mg of allicin induced a lower intestinal permeability compared to the ethanol diet alone. Allicin mediated the lipopolysaccharide (LPS)-CD14-toll-like receptor 4 (TLR4)-induced hepatic inflammation pathway by reducing LPS, CD14, TLR4, and pro-inflammatory cytokines-tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6. However, hepatic inflammation primarily resulted from alcohol toxicity rather than LPS production in the gut. The prediction of functional profiles from metagenomic 16S ribosomal RNA (rRNA) data revealed different functional profiles in each group. The predicted aldehyde dehydrogenase tended to increase in alcoholic mice administered allicin. The predicted LPS-related pathway and LPS biosynthesis protein results exhibited a similar trend as plasma LPS levels. Thus, alcohol and allicin intake shapes the gut microbiota and its functional profile and improves the CD14-TLR4 pathway to alleviate inflammation in the liver.

Alcohol-induced modulation of signaling pathways in liver parenchymal and nonparenchymal cells: implications for immunity.

Alcoholic liver injury involves a complex array of derangements in cellular signaling of hepatic parenchymal and nonparenchymal cells as well as cells of the immune system. In the hepatocyte, chronic ethanol abuse leads to lipid accumulation and liver steatosis. Multiple pathways are affected to promote lipid accumulation in the ethanol-exposed hepatocyte. Chronic ethanol renders Kupffer cells hyperresponsive to endotoxin, which results in production of inflammatory cytokines and the tumor necrosis factor-alpha via a toll-like receptor 4 dependent pathway, leading to inflammation and hepatic necrosis. Dysfunction of the innate and adaptive immune responses caused by ethanol contributes to impaired antiviral response, inflammatory injury, and autoimmune activation. Recent developments in the literature are reviewed in a model that suggests lipid accumulation, dysregulation of immunity, and impaired antiviral and autoimmune responses as three distinct, though interwoven, pathophysiological mechanisms of alcoholic liver injury.

sTRAIL levels and TRAIL gene polymorphisms in Chinese patients with fatty liver disease.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, and has been identified as a novel mediator of fatty liver disease (FLD). The aim of our study was to further investigate the relationship between TRAIL and FLD. We found that soluble TRAIL (sTRAIL) concentrations in non-alcoholic FLD (NAFLD) patients were significantly higher than those of controls, and that sTRAIL levels positively correlated with triglyceride concentrations in NAFLD patients. Our results also indicated that the AA/TT genotypes of TRAIL at 1525/1595 engendered a lower risk of FLD attack and a less severe form of steatosis for NAFLD patients in Chinese population. This study provides a means to test for susceptibility to FLD and may assist in the diagnosis of FLD. In addition, we found that 1525G/A and 1595C/T sites were in complete linkage disequilibrium in Chinese population. This might indicate a haplotype with high genetic frequency of TRAIL.

The influence of estrogen on hepatobiliary osteopontin (SPP1) expression in a female rodent model of alcoholic steatohepatitis.

Our recent studies suggest that higher neutrophil infiltration in females correlates with increased hepatobiliary expression of osteopontin (OPN) in alcoholic steatohepatitis (ASH). The objective of this study was to understand the role of alcohol in altering estrogen levels in females by examining the effect of ethanol (EtOH) on the estrous cycle and then investigate the potential relationship between estradiol (E2) and hepatobiliary OPN expression in a female rat ASH model. Ovariectomized (OVX) and E2-implanted OVX rats in the ASH group were evaluated for OPN mRNA and protein expression. Low doses of E2 resulted in significant down-regulation of OPN protein and mRNA as compared to the OVX group. However, with increasing doses of E2, there was up-regulation of both OPN mRNA and protein. Osteopontin was localized primarily to the biliary epithelium. Liver injury assessed by serum ALT and histopathology revealed a pattern similar to OPN expression. In all groups, hepatic neutrophilic infiltration correlated positively with OPN expression. Based on these data, we conclude that in our ASH model, low doses of E2 appear to be hepatoprotective, whereas the protective effect appears to diminish with increasing doses of E2, although additional cause and effect studies are needed for confirmation.

Inhibition of P2X7R-NLRP3 Inflammasome Activation by Pleurotus citrinopileatus: A Possible Protective Role in Alcoholic Hepatosteatosis.

