Intestinal barrier dysfunction and dysbiosis contribute to development of diseases in liver and other organs. Physical, immunologic, and microbiologic (bacterial, fungal, archaeal, viral, and protozoal) features of the intestine separate its nearly 100 trillion microbes from the rest of the human body. Failure of any aspect of this barrier can result in translocation of microbes into the blood and sustained inflammatory response that promote liver injury, fibrosis, cirrhosis, and oncogenic transformation. Alterations in intestinal microbial populations or their functions can also affect health. We review the mechanisms that regulate intestinal permeability and how changes in the intestinal microbiome contribute to development of acute and chronic liver diseases. We discuss individual components of the intestinal barrier and how these are disrupted during development of different liver diseases. Learning more about these processes will increase our understanding of the interactions among the liver, intestine, and its flora.

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Abbreviations: CEB, colonic epithelial barrier; MetS, Metabolic Syndrome; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis. AbstractThere is compelling evidence implicating intestinal permeability in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain poorly understood. Here we examined the role of bile acids (BA) in western diet (WD)-induced loss of colonic epithelial barrier (CEB) function in mice with a genetic impairment in intestinal epithelial barrier function, junctional adhesion molecule A knockout mice, F11r −/− . WD-fed knockout mice developed severe NASH, which was associated with increased BA concentration in the cecum and loss of CEB function. Analysis of cecal BA composition revealed selective increases in primary unconjugated BAs in the WD-fed mice, which correlated with increased abundance of microbial taxa linked to BA metabolism. In vitro permeability assays revealed that chenodeoxycholic acid (CDCA), which was elevated in the cecum of WD-fed mice, increased paracellular permeability, while the BA-binding resin sevelamer hydrochloride protected against CDCA-induced loss of barrier function. Sequestration of intestinal BAs by in vivo delivery of sevelamer to WD-fed knockout mice attenuated colonic mucosal inflammation and improved CEB. Sevelamer also reduced hepatic inflammation and fibrosis, and improved metabolic derangements associated with NASH. Collectively, these findings highlight a hitherto unappreciated role for BAs in WD-induced impairment of the intestinal epithelial barrier in NASH. K E Y W O R D S bile acids, intestinal permeability, microbiome, NAFLD, NASH, tight junction 7090 | GUPTA eT Al. | MATERIALS AND METHODS | MiceJunctional adhesion molecule A (JAM-A) knock out mice (F11r −/− ) were generated as previously described. 9 Mice were bred and maintained at Emory University and the University of Pittsburgh Divisions of Animal Resources. All animal studies were approved by the Institutional Animal Care and Use Committees. | BA feeding experimentC57BL/6 mice were fed CDCA (3mg/gm body weight, Millipore Sigma, St. Louis, MO) mixed with FITC-conjugated dextran (4 kDa) (0.6 mg/gm body weight, Millipore Sigma, St. Louis, MO) solution by oral gavage following a 6 hours fast. After 3 hours, blood was collected and fluorescence intensity was measured using Fluorescence Spectrophotometer (Synergy 2, BioTek, Winooski, VT) as described previously. 9

Adiponectin inhibits hepatic stellate cell (HSC) activation and subsequent development of liver fibrosis via multiple mechanisms. Phosphatase and tensin homolog deletion 10 (PTEN) plays a crucial role in suppression of HSC activation, but its regulation by adiponectin is not fully understood. Here, we investigated the effect of adiponectin on PTEN in LX-2 cells, a human cell line and examined the underlying molecular mechanisms involved in adiponectin-mediated upregulation of PTEN activity during fibrosis. PTEN expression was found to be significantly reduced in the livers of mice treated with CCl4, whereas its expression was rescued by adiponectin treatment. The DNA methylation proteins DNMT1, DNMT3A, and DNMT3B are all highly expressed in activated primary HSCs compared to quiescent HSCs, and thus represent additional regulatory targets during liver fibrogenesis. Expression of DNMT proteins was significantly induced in the presence of fibrotic stimuli; however, only DNMT3B expression was reduced in the presence of adiponectin. Adiponectin-induced suppression of DNMT3B was found to be mediated by enhanced miR-29b expression. Furthermore, PTEN expression was significantly increased by overexpression of miR-29b, whereas its expression was markedly reduced by a miR-29b inhibitor in LX-2 cells. These findings suggest that adiponectin-induced upregulation of miR-29b can suppress DNMT3B transcription in LX-2 cells, thus resulting in reduced methylation of PTEN CpG islands and ultimately suppressing the PI3K/AKT pathway. Together, these data suggest a possible new explanation for the inhibitory effect of adiponectin on HSC activation and liver fibrogenesis.

