Loss of hepatocyte identity following aberrant YAP activation: A key mechanism in alcoholic hepatitis.

BACKGROUND & AIMS: Alcoholic hepatitis (AH) is a life-threatening disease with limited therapeutic options, as the molecular mechanisms leading to death are not well understood. This study evaluates the Hippo/Yes-associated protein (YAP) pathway which has been shown to play a role in liver regeneration. METHOD: The Hippo/YAP pathway was dissected in explants of patients transplanted for AH or alcohol-related cirrhosis and in control livers, using RNA-seq, real-time PCR, western blot, immunohistochemistry and transcriptome analysis after laser microdissection. We transfected primary human hepatocytes with constitutively active YAP (YAPS127A) and treated HepaRG cells and primary hepatocytes isolated from AH livers with a YAP inhibitor. We also used mouse models of ethanol exposure (Lieber de Carli) and liver regeneration (carbon tetrachloride) after hepatocyte transduction of YAPS127A. RESULTS: In AH samples, RNA-seq analysis and immunohistochemistry of total liver and microdissected hepatocytes revealed marked downregulation of the Hippo pathway, demonstrated by lower levels of active MST1 kinase and abnormal activation of YAP in hepatocytes. Overactivation of YAP in hepatocytes in vitro and in vivo led to biliary differentiation and loss of key biological functions such as regeneration capacity. Conversely, a YAP inhibitor restored the mature hepatocyte phenotype in abnormal hepatocytes taken from patients with AH. In ethanol-fed mice, YAP activation using YAPS127A resulted in a loss of hepatocyte differentiation. Hepatocyte proliferation was hampered by YAPS127A after carbon tetrachloride intoxication. CONCLUSION: Aberrant activation of YAP plays an important role in hepatocyte transdifferentiation in AH, through a loss of hepatocyte identity and impaired regeneration. Thus, targeting YAP is a promising strategy for the treatment of patients with AH. LAY SUMMARY: Alcoholic hepatitis is characterized by inflammation and a life-threatening alteration of liver regeneration, although the mechanisms behind this have not been identified. Herein, we show that liver samples from patients with alcoholic hepatitis are characterized by profound deregulation of the Hippo/YAP pathway with uncontrolled activation of YAP in hepatocytes. We used human cell and mouse models to show that inhibition of YAP reverts this hepatocyte defect and could be a novel therapeutic strategy for alcoholic hepatitis.

Severe SARS-CoV-2 patients develop a higher specific T-cell response.

OBJECTIVES: Assessment of the adaptive immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for studying long-term immunity and vaccine strategies. We quantified IFNγ-secreting T cells reactive against the main viral SARS-CoV-2 antigens using a standardised enzyme-linked immunospot assay (ELISpot). METHODS: Overlapping peptide pools built from the sequences of M, N and S viral proteins and a mix (MNS) were used as antigens. Using IFNγ T-CoV-Spot assay, we assessed T-cell and antibody responses in mild, moderate and severe SARS-CoV-2 patients and in control samples collected before the outbreak. RESULTS: Specific T cells were assessed in 60 consecutive patients (mild, n = 26; moderate, n = 10; and severe patients, n = 24) during their follow-up (median time from symptom onset [interquartile range]: 36 days [28;53]). T cells against M, N and S peptide pools were detected in n = 60 (100%), n = 56 (93.3%), n = 55 patients (91.7%), respectively. Using the MNS mix, IFNγ T-CoV-Spot assay showed a specificity of 96.7% (95% CI, 88.5-99.6%) and a specificity of 90.3% (75.2-98.0%). The frequency of reactive T cells observed with M, S and MNS mix pools correlated with severity and with levels of anti-S1 and anti-RBD serum antibodies. CONCLUSION: IFNγ T-CoV-Spot assay is a reliable method to explore specific T cells in large cohorts of patients. This test may become a useful tool to assess the long-lived memory T-cell response after vaccination. Our study demonstrates that SARS-CoV-2 patients developing a severe disease achieve a higher adaptive immune response.

IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis.

