Cirrhosis-downregulated LSECtin can be retrieved by cytokines, shifts the TLR-induced LSECs secretome and correlates with the hepatic Th response.

BACKGROUND AND AIMS: We evaluated tolerogenic C-type lectin LSECtin loss in cirrhosis and its potential regulation by cytokines. METHODS: Liver tissue from patients with cirrhosis and healthy controls, immortalised and generated LSECtin-CRISPR immortalised LSECs, and murine primary LSECs from the CCl4 model were handled. RESULTS: LSECtin expression was reduced in liver tissue from cirrhotic patients, and it decreased from compensated to decompensated disease. Increased phosphorylation of MAPK, Akt and NFkB was observed upon LSECtin stimulation in LSEC murine cell line, showing a pattern of inflammatory and chemotactic cytokines either restrained (IL-10, CCL4) or unrestrained (TNF-α, IL-1ß, IL-6, CCL2). CD44 attenuated whereas LAG-3 increased all substrates phosphorylation in combination with TLR4 and TLR2 ligands except for NFkB. TNF-α, IL-1 ß, IL-6 and CCL2 were restrained by LSECtin crosslinking on TLRs studied. Conversely, IL-10 and CCL4 were upregulated, suggesting a LSECtin-TLRs synergistic effect. Also, LSECtin was significantly induced after IL-13 stimulation or combined with anti-inflammatory cytokines in cirrhotic and immortalised LSECs. Th17 and regulatory T cells were progressively increased in the hepatic tissue from compensated to decompensated patients. A significant inverse correlation was present between gene expression levels of CLEC4G/LSECtin and RORγT and FOXP3 in liver tissues. CONCLUSION: LSECtin restrains TLR proinflammatory secretome induced on LSECs by interfering immune response control, survival and MAPKs signalling pathways. The cytokine-dependent induction of LSECtin and the association between LSECtin loss and Th17 cell subset expansion in the liver, provides a solid background for exploring LSECtin retrieval as a mechanism to reprogram LSEC homeostatic function hampered during cirrhosis.

The 3Rs in Experimental Liver Disease.

Patients with cirrhosis present multiple physiological and immunological alterations that play a very important role in the development of clinically relevant secondary complications to the disease. Experimentation in animal models is essential to understand the pathogenesis of human diseases and, considering the high prevalence of liver disease worldwide, to understand the pathophysiology of disease progression and the molecular pathways involved, due to the complexity of the liver as an organ and its relationship with the rest of the organism. However, today there is a growing awareness about the sensitivity and suffering of animals, causing opposition to animal research among a minority in society and some scientists, but also about the attention to the welfare of laboratory animals since this has been built into regulations in most nations that conduct animal research. In 1959, Russell and Burch published the book "The Principles of Humane Experimental Technique", proposing that in those experiments where animals were necessary, everything possible should be done to try to replace them with non-sentient alternatives, to reduce to a minimum their number, and to refine experiments that are essential so that they caused the least amount of pain and distress. In this review, a comprehensive summary of the most widely used techniques to replace, reduce, and refine in experimental liver research is offered, to assess the advantages and weaknesses of available experimental liver disease models for researchers who are planning to perform animal studies in the near future.

Saturated fatty acid-enriched small extracellular vesicles mediate a crosstalk inducing liver inflammation and hepatocyte insulin resistance.