Acetaminophen (APAP)‐induced liver injury is the most common cause of acute liver failure (ALF) in the Western world. APAP toxicity progresses to multiorgan dysfunction and thus has broader whole‐body implications. Importantly, greater 30‐day mortality has been observed in liver transplant recipients following ALF due to APAP‐related versus non‐APAP‐related causes. Reasons for this discrepancy have yet to be determined. Extrahepatic toxicities of APAP overdose may represent underappreciated and unaddressed comorbidities within this patient population. In the present study, rapid induction of apoptosis following APAP overdose was observed in the intestine, an organ that greatly influences the physiology of the liver. Strikingly, apoptotic cells appeared to be strictly restricted to the intestinal crypts. The use of leucine‐rich repeat‐containing G protein–coupled receptor 5 (LGR5) reporter mice confirmed that the LGR5‐positive (+) crypt base stem cells were disproportionately affected by APAP‐induced cell death. Although the apoptotic cells were cleared within 24 hours after APAP treatment, potentially long‐lived consequences on the intestine due to APAP exposure were indicated by prolonged deficits in gut barrier function. Moreover, small intestinal cell death was found to be independent of tumor necrosis factor receptor signaling and may represent a direct toxic insult to the intestine by exposure to high concentrations of APAP. Conclusion: APAP induces intestinal injury through a regulated process of apoptotic cell death that disproportionately affects LGR5+ stem cells. This work advances our understanding of the consequences of APAP toxicity in a novel organ that was not previously considered as a significant site of injury and thus presents potential new considerations for patient management.

Our data establish relationships between resistin levels in the plasma and sputum of CF patients that correlate with disease status, and identify resistin as a novel mechanistic link between neutrophilic inflammation and lung disease in CF.

Alcohol consumption promotes loss of intestinal barrier function. However, mechanisms by which ethanol affects the tight junction (TJ), the cellular structure responsible for maintaining the gut epithelial barrier, are not well understood. Three classes of transmembrane proteins comprise TJs: occludin, claudins, and junctional adhesion molecules (JAMs). It has recently been postulated that JAM‐A (F11R), the most abundant JAM expressed in intestinal epithelium, regulates “leak” pathway flux, a paracellular route for the nonselective permeation of large solutes. Since transluminal flux of many gut‐derived antigens occurs through this pathway, we investigated the role of JAM‐A in ethanol‐induced disruption of the intestinal epithelial barrier. Using Caco‐2 and SK‐CO15 monolayers, we found that ethanol induced a dose‐ and time‐dependent decrease in JAM‐A protein expression to about 70% of baseline levels. Alcohol also reduced Ras‐related protein 2 (Rap2) activity, and enhanced myosin light chain kinase (MLCK) activity, changes consistent with impaired JAM‐A signaling. Stable overexpression and shRNA‐mediated knockdown of JAM‐A were employed to investigate the role of JAM‐A in paracellular‐mediated flux following alcohol exposure. The paracellular flux of 40‐kDa fluorescein isothiocynate (FITC)‐dextran following ethanol treatment was decreased by the overexpression of JAM‐A; conversely, flux was enhanced by JAM‐A knockdown. Thus, we conclude that ethanol‐mediated control of JAM‐A expression and function contributes to mechanisms by which this chemical induces intestinal epithelial leakiness.

Resistin, a protein initially cloned from mouse adipocytes, is a strong antagonist of insulin signaling. In humans, it is located in both the azurophil and specific granules of PMNs and as a result can be elevated in metabolic and PMN-driven inflammatory disorders. Cystic fibrosis (CF) combines PMN-dominated airway inflammation with profound metabolic anomalies, but the presence of resistin in CF patients has not been studied thus far. Methods: We measured resistin levels using an ELISA on platelet-free plasma and airway fluid from CF patients and measured the presence of neutrophil extracellular traps (NETs) a marker of neutrophil activation in CF airway fluid. Results: Resistin levels in platelet-free plasma were significantly higher in CF subjects than in HC subjects. Resistin levels in expectorated CF sputum were 2 to 3 orders (100-500 fold) of magnitude higher than in plasma. Interestingly, we found a strong negative correlation between sputum resistin and lung function (Spearman Rho=-0.81, P<10-4). Similarly resistin was also shown to positively correlate with NETs in CF sputum. Conclusions: Resistin levels are abnormal in CF plasma and strikingly elevated in CF airway fluid during chronic disease. These results show, as also suggested by prior studies, that extracellular resistin is associated with PMN-driven CF airway inflammation, including NET release. Mechanistic studies looking at the precise impact of resistin on metabolism and inflammation in CF are warranted.