BACKGROUND & AIMS: Severe alcoholic hepatitis (SAH) is associated with a high risk of infection. The IL-33/ST2 pathway is involved in sepsis control but data regarding its role in alcohol-related liver disease (ALD) are lacking. We aimed to characterize the role of IL-33/ST2 in the polymorphonuclear neutrophils (PMNs) of patients with ALD and SAH. METHODS: Serum and circulating neutrophils were collected from patients with SAH, alcoholic cirrhosis and healthy controls. We quantified IL-33/ST2 pathway activity and CXCR2 at baseline and after exposure to IL-33. We also determined the migration capacity of PMNs. RESULTS: The decoy receptor of IL-33 (soluble ST2 [sST2]) was increased in SAH vs. cirrhosis and controls, demonstrating the defect in this pathway during ALD. The sST2 level was associated with response to treatment, 2-month survival, infection-free survival and probability of infection in SAH. Endotoxemia was weakly correlated with sST2. GRK2, a negative regulator of CXCR2, was overexpressed in PMNs of patients with SAH and cirrhosis and was decreased by IL-33. CXCR2 levels on PMNs were lower in SAH vs. cirrhosis and controls. Treatment with IL-33 partially restored CXCR2 expression in SAH and cirrhosis. PMN migration upon IL-8 was lower in patients with SAH and cirrhosis vs. controls. Treatment with IL-33 partially restored migration in those with SAH and cirrhosis. Interestingly, the migration capacity of PMNs and the response to IL-33 were enhanced in responders to corticosteroids (Lille <0.45) compared to non-responders. CONCLUSION: The IL33/ST2 pathway is defective in SAH and predicts outcome. This defect is associated with decreased CXCR2 expression on the surface of PMNs and lower migration capacity, which can be corrected by IL-33, especially in patients responding to steroids. These results suggest that IL-33 has therapeutic potential for SAH and its infectious complications. LAY SUMMARY: The neutrophils of patients with severe alcoholic hepatitis are associated with a defect in the IL-33/ST2 pathway. This defect is associated with lower migration capacities in neutrophils and a higher probability of getting infected. Administration of IL-33 to the neutrophils at least partly restores this defect and may be effective at reducing the risk of infection in patients with severe alcoholic hepatitis.

Nucleotide-binding oligomerization domain 1 (NOD1) modulates liver ischemia reperfusion through the expression adhesion molecules.

BACKGROUND & AIMS: In liver transplantation, organ shortage leads to the use of marginal grafts that are more susceptible to ischemia-reperfusion (IR) injury. We identified nucleotide-binding oligomerization domain 1 (NOD1) as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in IR. Herein, we aimed to elucidate the role of NOD1 in IR injury, particularly focusing on its effects on the endothelium and hepatocytes. METHOD: Nod1 WT and KO mice were treated with NOD1 agonists and subjected to liver IR. Expression of adhesion molecules was analyzed in total liver, isolated hepatocytes and endothelial cells. Interactions between PMNs and hepatocytes were studied in an ex vivo co-culture model using electron microscopy and lactate dehydrogenase levels. We generated NOD1 antagonist-loaded nanoparticles (np ALINO). RESULTS: NOD1 agonist treatment increased liver injury, PMN tissue infiltration and upregulated ICAM-1 and VCAM-1 expression 20 hours after reperfusion. NOD1 agonist treatment without IR increased expression of adhesion molecules (ICAM-1, VCAM-1) in total liver and more particularly in WT hepatocytes, but not in Nod1 KO hepatocytes. This induction is dependent of p38 and ERK signaling pathways. Compared to untreated hepatocytes, a NOD1 agonist markedly increased hepatocyte lysis in co-culture with PMNs as shown by the increase of lactate dehydrogenase in supernatants. Interaction between hepatocytes and PMNs was confirmed by electron microscopy. In a mouse model of liver IR, treatment with np ALINO significantly reduced the area of necrosis, aminotransferase levels and ICAM-1 expression. CONCLUSION: NOD1 regulates liver IR injury through induction of adhesion molecules and modulation of hepatocyte-PMN interactions. NOD1 antagonist-loaded nanoparticles reduced liver IR injury and provide a potential approach to prevent IR, especially in the context of liver transplantation. LAY SUMMARY: Nucleotide-binding oligomerization domain 1 (NOD1) is as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in ischemia-reperfusion. Here, we show that the NOD1 pathway targets liver adhesion molecule expression on the endothelium and on hepatocytes through p38 and ERK signaling pathways. The early increase of adhesion molecule expression after reperfusion emphasizes the importance of adhesion molecules in liver injury. In this study we generated nanoparticles loaded with NOD1 antagonist. These nanoparticles reduced liver necrosis by reducing PMN liver infiltration and adhesion molecule expression.

Progenitor cell expansion and impaired hepatocyte regeneration in explanted livers from alcoholic hepatitis.