Background & Aims: Lipotoxicity triggers non-alcoholic fatty liver disease (NAFLD) progression owing to the accumulation of toxic lipids in hepatocytes including saturated fatty acids (SFAs), which activate pro-inflammatory pathways. We investigated the impact of hepatocyte- or circulating-derived small extracellular vesicles (sEV) secreted under NAFLD conditions on liver inflammation and hepatocyte insulin signalling. Methods: sEV released by primary mouse hepatocytes, characterised and analysed by lipidomics, were added to mouse macrophages/Kupffer cells (KC) to monitor internalisation and inflammatory responses. Insulin signalling was analysed in hepatocytes exposed to conditioned media from sEV-loaded macrophages/KC. Mice were i.v. injected sEV to study liver inflammation and insulin signalling. Circulating sEV from mice and humans with NAFLD were used to evaluate macrophage-hepatocyte crosstalk. Results: Numbers of sEV released by hepatocytes increased under NAFLD conditions. Lipotoxic sEV were internalised by macrophages through the endosomal pathway and induced pro-inflammatory responses that were ameliorated by pharmacological inhibition or deletion of Toll-like receptor-4 (TLR4). Hepatocyte insulin signalling was impaired upon treatment with conditioned media from macrophages/KC loaded with lipotoxic sEV. Both hepatocyte-released lipotoxic sEV and the recipient macrophages/KC were enriched in palmitic (C16:0) and stearic (C18:0) SFAs, well-known TLR4 activators. Upon injection, lipotoxic sEV rapidly reached KC, triggering a pro-inflammatory response in the liver monitored by Jun N-terminal kinase (JNK) phosphorylation, NF-κB nuclear translocation, pro-inflammatory cytokine expression, and infiltration of immune cells into the liver parenchyma. sEV-mediated liver inflammation was attenuated by pharmacological inhibition or deletion of TLR4 in myeloid cells. Macrophage inflammation and subsequent hepatocyte insulin resistance were also induced by circulating sEV from mice and humans with NAFLD. Conclusions: We identified hepatocyte-derived sEV as SFA transporters targeting macrophages/KC and activating a TLR4-mediated pro-inflammatory response enough to induce hepatocyte insulin resistance. Impact and Implications: Small extracellular vesicles (sEV) released by the hepatocytes under non-alcoholic fatty liver disease (NAFLD) conditions cause liver inflammation and insulin resistance in hepatocytes via paracrine hepatocyte-macrophage-hepatocyte crosstalk. We identified sEV as transporters of saturated fatty acids (SFAs) and potent lipotoxic inducers of liver inflammation. TLR4 deficiency or its pharmacological inhibition ameliorated liver inflammation induced by hepatocyte-derived lipotoxic sEV. Evidence of this macrophage-hepatocyte interactome was also found in patients with NAFLD, pointing to the relevance of sEV in SFA-mediated lipotoxicity in NAFLD.

Non-Alcoholic Fatty Liver Disease: Metabolic, Genetic, Epigenetic and Environmental Risk Factors.

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of chronic liver disease in the Western world, probably due to the growing prevalence of obesity, metabolic diseases, and exposure to some environmental agents. In certain patients, simple hepatic steatosis can progress to non-alcoholic steatohepatitis (NASH), which can sometimes lead to liver cirrhosis and its complications including hepatocellular carcinoma. Understanding the mechanisms that cause the progression of NAFLD to NASH is crucial to be able to control the advancement of the disease. The main hypothesis considers that it is due to multiple factors that act together on genetically predisposed subjects to suffer from NAFLD including insulin resistance, nutritional factors, gut microbiota, and genetic and epigenetic factors. In this article, we will discuss the epidemiology of NAFLD, and we overview several topics that influence the development of the disease from simple steatosis to liver cirrhosis and its possible complications.

Bacterial antigen translocation and age as BMI-independent contributing factors on systemic inflammation in NAFLD patients.

BACKGROUND & AIMS: Low-grade systemic inflammation is a crucial landmark in NAFLD favouring disease progression and comorbidities. We evaluated the input of circulating bacterial antigens on systemic markers of inflammation in NAFLD patients. PATIENTS & METHODS: Multicenter cross-sectional study including consecutive patients with biopsy-proven NAFLD. Demographic, metabolic and fibrosis-related variables were collected. Circulating bacterial antigens were quantified in blood. Toll-like receptor SNPs were genotyped. Serum cytokine levels were evaluated. Peripheral blood mononuclear cell response to bacterial antigens was evaluated in vitro. RESULTS: Three hundred and fifteen patients from five Spanish hospitals were distributed by BMI. At least, one bacterial antigenic type was found in 66 patients with BMI < 30 (63.4%) and 163 patients with BMI > 30 (77.3%) (P = .014). HOMA-IR was significantly higher in the presence of circulating antigens among patients with BMI < 30. NASH and significant fibrosis in non-obese patients were more frequent in the presence of at least two circulating antigenic types. Allelic frequencies of TLR variants were similar to controls and did not affect clinical or laboratory parameters. Pro-inflammatory cytokines were significantly increased in patients with bacterial antigens, regardless of BMI. TLR gene and protein expression levels were significantly increased in PBMCs from patients with bacterial antigens. Antigen concentrations independently influenced TNF-α and IL-6, in both BMI subgroups of patients. Age independently influenced TNF-α and IL-6 in non-obese patients, and TNF-α in obese patients. CONCLUSION: Serum circulating bacterial antigens as well as age were BMI-independent factors related to increased systemic inflammation in NAFLD and provides insight on the multifaceted sources of inflammation in these patients.

