Leticia Muñoz

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.

Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis.

Cirrhosis with ascites is associated with a high rate of gut bacterial translocation (GBT) and spontaneous bacterial infections of enteric origin. We addressed the activation state and role of intestinal dendritic cells (DCs) in experimental ascitic cirrhosis and their relationship with GBT. Cirrhosis with ascites was CCl4 induced in rats. To examine their activation state and functions, DCs (CD103+RT1B+CD3−CD45RA−) were isolated from the intestinal lamina propria and mesenteric lymph nodes (MLNs), and the following parameters were determined by flow cytometry: surface antigen expression; spontaneous or lipopolysaccharide‐stimulated tumor necrosis factor alpha (TNF‐α) production; and in vitro capacity to phagocytose latex beads and to migrate toward the chemokine (C‐C motif) ligand 21. GBT was defined as the growth of bacteria in MLNs culture. Bacterial DNA (Bact‐DNA) in MLNs was identified by polymerase chain reaction. In rats with Bact‐DNA in MLNs without GBT, intestinal and MLNs CD103+‐DCs showed features of activation, expansion of the proinflammatory CD4+‐DC subpopulation, augmented TNF‐α production, and increased phagocytic and migratory capacities. In contrast, in rats with GBT, CD103+‐DCs showed the absence of an activated phenotype, lowered TNF‐α production, and relatively deficient phagocytosis and migration capacities. The CD103+‐DC of rats without Bact‐DNA in MLNs or GBT were similar to controls. In cirrhotic rats, bowel decontamination with antibiotics eliminated Bact‐DNA in MLNs and GBT, normalized the activation state and functions of CD103+‐DCs, and increased their TNF‐α production. Conclusion: In experimental cirrhosis with ascites, continuous pressure of gut bacteria shapes the phenotypic and functional profile of intestinal DCs to produce effects that range from their activation and enhanced functions to their exhaustion and tolerance. (HEPATOLOGY 2012;56:1861–1869)

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.

The biological response modifier AM3 attenuates the inflammatory cell response and hepatic fibrosis in rats with biliary cirrhosis

Background An inflammatory immune system response ensues in the liver and in the systemic circulation in cirrhosis, where it contributes to hepatic fibrosis and peripheral vasodilation. Modulation of the inflammatory response without increasing susceptibility to infection is a therapeutic target in cirrhosis. AM3 is a low-toxicity biological response modifier with regulatory effects on innate and adaptative immunity, and the ability to normalise the production of tumour necrosis factor α (TNFα). Aims This was an experimental study to investigate the effects of oral AM3 on the systemic and hepatic inflammatory response, liver fibrosis and on the haemodynamic abnormalities of portal hypertension in rats with biliary cirrhosis. Design Bile-duct ligated rats received a 3-week oral course of AM3 or placebo. Results In cirrhotic rats, AM3 blunted the inflammatory switch of circulating and intrahepatic monocytes and T-cells to TNFα and interferon γ (IFNγ) production, respectively. AM3 modified the intrahepatic polarisation pattern of the regulatory cytokines, decreasing the mRNA expression of transforming growth factor β1 (TGFβ1), interleukin 4 (IL4), and IFNγ, and increasing that of IL10. Total and IFNγ-producing natural killer (NK) cells were lowered by AM3 in the peripheral blood and liver of cirrhotic rats. The immunomodulatory effects of AM3 led to reduced hepatic fibrogenesis in cirrhotic rats, as shown by decreased area of liver fibrosis, hydroxyproline content and mRNA expression of procollagen α1(I). Besides, AM3 lowered portal pressure and systemic hyperaemia. Conclusions The biological response modifier AM3 reverses the concurrent inflammatory immune system activation in peripheral blood and liver of experimental established cirrhosis, which results in reductions of hepatic fibrosis, portal pressure and peripheral vasodilation.

Systemic inflammation in absence of gut bacterial translocation in C57BL/6 mice with cirrhosis

We read with interest the article by Wiest et al about spontaneous bacterial peritonitis guidelines.1 The authors point out the relevance of pathological bacterial translocation (BT) from the gut to mesenteric lymph nodes (MLN) as the pathophysiological hallmark of spontaneous bacterial peritonitis in cirrhosis, and the need of further research in the factors involved in BT. The study of BT, ascites and related immune system abnormalities in carbon tetrachloride (CCl4) cirrhosis is usually conducted in rats. This model has proved useful for research into the pathogenesis and role of the systemic pro-inflammatory response associated with cirrhosis and its link to BT.2–4 We recently showed that modulation of the immune system response in cirrhotic rats …

Loss of surface antigens is a conserved feature of apoptotic lymphocytes from several mammalian species.