OBJECTIVE: In alcoholic hepatitis (AH), development of targeted therapies is crucial and requires improved knowledge of cellular and molecular drivers in liver dysfunction. The unique opportunity of using explanted livers from patients with AH having undergone salvage liver transplantation allowed to perform more in-depth molecular translational studies. DESIGN: We studied liver explants from patients with AH submitted to salvage transplantation (n=16), from patients with alcoholic cirrhosis without AH (n=12) and fragments of normal livers (n=16). Hepatic cytokine content was quantified. Hepatocyte function and proliferation and the presence of hepatic progenitor cells (HPCs) were evaluated by immunohistochemistry, western blot or quantitative PCR. Mitochondrial morphology was evaluated by electron microscopy. RESULTS: Livers from patients with AH showed decreased cytokine levels involved in liver regeneration (tumour necrosis factor α and interleukin-6), as well as a virtual absence of markers of hepatocyte proliferation compared with alcoholic cirrhosis and normal livers. Electron microscopy revealed obvious mitochondrial abnormalities in AH hepatocytes. Importantly, livers from patients with AH showed substantial accumulation of HPCs that, unexpectedly, differentiate only into biliary cells. AH livers predominantly express laminin (extracellular matrix protein favouring cholangiocyte differentiation); consequently, HPC expansion is inefficient at yielding mature hepatocytes. CONCLUSIONS: AH not responding to medical therapy is associated with lack of expression of cytokines involved in liver regeneration and profound mitochondrial damage along with lack of proliferative hepatocytes. Expansion of HPCs is inefficient to yield mature hepatocytes. Manoeuvres aimed at promoting differentiation of HPCs into mature hepatocytes should be tested in AH.

Glugacon-like peptide-2: broad receptor expression, limited therapeutic effect on intestinal inflammation and novel role in liver regeneration.

The glucagon-like peptide 2 (GLP-2) is an intestinotrophic hormone with growth promoting and anti-inflammatory actions. However, the full biological functions of GLP-2 and the localization of its receptor (GLP-2R) remain controversial. Among cell lines tested, the expression of GLP-2R transcript was detected in human colonic myofibroblasts (CCD-18Co) and in primary culture of rat enteric nervous system but not in intestinal epithelial cell lines, lymphocytes, monocytes, or endothelial cells. Surprisingly, GLP-2R was expressed in murine (GLUTag), but not human (NCI-H716) enteroendocrine cells. The screening of GLP-2R mRNA in mice organs revealed an increasing gradient of GLP-2R toward the distal gut. An unexpected expression was detected in the mesenteric fat, mesenteric lymph nodes, bladder, spleen, and liver, particularly in hepatocytes. In two mice models of trinitrobenzene sulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced colitis, the colonic expression of GLP-2R mRNA was decreased by 60% compared with control mice. Also, GLP-2R mRNA was significantly downregulated in intestinal tissues of inflammatory bowel disease patients. Therapeutically, GLP-2 showed a weak restorative effect on intestinal inflammation during TNBS-induced colitis as assessed by macroscopic score and inflammatory markers. Finally, GLP-2 treatment accelerated mouse liver regeneration following partial hepatectomy as assessed by histological and molecular analyses. In conclusion, the limited therapeutic effect of GLP-2 on colonic inflammation dampens its utility in the management of severe inflammatory intestinal disorders. However, the role of GLP-2 in liver regeneration is a novelty that might introduce GLP-2 into the management of liver diseases and emphasizes on the importance of elucidating other extraintestinal functions of GLP-2.

Colonic inflammation in mice is improved by cigarette smoke through iNKT cells recruitment.