Liver Sinusoidal Endothelial Cells Contribute to Hepatic Antigen-Presenting Cell Function and Th17 Expansion in Cirrhosis.

Hepatic immune function is compromised during cirrhosis. This study investigated the immune features of liver sinusoidal endothelial cells (LSECs) in two experimental models of cirrhosis. Dendritic cells, hepatic macrophages, and LSECs were isolated from carbon tetrachloride and bile duct-ligated rats. Gene expression of innate receptors, bacterial internalization, co-stimulatory molecules induction, and CD4+ T cell activation and differentiation were evaluated. Induced bacterial peritonitis and norfloxacin protocols on cirrhotic rats were also carried out. LSECs demonstrated an active immunosurveillance profile, as shown by transcriptional modulation of different scavenger and cell-adhesion genes, and their contribution to bacterial internalization. LSECs significantly increased their expression of CD40 and CD80 and stimulated CD4+ T cell activation marker CD71 in both models. The pro-inflammatory Th17 subset was expanded in CCl4-derived LSECs co-cultures. In the bile duct ligation (BDL) model, CD4+ T cell differentiation only occurred under induced bacterial peritonitis conditions. Differentiated pro-inflammatory Th cells by LSECs in both experimental models were significantly reduced with norfloxacin treatment, whereas Foxp3 tolerogenic Th CD4+ cells were expanded. Conclusion: LSECs' participation in the innate-adaptive immune progression, their ability to stimulate pro-inflammatory CD4+ T cells expansion during liver damage, and their target role in norfloxacin-induced immunomodulation granted a specific competence to this cell population in cirrhosis.

Improved hemodynamic and liver function in portal hypertensive cirrhotic rats after administration of B. pseudocatenulatum CECT 7765.

PURPOSE: Evaluating whether changes in gut microbiota induced by a bifidobacterial strain may have an effect on the hepatic vascular function in portal hypertensive cirrhotic rats. METHODS: Bile duct ligation (BDL) was performed in rats. A subgroup of animals received B. pseudocatenulatum CECT7765 (109 cfu/daily ig.) for 1 week prior to laparotomy. Hemodynamic, biochemical and inflammatory markers were evaluated. Ileal microbiota composition was identified. Statistical analysis was performed. RESULTS: Sham-operated (n = 6), BDL (n = 6) and BDL treated with bifidobacteria (n = 8) rats were included. B. pseudocatenulatum CECT7765 significantly decreased proteobacteria (p = 0.001) and increased Bacteroidetes (p = 0.001) relative abundance. The bifidobacteria decreased the Firmicutes/Bacteroidetes ratio in the BDL model (p = 0.03). BDL with bifidobacteria vs BDL rats showed: significantly reduced portal vein area, portal flow, congestion index, alkaline phosphatase and total bilirubin, significantly increased serum cytokines and nitric oxide levels, gene expression levels of bile acids receptor FXR and endothelial nitric oxide synthase. Quantitative changes in the Clostridiales and Bacteroidales orders were independently associated with variations in portal vein area and portal flow, while changes in the Proteobacteria phylum were independently associated with congestion. Variations in all liver function markers significantly correlated with total OTUs mainly in the Firmicutes, but only changes in the Clostridiales were independently associated with alkaline phosphatase in the ANCOVA analysis. CONCLUSION: Hemodynamic alterations and liver dysfunction induced by BDL in rats are partially restored after oral administration of B. pseudocatenulatum CECT7765. Results provide a proof-of-concept for the beneficial effect of this bifidobacterial strain in reducing complications derived from portal hypertension in cirrhosis.

Regulatory T Cells Restrict Permeability to Bacterial Antigen Translocation and Preserve Short-Chain Fatty Acids in Experimental Cirrhosis.

