M.J. Borrero

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.

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

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 (CCl4)‐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 (Th) 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 Th 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. (HEPATOLOGY 2010;52:2086‐2095)

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.

609 SMALL INTESTINE INFLAMMATION IN RATS WITH CCL4 CIRRHOSIS: ROLE OF ENTERIC BACTERIA

macrophages during conditions of excess iron by blocking the iron exporter ferroportin. In addition, IL-6 mediated upregulation of hepcidin during inflammation is the major mechanism of the socalled ‘anemia of chronic disease’. We here identify H2O2 which is co-released by inflammatory cells as potent transcriptional activator of hepcidin independent of upstream regulators IL-6 and BMP6. Material andmethods: H2O2 release (1–8mM) by immune cells was mimicked using purified enzymes glucose oxidase and catalase as recently described (GOX/CAT system). Huh7 hepatoma cells were treated with either IL-6 or BMP6 alone, or in combination with steady state H2O2 over 24h. Hepcidin regulation was assessed by quantitative real time PCR. Members of the intracellular signaling cascade such as STAT3 were assessed by Western blotting. Promotor studies were performed using hepcidin-promoter constructs with various deletions fused to luciferase as reporter gene with Renilla as control reporter gene. Results: Steady state non-toxic H2O2 concentrations comparable to those by inflammatory cells rapidly and drastically upregulate hepcidin in a dose-dependent way in Huh7 cells by a factor of 10. In addition, H2O2 further potentiates IL6 and BMP6 mediated upregulation of hepcidin 5-fold. Interestingly, induction was also not blunted by confounding hypoxia (2%). The H2O2mediated hepcidin response is an early response reaching the maximum mRNA-concentration already after 6h. Promoter studies identified the STAT3 element as major promoter region of H2O2 mediated hepcidin induction. Indeed, phosphorylation of STAT3 was confirmed under these conditions using Western blotting. Conclusions: Our studies establish H2O2 as as an important regulatory link between systemic iron regulation and oxidative stress. Impaired H2O2-hepcidin signaling in chronic liver diseases could contribute to hepatic accumulation of iron in cirrhotic patients.

528 THE DEFECTIVE FUNCTION OF GUT DENDRITIC CELLS OF RATS WITH CIRRHOSIS AND BACTERIAL TRANSLOCATION IS CORRECTED BY BOWEL DECONTAMINATION WITH ANTIBIOTICS

528 THE DEFECTIVE FUNCTION OF GUT DENDRITIC CELLS OF RATS WITH CIRRHOSIS AND BACTERIAL TRANSLOCATION IS CORRECTED BY BOWEL DECONTAMINATION WITH ANTIBIOTICS L. Munoz, M. Ubeda, M.J. Borrero, M. Lario, R. Frances, D. Diaz, J. Monserrat, L. Lledo, J. Such, M. Alvarez-Mon, A. Albillos. University of Alcala, Ciberehd, Alcala de Henares, Madrid, Hospital General Universitario de Alicante, Ciberhed, Alicante, Hospital Universitario Pŕincipe de Asturias, University of Alcala, Ciberehd, Alcala de Henares, Hospital Universitario Ramon y Cajal, University of Alcala, Ciberehd, Madrid, Spain E-mail: leticia.munoz@uah.es