Pleurotus citrinopileatus (golden oyster mushroom) is a widely used edible mushroom. We investigated the inhibitory effect of P. citrinopileatus aqueous extract against alcoholic steatohepatitis and its underlying mechanism. Acute and chronic ethanol-feeding murine models were established by intragastrically administering ethanol or feeding an ethanol-containing Lieber-DeCarli liquid diet to male C57BL/6 mice. In both models, P. citrinopileatus decreased serum alanine aminotransferase (ALT), aspartate transaminase (AST), triglyceride (TG), and hepatic TG levels. Hematoxylin and eosin (HE) and Oil Red O staining confirmed that P. citrinopileatus ameliorated both acute and chronic alcoholic hepatosteatosis, characterized by regulation of lipid-metabolism-related proteins, including sirtuin 1 (SIRT1), AMP-activated kinase (AMPK), and sterol regulatory element-binding protein (SREBP1). P. citrinopileatus reversed inflammatory response via modulating purinergic receptor P2X ligand-gated ion channel 7 (P2X7R)-NOD-like receptor pyrin domain 3 (NLRP3) inflammasome. P. citrinopileatus restored the expressions of those proteins to a normal level. In addition, HepG2 cells were incubated with P. citrinopileatus prior to ethanol stimulation. P. citrinopileatus reduced ethanol exposure-induced lipid deposition. Concomitantly, P. citrinopileatus increased AMPK and SIRT1 expressions, which were reduced by ethanol treatment. P. citrinopileatus ameliorated alcoholic hepatic steatosis and accompanied inflammatory response via regulating SIRT1-AMPK and P2X7R-NLRP3 inflammasome activation, highlighting a promising strategy and utility of P. citrinopileatus for alcoholic steatohepatitis as dietary health supplements.

Immunohistochemical demonstration of acetaldehyde-modified epitopes in human liver after alcohol consumption.

Acetaldehyde, the toxic product of ethanol metabolism in the liver, covalently binds to a variety of proteins. Recent studies indicate that such binding can stimulate the production of antibodies against the acetaldehyde adducts. We raised rabbit antibodies which recognized various protein-acetaldehyde conjugates but not the corresponding control proteins. Such antibodies were used in immunohistochemical studies to find out whether acetaldehyde-generated epitopes can be detected from liver specimens of 13 human subjects with different degrees of alcohol consumption. While the specimens obtained from alcohol abusers (n = 4) and alcoholics (n = 3) exhibited marked positive staining for acetaldehyde adducts inside the hepatocytes in a granular uneven pattern, the control samples (n = 6) were almost devoid of immunoreactivity. In the alcohol abusers with an early stage of alcohol-induced liver damage, staining was detected exclusively around the central veins. The data indicate that intracellular acetaldehyde adducts occur in the centrilobular region of the liver of individuals consuming excessive amounts of alcohol. Immunohistochemical detection of such adducts may prove to be of value in the early identification of alcohol abuse and in elucidating the mechanisms of alcohol-induced organ damage.

Effect of the inducer of interleukin-6 (ME3738) on rat liver treated with ethanol.

BACKGROUND: Recently, ME3738, a derivative of soyasapogenol B, was developed as an inducer of interleukin (IL)-6. It has been demonstrated that ME3738 is stimulate to produce IL-6 and that it protects against concanavalin A-induced liver failure. It has also been reported that IL-6 prevents alcoholic fatty liver in mice. These results suggest that ME3738 may prevent alcoholic liver injury. In the present study, we investigated whether ME3738 prevents fatty liver in ethanol-fed rats. METHODS: Twenty-four male rats were fed with liquid diets containing ethanol or carbohydrates for 8 weeks. Liquid diets were prepared with or without ME3738 (0.8 mg/mL). Liver sections were stained for histology and IL-6 expression. Fatty changes of liver were classified into 4 grades: 0, 1+, 2+, and 3+. Plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), triglyceride, total cholesterol, and IL-6 were measured, as was hepatic ATP content. RESULTS: The extent of fatty degeneration in ethanol-fed rats was significantly greater (p=0.023) than that in controls. Fatty changes in rats fed ethanol containing ME3738 decreased, but were not significantly different from those in rats fed ethanol. Immunohistochemical staining of IL-6 was observed in perivenular hepatocytes of all rats, with its intensity becoming stronger in the order of controls, controls containing ME3738, ethanol, and ethanol-containing ME3738-fed rats. Plasma levels of AST and ALT in rats fed ethanol were significantly higher than those in controls. In rats fed ethanol-containing ME3738, these levels decreased to those of control-fed rats, but were not significantly different from those in rats fed ethanol. Plasma IL-6 was not detected in any rats. Hepatic ATP content in rats fed ethanol was significantly (p<0.05) lower than that in control-fed rats; however, in rats fed ethanol-containing ME3738, it increased to that in control-fed rats. CONCLUSIONS: Oral administration of ME3738, inducer of IL-6 may prevent the development of fatty liver caused by chronic ethanol consumption.