Intestinal permeability to translocation of bacterial products is increased in cirrhosis. Regulatory T cells (Tregs) remain central to the interplay between the host and microbial milieu. We propose that Tregs are involved in promoting gut barrier integrity and a balanced interaction with gut microbiota-derived short-chain fatty acids (SCFAs). Carbon tetrachloride cirrhosis was induced in wild-type and recombination activating gene 1 (Rag1)-/- mice. Naive T cells and Treg cells were transferred into Rag1 -/- mice. Intestinal permeability was assessed in vivo after lipopolysaccharide (LPS) oral administration, and bacterial DNA presence was evaluated in mesenteric lymph nodes. Transcript and protein levels of tight-junction (TJ) proteins were measured in colonic tissue. Intestinal T helper profile in response to Escherichia coli (E. coli) was determined by flow cytometry. SCFAs were measured by gas chromatography-mass spectrometry in colonic content before and after E. coli challenge. Rag1 -/- mice showed significantly increased permeability to LPS and bacterial DNA translocation rate compared with control mice. Naive T and Treg cotransfer significantly reduced gut permeability to bacterial antigen translocation and restored TJ protein expression in Rag1 -/- mice. Naive T and Treg replenishment in Rag1 -/- mice restrained proinflammatory differentiation of intestinal lymphocytes in response to E. coli. The main SCFA concentration resulted in significant reduction in Rag1 -/- mice after E. coli administration but remained unaltered after naive T and Tregs cotransfer. The reduced expression of SCFA receptors induced by E. coli was reestablished following naive T and Treg reconstitution in Rag1 -/- mice. Conclusion: The restriction of gut permeability, local inflammatory differentiation, and loss of bacteria-derived SCFAs foster the value of Tregs in preventing bacterial translocation in cirrhosis.

AIM2 deficiency reduces the development of hepatocellular carcinoma in mice.

Chronic liver inflammation is crucial in the pathogenesis of hepatocellular carcinoma (HCC). Activation of the inflammasome complex is a key inflammatory process that has been associated with different liver diseases, but its role in HCC development remains largely unexplored. Here we analyzed the impact of different inflammasome components, including absent in melanoma 2 (AIM2) and NOD-like receptor family pyrin domain containing 3 (NLRP3), in the development of diethylnitrosamine (DEN)-induced HCC in mice. Genetic inactivation of AIM2, but not NLRP3, reduces liver damage and HCC development in this model. AIM2 deficiency ameliorates inflammasome activation, liver inflammation and proliferative responses during HCC initiation. We also identified that AIM2 is highly expressed in Kupffer cells, and that AIM2-mediated production of IL-1ß by these cells is enhanced after DEN-induced liver damage. Our data indicate that AIM2 promotes inflammation during carcinogenic liver injury and that it contributes to genotoxic HCC development in mice, thereby recognizing AIM2 as a potential therapeutic target in this disease.

Norfloxacin is more effective than Rifaximin in avoiding bacterial translocation in an animal model of cirrhosis.

BACKGROUND & AIMS: Norfloxacin administration is useful in preventing bacterial infections in cirrhosis but associated to the generation of resistant species. Rifaximin is known to reach high concentrations in the intestinal lumen without generating relevant resistance in the intestinal flora. Our aim was to compare the effect of Norfloxacin and Rifaximin on intestinal flora composition, bacterial translocation and survival in cirrhotic rats. METHODS: Cirrhosis was induced in rats by oral administration of CCl4 . Animals were divided into three groups: only CCl4 (group I, n = 10); CCl4 + Norfloxacin (group II, n = 17) and CCl4 + Rifaximin (group III, n = 14). Gut bacterial composition, bacterial translocation and cytokine levels were measured. RESULTS: Forty-one rats were finally included. The incidence of viable and non-viable bacterial translocation was significantly reduced in animals receiving Norfloxacin; Rifaximin also decreased the incidence of viable and non-viable bacterial translocation, but did not reach statistical significance. Serum TNF-α levels were significantly lower in antibiotic groups. Norfloxacin modified intestinal microbiota, depleting significantly more pathobionts than Rifaximin. CONCLUSION: Norfloxacin is more effective than Rifaximin in preventing bacterial translocation in rats with cirrhosis probably because of its capacity to reduce pathobionts from intestinal microbiota.

Selective intestinal decontamination with norfloxacin enhances a regulatory T cell-mediated inflammatory control mechanism in cirrhosis.