Cigarette smoke (CS) protects against intestinal inflammation during ulcerative colitis. Immunoregulatory mechanisms sustaining this effect remain unknown. The aim of this study was to assess the effects of CS on experimental colitis and to characterize the intestinal inflammatory response at the cellular and molecular levels. Using the InExpose® System, a smoking device accurately reproducing human smoking habit, we pre-exposed C57BL/6 mice for 2 weeks to CS, and then we induced colitis by administration of dextran sodium sulfate (DSS). This system allowed us to demonstrate that CS exposure improved colonic inflammation (significant decrease in clinical score, body weight loss and weight/length colonic ratio). This improvement was associated with a significant decrease in colonic proinflammatory Th1/Th17 cytokine expression, as compared to unexposed mice (TNF (p=0.0169), IFNγ (p<0.0001), and IL-17 (p=0.0008)). Smoke exposure also induced an increased expression of IL-10 mRNA (p=0.0035) and a marked recruitment of iNKT (invariant Natural Killer T; CD45+ TCRß+ CD1d tetramer+) cells in the colon of DSS-untreated mice. Demonstration of the role of iNKT cells in CS-dependent colitis improvement was performed using two different strains of NKT cells deficient mice. Indeed, in Jα18KO and CD1dKO animals, CS exposure failed to induce significant regulation of DSS-induced colitis both at the clinical and molecular levels. Thus, our study demonstrates that iNKT cells are pivotal actors in the CS-dependent protection of the colon. These results highlight the role of intestinal iNKT lymphocytes and their responsiveness to environmental stimuli. Targeting iNKT cells would represent a new therapeutic way for inflammatory bowel diseases.

Modulation of intestinal inflammation by yeasts and cell wall extracts: strain dependence and unexpected anti-inflammatory role of glucan fractions.

Yeasts and their glycan components can have a beneficial or adverse effect on intestinal inflammation. Previous research has shown that the presence of Saccharomyces cerevisiae var. boulardii (Sb) reduces intestinal inflammation and colonization by Candida albicans. The aim of this study was to identify dietary yeasts, which have comparable effects to the anti-C. albicans and anti-inflammatory properties of Sb and to assess the capabilities of yeast cell wall components to modulate intestinal inflammation. Mice received a single oral challenge of C. albicans and were then given 1.5% dextran-sulphate-sodium (DSS) for 2 weeks followed by a 3-day restitution period. S. cerevisiae strains (Sb, Sc1 to Sc4), as well as mannoprotein (MP) and ß-glucan crude fractions prepared from Sc2 and highly purified ß-glucans prepared from C. albicans were used in this curative model, starting 3 days after C. albicans challenge. Mice were assessed for the clinical, histological and inflammatory responses related to DSS administration. Strain Sc1-1 gave the same level of protection against C. albicans as Sb when assessed by mortality, clinical scores, colonization levels, reduction of TNFα and increase in IL-10 transcription. When Sc1-1 was compared with the other S. cerevisiae strains, the preparation process had a strong influence on biological activity. Interestingly, some S. cerevisiae strains dramatically increased mortality and clinical scores. Strain Sc4 and MP fraction favoured C. albicans colonization and inflammation, whereas ß-glucan fraction was protective against both. Surprisingly, purified ß-glucans from C. albicans had the same protective effect. Thus, some yeasts appear to be strong modulators of intestinal inflammation. These effects are dependent on the strain, species, preparation process and cell wall fraction. It was striking that ß-glucan fractions or pure ß-glucans from C. albicans displayed the most potent anti-inflammatory effect in the DSS model.

Murine model of dextran sulfate sodium-induced colitis reveals Candida glabrata virulence and contribution of ß-mannosyltransferases.

Candida glabrata, like Candida albicans, is an opportunistic yeast pathogen that has adapted to colonize all segments of the human gastrointestinal tract and vagina. The C. albicans cell wall expresses ß-1,2-linked mannosides (ß-Mans), promoting its adherence to host cells and tissues. Because ß-Mans are also present in C. glabrata, their role in C. glabrata colonization and virulence was investigated in a murine model of dextran sulfate sodium (DSS)-induced colitis. Five clustered genes of C. glabrata encoding ß-mannosyltransferases, BMT2-BMT6, were deleted simultaneously. ß-Man expression was studied by Western blotting, flow cytometry, and NMR analysis. Mortality, clinical, histologic, and colonization scores were determined in mice receiving DSS and different C. glabrata strains. The results show that C. glabrata bmt2-6 strains had a significant reduction in ß-1,2-Man expression and a disappearance of ß-1,2-mannobiose in the acid-stable domain. A single gavage of C. glabrata wild-type strain in mice with DSS-induced colitis caused a loss of body weight, colonic inflammation, and mortality. Mice receiving C. glabrata bmt2-6 mutant strains had normal body weight and reduced colonic inflammation. Lower numbers of colonies of C. glabrata bmt2-6 were recovered from stools and different parts of the gastrointestinal tract. Histopathologic examination revealed that the wild-type strain had a greater ability to colonize tissue and cause tissue damage. These results showed that C. glabrata has a high pathogenic potential in DSS-induced colitis, where ß-Mans contribute to colonization and virulence.