Intestinal Immune Dysregulation Driven by Dysbiosis Promotes Barrier Disruption and Bacterial Translocation in Rats With Cirrhosis.

In cirrhosis, intestinal dysbiosis, intestinal barrier impairment, and systemic immune system abnormalities lead to gut bacterial translocation (GBT) and bacterial infection. However, intestinal immune system dysfunction and its contribution to barrier damage are poorly understood. This study correlates immune system dysregulation in the intestines of rats at different stages of CCl4 -induced cirrhosis with barrier function and pathogenic microbiota. The following variables were addressed in the small intestine: intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) activation status and cytokine production (flow cytometry), cytokine mRNA and protein expression (quantitative real-time PCR and immunofluorescence), microbiota composition of ileum content (16S recombinant DNA massive sequencing), permeability (fecal albumin loss), and epithelial junctions (immunohistochemistry and immunofluorescence). The intestinal mucosa in rats with cirrhosis showed a proinflammatory pattern of immune dysregulation in IELs and LPLs, which featured the expansion of activated lymphocytes, switch to a T helper 1 (Th1) regulatory pattern, and Th17 reduction. In rats with cirrhosis with ascites, this state was associated with epithelial junction protein disruption, fecal albumin loss, and GBT. Direct correlations (P < 0.01) were observed between elevated interferon gamma (IFNγ)-expressing T cytotoxic LPLs and fecal albumin and between inflammatory taxa abundance and IFNγ-producing immune cells in the ileum. Bowel decontamination led to redistributed microbiota composition, reduced proinflammatory activation of mucosal immune cells, normalized fecal albumin levels, and diminished GBT; but there were no modifications in Th17 depletion. Conclusion: The intestinal mucosa of rats with cirrhosis acquires a proinflammatory profile of immune dysregulation that parallels the severity of cirrhosis; this impaired intestinal immune response is driven by gut dysbiosis and leads to disrupted barrier function, promoting GBT.

Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats.

BACKGROUND & AIMS: In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. METHODS: Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. RESULTS: Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. CONCLUSIONS: In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis.

Defective thymopoiesis and poor peripheral homeostatic replenishment of T-helper cells cause T-cell lymphopenia in cirrhosis.

BACKGROUND & AIMS: Depletion of circulating CD4(+) T-helper (Th) lymphocytes, especially naive Th cells, is common in cirrhosis. Little is known about the pathogenetic mechanisms involved in Th-cell depletion in cirrhosis. We investigated the mechanisms involved in circulating Th-cell lymphopenia in cirrhosis. METHODS: Circulating naive and memory Th cells were analyzed by flow cytometry in 60 patients with cirrhosis and 40 sex- and age-matched healthy controls. Thymopoiesis, apoptosis, cell activation, and proliferation were assessed through CD31, annexin-V, HLA-DR and Ki-67 expression, respectively. Lipopolysaccharide (LPS)-binding protein (LBP) and spleen size were measured as indicators of bacterial translocation and splenic pooling, respectively. RESULTS: Compared to controls, patients showed reduced numbers of Th cells involving a greater depletion of the naive than memory Th-cell compartment (2.7- vs. 1.5-fold, respectively). Recent thymic emigrants were diminished (p < 0.01), and each patient had a lower number of CD31(+) naive Th cells than the matched-control. Spontaneous and induced apoptosis (Annexin-V(+)) of Th cells was increased in patients. Activated (HLA-DR(+)) and proliferating (Ki-67(+)) memory Th cells were increased in patients (p < 0.01), and they directly correlated with plasma LBP (p < 0.05) and negatively with naive Th cells (p < 0.01), respectively. Naive Th cells were inversely correlated (p < 0.01) with their frequencies of apoptosis and of activated memory Th cells, LBP, and spleen size. On multivariate analysis, defective thymic generation of naive Th cells, increased memory Th-cell activation, and splenomegaly were independently associated with Th-cell depletion. CONCLUSIONS: Th-cell immunodeficiency in cirrhosis is explained by a universal defect in thymopoiesis exacerbated by splenic pooling and activation-driven cell-death induced by bacterial translocation.

Critical role of the liver in the induction of systemic inflammation in rats with preascitic cirrhosis.

UNLABELLED: Systemic activation of the inflammatory immune system contributes to the progression of cirrhosis with ascites. Immune cells become activated after interacting at the mesenteric lymph nodes (MLNs) with bacteria translocated from the gut, and thereafter reach the bloodstream through recirculation. It is unknown whether systemic activation of the immune system is present in pre-ascitic cirrhosis, in which gut bacterial translocation has not been described. The purpose of this study was to determine whether systemic activation of the immune system initiates in rats with compensated carbon tetrachloride (CCl(4))-induced cirrhosis, and if so to establish the activation site of immune cells. We studied the activation status of immune cells in peripheral blood, MLNs, and hepatic lymph nodes (HLNs). Systemic inflammation was present in rats with cirrhosis, as shown by expansion (P < 0.01) of circulating total and inflammatory monocytes and recently activated CD134(+) T helper (T(h)) cells. The same populations of cells were increased (P < 0.01) in MLNs and HLNs. Bacterial translocation was absent in rats with cirrhosis or control rats, but bacterial DNA fragments were present in the MLNs of 54% of rats with cirrhosis. The liver was the source of activated immune cells present in the blood, as shown by the direct correlation between activated T(h) cells in the blood and HLNs, but not in MLNs, and the normalization by gut decontamination with antibiotics of activated cells in MLNs, but not in the blood or HLNs. CONCLUSION: In experimental cirrhosis, systemic activation of the immune system occurs before ascites development and is driven by recirculation of cells activated in HLNs. In addition, in compensated cirrhosis, bacterial DNA fragments reach the MLNs, where they elicit a local inflammatory response.