BACKGROUND & AIMS: Norfloxacin exerts immunomodulatory effects in cirrhosis beyond its bactericidal activity. We aimed at identifying the role of regulatory T (Treg) cells in the norfloxacin mechanism that compensates the inflammatory environment in cirrhosis. PATIENTS & METHODS: Consecutively admitted patients with cirrhosis and ascitic fluid (AF) with: spontaneous bacterial peritonitis (SBP), non-infected AF, and norfloxacin as secondary SBP prophylaxis (SID group). Tregs were defined by flow-cytometry as CD4+ CD25+ FoxP3+ cells. Dendritic cells (DCs) were purified for co-stimulatory signalling evaluation and norfloxacin and IL-10 levels were measured in serum. Wildtype and recombination activating gene 1 (Rag1)-deficient mice with CCl4 -induced cirrhosis were used for adoptive-transfer experiments using naïve CD4+ T cells and Tregs. RESULTS: Eighty-four patients were included. Treg percentage was significantly increased in SID patients compared with SBP or non-infected AF patients. A positive correlation was observed between Tregs and serum norfloxacin and IL-10 levels. DCs from SID patients showed a significantly decreased expression of CD80 and CD86 compared with SBP and non-infected AF patients and correlated with norfloxacin levels. Modulation of co-stimulatory signalling by norfloxacin was not detected in Rag1-deficient mice and Rag1-deficient mice reconstituted with naïve T-cells. However, reconstitution with naïve T-cells and Tregs was associated with significantly downregulated CD80 and CD86 expression in the presence of norfloxacin. Norfloxacin immunomodulatory effect on IL-2 and IFN-gamma reduction and on the increase of IL-10 was significantly achieved only when the Tregs were restored in Rag1-deficient mice. CONCLUSIONS: These results provide a plausible mechanism for the immunomodulatory effects of norfloxacin in cirrhosis beyond its bactericidal effect.

Microbiome and bacterial translocation in cirrhosis. / Microbioma y traslocación bacteriana en la cirrosis.

Qualitative and quantitative changes in gut microbiota play a very important role in cirrhosis. Humans harbour around 100 quintillion gut bacteria, thus representing around 10 times more microbial cells than eukaryotic ones. The gastrointestinal tract is the largest surface area in the body and it is subject to constant exposure to these living microorganisms. The existing symbiosis, proven by the lack of proinflammatory response against commensal bacteria, implies the presence of clearly defined communication lines that contribute to the maintenance of homeostasis of the host. Therefore, alterations of gut flora seem to play a role in the pathogenesis and progress of multiple liver and gastrointestinal diseases. This has made its selective modification into an area of high therapeutic interest. Bacterial translocation is defined as the migration of bacteria or bacterial products from the intestines to the mesenteric lymph nodes. It follows that alteration in gut microbiota have shown importance, at least to some extent, in the pathogenesis of several complications arising from terminal liver disease, such as hepatic encephalopathy, portal hypertension and spontaneous bacterial peritonitis. This review sums up, firstly, how liver disease can alter the common composition of gut microbiota, and secondly, how this alteration contributes to the development of complications in cirrhosis.

Bifidobacterium pseudocatenulatum CECT7765 induces an M2 anti-inflammatory transition in macrophages from patients with cirrhosis.

BACKGROUND & AIMS: Patients with cirrhosis show recurrent access of bacterial products into the bloodstream inducing a multi-altered immunological status leading to relevant complications. We aimed at evaluating Bifidobacterium pseudocatenulatum CECT7765 effect on the host's macrophage function. PATIENTS & METHODS: Patients with cirrhosis and ascites were included. Granulocyte-macrophage colony-stimulating factor (GM-CSF) monocyte-derived and ascitic fluid (AF) macrophages were cultured with M-CSF, lipopolysaccharide (LPS) and/or the bifidobacterial strain. Pellets and supernatants were evaluated for gene expression of M1 and M2-related genes and cytokine secretion. Cell surface expression molecules were evaluated by flow cytometry. Kupffer cells from bile duct ligated and CCl4 rats were also evaluated. RESULTS: Experiments were run on GM-CSF blood-derived and AF macrophages from 10 patients with cirrhosis and 10 healthy donors. Different macrophage morphology was observed by optical microscopy in cells stimulated with bifidobacteria vs. LPS. M2-like expression of CD206, CD163 and CD16 was significantly increased in macrophages after stimulation with the bifidobacterial strain vs. LPS. B. pseudocatenulatum CECT7765 was able to significantly change the cytokine secretion pattern of blood-derived and AF macrophages and Kupffer cells from bile duct ligated and CCl4 cirrhotic rats compared to that induced by LPS. B. pseudocatenulatum CECT7765 was also effective in inducing a phenotype transition and a functional change from an M1- to an M2-related gene expression and cytokine secretion pattern in AF macrophages even after LPS-pretreatment. B. pseudocatenulatum CECT7765 did not reduce AF macrophage bacterial killing capacity. CONCLUSION: B. pseudocatenulatum CECT7765 induces a morphologic, phenotypic and functional transition towards an anti-inflammatory profile in GM-CSF monocyte-derived and AF macrophages from patients with cirrhosis that may help in controlling sustained inflammation in decompensated cirrhosis.

