FOXO4 ameliorates alcohol-induced chronic liver injury via inhibiting NF-κB and modulating gut microbiota in C57BL/6J mice.

BACKGROUND: Intestinal mucosa barrier function and gut-liver axis are impaired by ethanol in chronic alcoholic liver disease (ALD). However, the possible mechanism is not clear. This study aimed to investigate the effects of Forkhead Box O4 (FOXO4) on alcohol-induced chronic liver injury and its molecular mechanism(s). METHODS: Male C57BL/6J mice were injected with or without FOXO4-WT, FOXO4-TB or NF-κB vectors, and fed with Lieber-DeCarli liquid diets containing 36% ethanol for eight weeks to induce chronic ALD. Thereafter, blood, liver, colon and fecal samples were collected. Biochemical parameters, endotoxin and inflammatory cytokines in the blood and antioxidant enzymes in the liver were tested by commercial kits. Histopathological changes in the liver were evaluated by HE staining. In addition, the mRNA and protein expression of FOXO4, NF-κB, ZO-1 and Occluding in the colon were measured by quantitative real-time PCR and Western blot, respectively. Furthermore, gut microbiota composition in the fecal samples was investigated with 16S rDNA sequencing. RESULTS: FOXO4 significantly ameliorated liver histopathological damage. Moreover, FOXO4 reduced the serum endotoxin, biochemical parameters (ALT, AST, ALP and TG), antioxidant enzymes (ROS and MDA), inflammatory cytokines (IL-6, IL-1ß, and TNF-α), but restored the levels of GSH, SOD and IL-10. Furthermore, FOXO4 significantly inhibited the expression of NF-κB, p-NF-κB p65, p-IKKα and p-IKKß, and up-regulated the expression of ZO-1 and Occludin. Additionally, FOXO4 modulated the gut microbiota composition and certain bacteria including Odoribacter, Parasutterella and Psychrobacter. CONCLUSION: These findings suggest that FOXO4 protects against alcohol-induced chronic liver injury via inhibiting NF-κB and modulating gut microbiota in C57BL/6J mice.

Binge Drinking's Effects on the Body.

Studies have focused on the effects of chronic alcohol consumption and the mechanisms of tissue injury underlying alcoholic hepatitis and cirrhosis, with less focus on the pathophysiological consequences of binge alcohol consumption. Alcohol binge drinking prevalence continues to rise, particularly among individuals ages 18 to 24. However, it is also frequent in individuals ages 65 and older. High blood alcohol levels achieved with this pattern of alcohol consumption are of particular concern, as alcohol can permeate to virtually all tissues in the body, resulting in significant alterations in organ function, which leads to multisystemic pathophysiological consequences. In addition to the pattern, amount, and frequency of alcohol consumption, additional factors, including the type of alcoholic beverage, may contribute differentially to the risk for alcohol-induced tissue injury. Preclinical and translational research strategies are needed to enhance our understanding of the effects of binge alcohol drinking, particularly for individuals with a history of chronic alcohol consumption. Identification of underlying pathophysiological processes responsible for tissue and organ injury can lead to development of preventive or therapeutic interventions to reduce the health care burden associated with binge alcohol drinking.

Susceptibility to Alcohol Hangovers: The Association with Self-Reported Immune Status.

Increasing evidence points at a role for the immune system in the genesis of the alcohol hangover. This study investigated the association between self-reported immune function and experiencing hangovers. Dutch students aged 18 to 30 years old were invited to complete an online survey. Eighteen items on immune-related complaints were completed to assess self-reported immune function. Alcohol consumption in the past month (with respect to usual consumption and the occasion of heaviest drinking) was also recorded. Subjects with an estimated blood alcohol concentration (eBAC) of 0.18% or higher on their heaviest drinking occasion in the prior month were included in the analyses. Self-reported immune function was compared between drinkers with a hangover and those who claimed to be hangover resistant. In total, of 481 subjects (79.2% women) with a mean (SD) age of 21.1 (1.9) years old were included in the analysis. Of these, 83.3% (n = 400) reported having hangovers and 16.8% (n = 81) claimed to be hangover resistant. Drinkers with hangovers had significantly higher self-reported overall immune function scores when compared to hangover-resistant drinkers (mean ± SD = 10.5 ± 3.6 versus 13.1 ± 4.9, p = 0.0001), indicating a poorer immune status. In conclusion, experiencing alcohol hangovers is associated with significantly poorer self-reported immune function.

IL-23 restoration of Th17 effector function is independent of IL-6 and TGF-ß in a mouse model of alcohol and burn injury.