Human lymphocytes lose the expression of lineage antigens (LAgs) along apoptosis. Our aim was to extent our previous studies of LAg loss to rodent species, quantifying LAg expression on apoptotic murine lymphocytes using flow cytometry to measure alterations in cell permeability, phosphatidylserine exposure and caspase activation of CD3, CD5, CD4, CD8, CD19 and CD28 LAgs in highly purified lymphocyte populations. We found loss of expression by apoptotic cells of all LAgs studied in the three species analyzed except for CD3 antigen in mouse. We also found an early, rapid and dramatic reduction in the expression of CD28 by early apoptotic cells. We found several homologies across the three species in the kinetic of loss of several LAgs such as CD5, CD4 and CD28. These data suggest that the loss of expression of LAgs by apoptotic lymphocytes is a common and conserved feature of lymphocytes undergoing apoptosis in several mammalian species.

The firing frequency of spontaneous action potentials and their corresponding evoked exocytosis are increased in chromaffin cells of CCl4-induced cirrhotic rats with respect to control rats

High catecolamine plasma levels because of sympathetic nervous system over‐activity contribute to cirrhosis progression. The aim of this study was to investigate whether chromaffin cells of the adrenal gland might potentiate the deleterious effect exerted by this over‐activity. Electrophysiological patch‐clamp and amperometric experiments with carbon‐fibre electrodes were conducted in single chromaffin cells of control and CCl4‐induced cirrhotic rats. The spontaneous action potential firing frequency was increased in chromaffin cells of cirrhotic rats with respect to control rats. The exocytosis evoked by that firing was also increased. However, exocytosis elicited by ACh did not vary between control and cirrhotic rats. Exocytosis triggered by depolarizing pulses was also unchanged. Amperometric recordings confirmed the lack of increased catecholamine charge released in cirrhosis after ACh or depolarization stimuli. However, the amperometric spikes exhibited faster kinetics of release. The overall Ca2+ entry through voltage‐dependent Ca2+ channels (VDCC), or in particular through Cav1 channels, did not vary between chromaffin cells of control and cirrhotic rats. The inhibition of VDCC by methionine‐enkephaline or ATP was not either altered, but it was increased by adrenaline in cells of cirrhotic rats. When a cocktail composed by the three neurotransmitters was tested in order to approach a situation closer to the physiological condition, the inhibition of VDCC was similar between both types of cells. In summary, chromaffin cells of the adrenal gland might contribute to exacerbate the sympathetic nervous system over‐activity in cirrhosis because of an increased exocytosis elicited by an enhanced spontaneous electrical activity.

Effects of aging on liver microcirculatory function and sinusoidal phenotype

The socioeconomic and medical improvements of the last decades have led to a relevant increase in the median age of worldwide population. Although numerous studies described the impact of aging in different organs and the systemic vasculature, relatively little is known about liver function and hepatic microcirculatory status in the elderly. In this study, we aimed at characterizing the phenotype of the aged liver in a rat model of healthy aging, particularly focusing on the microcirculatory function and the molecular status of each hepatic cell type in the sinusoid. Moreover, major findings of the study were validated in young and aged human livers. Our results demonstrate that healthy aging is associated with hepatic and sinusoidal dysfunction, with elevated hepatic vascular resistance and increased portal pressure. Underlying mechanisms of such hemodynamic disturbances included typical molecular changes in the cells of the hepatic sinusoid and deterioration in hepatocyte function. In a specific manner, liver sinusoidal endothelial cells presented a dysfunctional phenotype with diminished vasodilators synthesis, hepatic macrophages exhibited a proinflammatory state, while hepatic stellate cells spontaneously displayed an activated profile. In an important way, major changes in sinusoidal markers were confirmed in livers from aged humans. In conclusion, our study demonstrates for the first time that aging is accompanied by significant liver sinusoidal deregulation suggesting enhanced sinusoidal vulnerability to chronic or acute injuries.

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.