Bifidobacterium pseudocatenulatum CECT7765 promotes a TLR2-dependent anti-inflammatory response in intestinal lymphocytes from mice with cirrhosis.

BACKGROUND: Intestinal homeostasis plays an important role in bacteria-derived complications in cirrhosis. Intestinal lymphocytes are responsible for immune effector functions and can be modulated by certain probiotics. We evaluate the interaction between Bifidobacterium pseudocatenulatum CECT7765 and intestinal lymphocytes in mice with cirrhosis. ANIMALS AND METHODS: Cirrhosis was induced by intragastrical administration of carbon tetrachloride in Balb/C mice. One week prior to laparotomy, animals received B. pseudocatenulatum CECT7765 (10(7), 10(9) or 10(10) cfu/daily) or placebo. Chemokine receptor and cytokine expression were evaluated in intestinal lymphocytes. Gut permeability was studied by FITC-LPS recovery in vivo. Luminal antigens, inflammation and functional markers were evaluated in liver samples. RESULTS: Bifidobacterium pseudocatenulatum CECT7765 decreased the expression of pro-inflammatory chemokine receptors CCR6, CCR9, CXCR3 and CXCR6 in intestinal lymphocytes from cirrhotic mice in a concentration-dependent manner. The bifidobacterial strain induced a shift towards an anti-inflammatory cytokine profile in this cell subset. B. pseudocatenulatum CECT7765-induced inflammatory modulation was TLR2-mediated, as in vitro TLR2 blockade inhibited the reduction of TNF-alpha and its receptors and the increase of IL-10 and IL-10 receptor secretion. The recovery rate of administered fluorescence-labelled endotoxin was significantly and dose-dependently lowered with the bifidobacterial strain. The reduced intestinal permeability was associated with a decreased burden of bacterial antigens in the liver of mice treated with B. pseudocatenulatum CECT7765. Liver function and inflammation were improved with the use of the bifidobacterial strain at the highest dose tested (10(10) cfu). CONCLUSION: Bifidobacterium pseudocatenulatum CECT7765 improves gut homeostasis and prevents gut-derived complications in experimental chronic liver disease.

Use of proton pump inhibitors decrease cellular oxidative burst in patients with decompensated cirrhosis.

BACKGROUND AND AIMS: Proton pump inhibitors (PPIs) are commonly used antisecretory drugs and have been linked to an increased risk of bacterial infections in cirrhosis. We investigated whether the treatment with PPIs in cirrhosis affects the oxidative burst activity of granulocytes and monocytes and its possible interference with serum norfloxacin (Nflx) levels in these patients. METHODS: 70 patients with cirrhosis and ascitic fluid and 24 healthy controls were included in the study and distributed into groups according to the regular use of PPIs and/or norfloxacin. The blood granulocyte and monocyte's phagocytic activity and oxidative burst were evaluated by flow cytometry. Blood levels of norfloxacin were measured by HPLC and bacterial translocation was evaluated by detection of bacterial DNA in blood. RESULTS: Use of PPIs was associated with a decreased granulocyte and monocyte oxidative burst, but not of phagocytic activity, as compared with patients not receiving PPIs. PPIs use did not affect serum norfloxacin levels in patients. A not significant trend to an increased bacterial DNA translocation was observed in patients receiving PPIs, including patients simultaneously receiving norfloxacin. CONCLUSIONS: PPIs significantly decrease cellular oxidative burst in cirrhosis. This fact may provide a pathogenic explanation to the reported high rates of bacterial infections in this setting, and strongly suggests that PPIs should only be used in patients with cirrhosis when clinically indicated.

Absent in melanoma 2 triggers a heightened inflammasome response in ascitic fluid macrophages of patients with cirrhosis.