T cells play a critical role in host defense against intestinal bacteria. We have shown that ethanol combined with burn injury suppresses Peyer's patch (PP) Th17 cytokines 1 d after injury. We assessed the mechanism of suppressed Th17 effector functions. Mice were gavaged with ethanol 4 h before burn injury and euthanized 1, 3, and 7 d after injury. Mesenteric lymph nodes (MLNs), PPs, and spleen Th1 and Th17 cytokines were assessed. A significant decrease in IL-17, IL-22, IL-2, and IFN-γ were observed in all 3 lymphoid organs 1 and 3 d after injury. We used splenic cells to study the role of IL-6, IL-23, TGF-ß, and aryl hydrocarbon receptor (AHR) in suppressing Th17 cytokines. We also assessed whether the AHR agonist 6-formylindolo (3, 2-b) carbazole (FICZ) modulates Th17 cytokines. We found a significant decrease in IL-6 and TGF-ß after ethanol and burn; IL-23 was undetectable. The reconstitution of IL-23 in culture medium increased IL-17 by 2-fold and IL-22 by 20-fold in cells from burn ethanol mice. The restoration of IL-6 and TGF-ß combined did not influence the release of Th17 cytokines. We observed that AHR was necessary for IL-23 restoration of IL-22 after ethanol and burn injury. The AHR agonist FICZ enhanced IL-22, but not IL-17. None of these treatments influenced the release of Th1 cytokines. Together, these results suggest that IL-23 plays a critical role in regulation of Th17 cytokines. Furthermore, IL-6 and TGF-ß do not appear to influence IL-23-mediated restoration of Th17 cytokines after ethanol and burn injury.

Gastroprotective effect of 2-mercaptoethane sulfonate against acute gastric mucosal damage induced by ethanol.

Gastric mucosal damage induced by ethanol is a serious medical problem. Recent evidences suggest that reactive oxygen species and inflammatory mediators play a key role in the destruction of gastric mucosa. The present study was aimed to evaluate the potential beneficial effect of MESNA (2-mercaptoethane sulfonate) against ethanol-induced gastric mucosal damage in mice. The animals were orally pretreated with vehicle or MESNA and then treated with acidified ethanol to induce gastric mucosal damage. One hour after ethanol ingestion mice were euthanized and stomach samples were collected for biochemical analysis. Macroscopic and histopathological evaluation of gastric mucosa showed that pretreatment with MESNA attenuated gastric lesions induced by ethanol. Administration of MESNA significantly increased glutathione content and superoxide dismutase and catalase activity in the gastric tissues. In addition, MESNA markedly reduced ethanol-induced lipid peroxidation, myeloperoxidase activity, tumor necrosis factor-alpha, interleukin (IL)-1ß, IL-6, and monocyte chemotactic protein-1 levels. These findings suggest that the thiol-containing compound MESNA is able to decrease alcohol-induced oxidative stress and inflammation in the gastric tissue. It seems that MESNA may have a protective effect against ethanol-induced gastric mucosal damage.

Peripheral blood dendritic cells in alcoholic and autoimmune liver disorders.

Little is known about effects of alcohol consumption on dendritic cell (DC) function and resultant immune response. However, quantitative and qualitative disturbances of DCs are speculated to be involved in alcohol-related as well as in other liver pathology. The present study aimed to evaluate changes in circulating DC subsets in alcoholic liver disease (N = 43), autoimmune hepatitis (N = 26) and primary biliary cirrhosis (N = 20). DCs isolated from the peripheral blood of recruited participants were stained with monoclonal antibodies against blood dendritic cell antigens (BDCAs) and estimated using the flow cytometry. Myeloid DCs were defined as BDCA-1(+)/CD19(-) cells, and lymphoid DCs as BDCA-2(+)/CD123(+) cells. Total numbers of circulating DCs in subjects with some liver diseases were markedly lower than in the healthy participants (p = 0.03). There was a significantly lower percentage of circulating BDCA-2(+)/CD123(+) (p = 0.02), and a tendency for the percentage of circulating BDCA-1(+)/CD19(-) cells to decrease in patients with liver diseases compared to the controls (p = 0.09). These results may suggest that decreased numbers of DCs may be responsible for reduced adaptive immune responses and increased susceptibility to infections and cancer development observed in patients exposed to alcohol. Moreover, numerical abnormalities of DCs may contribute to the breakdown of self-tolerance, a feature of autoimmune diseases.

Mesenchymal stem cells and inflammatory lung diseases.

Mesenchymal stem cells (MSCs) are emerging as a therapeutic modality in various inflammatory disease states. A number of ongoing randomized Phase I/II clinical trials are evaluating the effects of allogeneic MSC infusion in patients with multiple sclerosis, graft-versus-host disease, Crohn's disease, and severe chronic myocardial ischemia. MSCs are also being considered as a potential therapy in patients with inflammatory lung diseases. Several studies, including our own, have demonstrated compelling benefits from the administration of MSCs in animal models of lung injury. These studies are leading to growing interest in the therapeutic use of MSCs in inflammatory lung diseases. In this Review, we describe how the immunoregulatory effects of MSCs can confer substantial protection in the setting of lung diseases such as acute lung injury, chronic obstructive pulmonary disease, asthma, and pulmonary hypertension. We also address potential pitfalls related to the therapeutic use of MSCs in fibrotic lung diseases such as idiopathic pulmonary fibrosis. In addition, we identify emerging areas for MSC- based therapies in modulating oxidative stress and in attenuating inflammation in alcohol-related acute lung injury.