BACKGROUND & AIMS: Inflammation is a common event in the pathogenesis of liver cirrhosis. The inflammasome pathway has acquired significant relevance in the pathogenesis of inflammation, but its role in the inflammatory response in patients with decompensated cirrhosis remains unexplored. METHODS: We performed a prospective study in which 44 patients with decompensated cirrhosis and 12 healthy volunteers were included. We isolated macrophages from blood and ascitic fluid and assessed the expression and activation of the inflammasome, its response to priming by bacterial products, and its association with the degree of liver disease. RESULTS: Macrophages from sterile ascitic fluids showed constitutive activation of caspase-1 and a marked increase in the expression of IL-1ß, IL-18, and absent in melanoma 2 (AIM2) when compared to blood macrophages. Pre-stimulation of blood-derived macrophages from cirrhotic patients with bacterial DNA increased the expression of AIM2 and induced a higher AIM2-mediated inflammasome response than priming with other bacterial products such as lipopolysaccharide. By contrast, activation of the AIM2 inflammasome did not require a priming signal in ascitic fluid-derived macrophages, demonstrating the preactivated state of the inflammasome in these cells. Last, higher IL-1ß and IL-18 production by ascitic fluid macrophages correlated with a more advanced Child-Pugh score. CONCLUSIONS: The inflammasome is highly activated in the ascitic fluid of cirrhotic patients, which may explain the exacerbated inflammatory response observed in these patients under non-infected conditions. Clinically, activation of the inflammasome is associated with a higher degree of liver disease.

Gut microbiota-related complications in cirrhosis.

Gut microbiota plays an important role in cirrhosis. The liver is constantly challenged with commensal bacteria and their products arriving through the portal vein in the so-called gut-liver axis. Bacterial translocation from the intestinal lumen through the intestinal wall and to mesenteric lymph nodes is facilitated by intestinal bacterial overgrowth, impairment in the permeability of the intestinal mucosal barrier, and deficiencies in local host immune defences. Deranged clearance of endogenous bacteria from portal and systemic circulation turns the gut into the major source of bacterial-related complications. Liver function may therefore be affected by alterations in the composition of the intestinal microbiota and a role for commensal flora has been evidenced in the pathogenesis of several complications arising in end-stage liver disease such as hepatic encephalopathy, splanchnic arterial vasodilatation and spontaneous bacterial peritonitis. The use of antibiotics is the main therapeutic pipeline in the management of these bacteria-related complications. However, other strategies aimed at preserving intestinal homeostasis through the use of pre-, pro- or symbiotic formulations are being studied in the last years. In this review, the role of intestinal microbiota in the development of the most frequent complications arising in cirrhosis and the different clinical and experimental studies conducted to prevent or improve these complications by modifying the gut microbiota composition are summarized.

Role of interleukin 10 in norfloxacin prevention of luminal free endotoxin translocation in mice with cirrhosis.

BACKGROUND & AIMS: Bacterial endotoxin is present in patients with advanced cirrhosis and can induce an immunogenic response without an overt infection. Norfloxacin is a gram-negative bactericidal drug able to maintain low endotoxin levels and stimulate IL-10 production. We aimed at investigating the role of IL-10 in decreasing endotoxin absorption in cirrhotic mice treated with norfloxacin. METHODS: Cirrhosis was induced by carbon tetrachloride or bile duct ligation in wild type and IL10-deficient mice with or without norfloxacin prior to an intragastrical administration of E. coli, K. pneumonia or E. faecalis. Spontaneous and induced bacterial translocation, free endotoxin and cytokine levels were evaluated in mesenteric lymph nodes. Intestinal permeability was followed by fluorimetry and barrier integrity markers were measured in disrupted intestinal samples. The inflammatory-modulating mechanism was characterized in purified intestinal mononuclear cells. RESULTS: Norfloxacin reduced spontaneous and induced MLN positive-cultures in wild type and IL-10-deficient animals. However, reduction of free endotoxin levels was associated with norfloxacin in wild type but not in IL-10-deficient mice. Wild type but not IL-10-deficient mice treated with norfloxacin significantly normalized intestinal permeability and improved gut barrier integrity markers. The toll-like receptor 4-mediated pro-inflammatory milieu was modulated by norfloxacin in a concentration-dependent manner in cultured intestinal mononuclear cells of wild type mice but not of IL-10-deficient mice. The restoration of IL-10 levels in IL-10-deficient animals reactivated the norfloxacin effect on inflammatory-modulation, gut barrier permeability, and luminal endotoxin absorption. CONCLUSION: Norfloxacin not only reduces gram-negative intestinal flora but also participates in an IL-10-driven modulation of gut barrier permeability, thus reducing luminal free endotoxin absorption in experimental cirrhosis.

Protective effect of Bifidobacterium pseudocatenulatum CECT7765 against induced bacterial antigen translocation in experimental cirrhosis.

BACKGROUND & AIMS: Intervention in the gut ecosystem is considered as a potential strategy to treat liver diseases and their complications. We have evaluated the effects of Bifidobacterium pseudocatenulatum CECT7765 on bacterial translocation and the liver status in experimental cirrhosis. ANIMALS & METHODS: Liver damage was induced in Balb/c mice by weight-controlled oral administration of carbon tetrachloride. Laparotomies were performed at week 12. One week prior to laparotomy, animals received B. pseudocatenulatum CECT7765 (10(9) cfu/daily) or placebo intragastrically. All animals received Escherichia coli (10(7) cfu/single dose) intragastrically 24 hours before laparotomy. A group of naïve non-treated animals was included as control. Liver tissue specimens, mesenteric lymph nodes, intestinal content and blood were collected. Liver histology, profibrogenic genes expression, bacterial DNA translocation, serum endotoxaemia and liver cytokine levels were measured. RESULTS: Bifidobacterium pseudocatenulatum CECT7765 showed no significant effect on structural liver damage, as determined by histological evaluation, alpha-smooth muscle actin distribution, profibrogenic gene expression levels, total hydroxyproline levels and malon dialdehyde production compared with mice receiving placebo. Interestingly, bacterial DNA translocation and serum endotoxin levels were significantly decreased in mice receiving the Bifidobacterium strain compared with placebo. Gut barrier integrity markers were up-regulated in mice receiving B. pseudocatenulatum CECT7765 and quantitatively correlated with intestinal gene copy numbers of the bifidobacterial strain. Gene expression levels of several anti-inflammatory mediators were also increased in mice receiving B. pseudocatenulatum CECT7765 compared with placebo. CONCLUSION: Oral administration of B. pseudocatenulatum CECT7765 is associated with improved gut barrier integrity and shows a beneficial effect against induced bacterial antigen translocation in the CCl4 -model of cirrhosis.

Beta-adrenergic receptor 1 selective antagonism inhibits norepinephrine-mediated TNF-alpha downregulation in experimental liver cirrhosis.

BACKGROUND: Bacterial translocation is a frequent event in cirrhosis leading to an increased inflammatory response. Splanchnic adrenergic system hyperactivation has been related with increased bacterial translocation. We aim at evaluating the interacting mechanism between hepatic norepinephrine and inflammation during liver damage in the presence of bacterial-DNA. ANIMALS AND METHODS: Forty-six mice were included in a 16-week protocol of CCl(4)-induced cirrhosis. Laparotomies were performed at weeks 6, 10, 13 and 16. A second set of forty mice injected with a single intraperitoneal dose of CCl(4) was treated with saline, 6-hydroxidopamine, Nebivolol or Butoxamine. After 5 days, mice received E. coli-DNA intraperitoneally. Laparotomies were performed 24 hours later. Liver bacterial-DNA, norepinephrine, TNF-alpha, IL-6 and beta-adrenergic receptor levels were measured. RESULTS: Bacterial-DNA translocation was more frequent in CCl(4)-treated animals compared with controls, and increased as fibrosis progressed. Liver norepinephrine and pro-inflammatory cytokines were significantly higher in mice with vs without bacterial-DNA (319.7 ± 120.6 vs 120.7 ± 68.6 pg/g for norepinephrine, 38.4 ± 6.1 vs 29.7 ± 4.2 pg/g for TNF-alpha, 41.8 ± 7.4 vs 28.7 ± 4.3 pg/g for IL-6). Only beta-adrenergic receptor-1 was significantly increased in treated vs control animals (34.6 ± 7.3 vs 12.5 ± 5.3, p=0.01) and correlated with TNF-alpha, IL-6 and norepinephrine hepatic levels in animals with bacterial-DNA. In the second set of mice, cytokine levels were increased in 6-hydroxidopamine and Nebivolol (beta-adrenergic receptor-1 antagonist) treated mice compared with saline. Butoxamine (beta-adrenergic receptor-2 antagonist) didn't inhibit liver norepinephrine modulation of pro-inflammatory cytokines. CONCLUSIONS: Beta-adrenergic receptor-1 mediates liver norepinephrine modulation of the pro-inflammatory response in CCl(4)-treated mice with bacterial-DNA.