Chronic ethanol exposure affects in vivo migration of hepatic dendritic cells to secondary lymphoid tissue.

The mechanisms by which chronic ethanol (EtOH) consumption results in an immune-compromised state have not been fully elucidated. No studies to date have ascertained whether EtOH affects the migratory capacity of dendritic cells (DC), potent immune regulators. We hypothesized that EtOH exposure might affect hepatic and splenic DC trafficking to secondary lymphoid tissues and the resulting immune response. Hepatic DC from EtOH-treated animals migrated in greater numbers to draining lymphoid tissue than controls, whereas spleen DC were unaffected. Moreover, hepatic EtOH-exposed (E) DC induced more vigorous priming of allogeneic T cells in vivo compared with splenic EDC or controls. Altered hepatic EDC migration was independent of either CCR7 or CD11a expression, with no striking changes in surface expression of other adhesion molecules analyzed. The modified trafficking to secondary lymphoid tissue observed for hepatic EDC may play a role in the altered immune response to microbial pathogens in chronic alcohol users.

Ethanol affects the generation, cosignaling molecule expression, and function of plasmacytoid and myeloid dendritic cell subsets in vitro and in vivo.

The influence of ethanol (EtOH) on multiple dendritic cell (DC) subsets, in the steady state or following their mobilization in vivo, has not been characterized. Herein, generation of mouse bone marrow-derived DC (BMDC) in response to fms-like tyrosine kinase 3 ligand was inhibited by physiologically relevant concentrations of EtOH with selective suppression of plasmacytoid (p)DC. EtOH reduced surface expression of costimulatory molecules (CD40, CD80, CD86) but not that of coinhibitory CD274 (B7-H1) on resting or CpG-stimulated DC subsets. Interleukin (IL)-12p70 production by activated DC was impaired. Consistent with these findings, EtOH-exposed BMDC exhibited a reduced capacity to induce naïve, allogeneic T cell proliferation and impaired ability to prime T cells in vivo. DC subsets freshly isolated from EtOH-fed mice were also examined. Liver DC, inherently immature and resistant to maturation, exhibited little change in their low surface cosignaling molecule expression, whereas splenic DC showed reduced expression of surface costimulatory molecules in response to CpG stimulation in vivo. These splenic DC elicited reduced naïve, allogeneic T cell proliferation in vitro, and the stimulatory capacity of resting but not CpG-activated liver DC was reduced by chronic EtOH administration. T cells from animals primed with EtOH-exposed DC produced elevated levels of IL-10 following ex vivo challenge with donor alloantigen. Thus, EtOH impairs cytokine-driven differentiation and function of myeloid DC and pDC in vitro. Hepatic DC from chronic EtOH-fed mice are less affected than splenic DC, which exhibit impaired functional maturation following CpG stimulation. These results indicate a potential mechanism by which alcohol consumption is associated with immunosuppression.

A role for interleukin-10 in alcohol-induced liver sensitization to bacterial lipopolysaccharide.

BACKGROUND: Proinflammatory cytokines play an important role in alcohol-induced liver injury. The role of anti-inflammatory cytokines in the initiation and progression of alcoholic liver disease has received little attention. This study tested the hypothesis that an imbalance exists between pro- and anti-inflammatory cytokines in the liver during chronic exposure to alcohol. Alcohol exposure results in predominantly proinflammatory cytokine secretion and liver injury. METHODS: IL-10 knock-out and their C57BL/6J counterpart wild-type mice were fed alcohol in drinking water for 7 weeks. At the end of alcohol feeding, Gram-negative bacterial lipopolysaccharide (LPS) was administered, and the animals were killed after 3 and 8 hr. Liver histology, plasma alanine aminotransferase and aspartate aminotransferase activity, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-10 levels, and liver cytokine messenger RNA levels were measured. RESULTS: Alcohol feeding and LPS treatment did not change plasma enzyme activity levels in wild-type mice. In the IL-10 knock-out mice, LPS alone increased aspartate aminotransferase and alanine aminotransferase enzyme activity, and this was potentiated by alcohol. Alcohol induced liver steatosis in both wild-type and knock-out mice. LPS markedly enhanced the histological effects further, especially in the knock-out mice, with the emergence of focal necrosis, polymorphonuclear infiltration, and microabscesses in the liver. Plasma tumor necrosis factor-alpha and IL-1beta levels were not affected by alcohol alone. Proinflammatory cytokine levels were increased by LPS and further enhanced by alcohol treatment, particularly in the IL-10 knock-out mice. IL-10 plasma levels in the wild-type animals were down-regulated by alcohol. Changes in liver cytokine messenger RNA paralleled those seen in plasma cytokine levels. CONCLUSIONS: Alcohol-induced liver sensitization to LPS in wild-type mice may involve down-regulation of IL-10. This anti-inflammatory cytokine, known for its hepatoprotective effects, is secreted simultaneously with proinflammatory cytokines. IL-10 may also limit alcohol-induced liver damage by counteracting the effects of proinflammatory cytokines.