Escherichia coli of sequence type (ST) 131 resistant to fluoroquinolones and producer of CTX-M-15 is globally one of the major extraintestinal pathogenic E. coli (ExPEC). ST131 phylogenesis showed that multidrug-resistant ST131 strains belong to a clade called C, descending from an ancestral clade called B, comprising mostly antibiotic-susceptible strains. Antibiotic resistance could appear as one of the keys of the clade C global success. We hypothesized that other features of ST131 clade C could contribute to this success since other major global ExPEC clones (ST73, ST95) are mostly antibiotic-susceptible. To test this hypothesis, we measured the growth abilities, early biofilm formation and virulence-factor content of a collection of clade B and clade C strains. Moreover, using competition assays, we measured the capacity of selected representative strains of clades B and C to colonize the mouse intestine and urinary tract, and to kill mice in a septicemia model. Clade B and C strains had similar growth ability. However, clade B strains were more frequently early biofilm producers, expressed mostly faster their type 1 fimbriae and displayed more virulence factor-encoding genes than clade C strains. Clade B outcompeted clade C in the gut and/or urinary tract colonization models and in the septicemia model. These results strongly suggest that clade C strain evolution includes a loss of virulence, i.e . a process that could enhance micro-organism persistence in a given host and thus optimize transmission. This process, associated with acquired antibiotic-resistance, could ensure clade C strain survival in environments under antibiotic pressure. Importance Extraintestinal pathogen Escherichia coli (ExPEC) are virulent but mostly antibiotic-susceptible. One worrying exception is ST131, a major multidrug resistant ExPEC clone that has spread worldwide since the 2000s. To contain the emergence of this threatening clone, we need to understand what factors favored its emergence and dissemination. Here, we investigated whether multidrug-resistant ST131 had advantageous phenotypic properties beyond multidrug resistance. To this end, we competed the emergent multidrug-resistant ST131 with its antibiotic-susceptible ancestor in different conditions: biofilm production, in vivo colonization and virulence experiments. In all in vivo competitions, we found that multidrug-resistant ST131 was losing to its ancestor, suggesting a lesser virulence of multidrug-resistant ST131. It was previously described that losing virulence can increase micro-organism persistence in some populations and subsequently its level of transmissibility. Thus, a decreased level of virulence, associated with multidrug resistance, could explain the global success of ST131.

Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells over-expressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients' specific mutation.

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22 PHOX , and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFα and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IκBζ, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn’s disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.

Liver fibrosis is the common response to chronic liver injury, and leads to cirrhosis and its complications. Persistent inflammation is a driving force of liver fibrosis progression. Mucosal-associated invariant T (MAIT) cells are non-conventional T cells that display altered functions during chronic inflammatory diseases. Here, we show that circulating MAIT cells are reduced in patients with alcoholic or non-alcoholic fatty liver disease-related cirrhosis while they accumulate in liver fibrotic septa. Using two models of chronic liver injury, we demonstrate that MAIT cell-enriched mice show increased liver fibrosis and accumulation of hepatic fibrogenic cells, whereas MAIT cell-deficient mice are resistant. Co-culture experiments indicate that MAIT cells enhance the proinflammatory properties of monocyte-derived macrophages, and promote mitogenic and proinflammatory functions of fibrogenic cells, via distinct mechanisms. Our results highlight the profibrogenic functions of MAIT cells and suggest that targeting MAIT cells may constitute an attractive antifibrogenic strategy during chronic liver injury.

Objective Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury. Design C57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells. Results MAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited. Conclusion MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

Objective Cirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients’ susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility to infections, we investigated the expression of NOX2 and partners in neutrophils of patients with severe alcoholic cirrhosis and have provided a novel approach to restore superoxide production capacity in patients’ neutrophils and blood. Design Neutrophils were isolated from patients with decompensated alcoholic cirrhosis. NOX2 activity was assessed after stimulation of purified neutrophils or whole blood with the bacterial-derived peptide fMet-Leu-Phe. The expression of NOX2 and partners was studied by western blot analysis, flow cytometry and reverse transcription-PCR. Results The impaired superoxide production by patients’ neutrophils was associated with a severe deficient expression of the NADPH oxidase catalytic core flavocytochrome-b558 (gp91 phox /NOX2 and p22 phox ), its cytosolic partner p47 phox but not p67 phox . NOX2 expression decreased rapidly by protein degradation involving elastase released during degranulation of healthy neutrophils stimulated with fMet-Leu-Phe, or highly present in patients’ plasma. Interestingly, the deficient superoxide production was reversed by treatment of patients’ neutrophils and whole blood with toll-like receptor 7/8 (TLR7/8) agonists. This treatment stimulated a rapid NOX2 transcription and translation through a process involving mammalian target of rapamycin (mTOR) whose expression was also deficient in patients’ neutrophils. NOX2 expression was also increased by the TLR4 agonist lipopolysaccharide but with only a modest improvement of reactive oxygen species production. Conclusion Impairment of neutrophil oxidants production in alcoholic cirrhosis is associated with NOX2 degradation and deficient mTOR-dependent translational machinery. The NOX2 depletion can be reversed via TRL7/8 activation and might be used to restore antimicrobial responses of immunocompromised patients.

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis.

Objective: Olive mill wastewater (OMWW) is a by-product of olive oil production and contains biologically active compounds. The aim of this study was to investigate the effect of OMWW on intestinal glucose absorption in mice, a target for treating diabetes. Methods: The effect oral administration of OMWW on mice glycemia was evaluated by oral glucose tolerance tests (OGTT), and mice jejunal mucosal strips were mounted in Ussing chambers to evaluate the activity of sodium-dependent glucose transporter (SGLT-1). Western blot analysis was performed to study SGLT-1 and protein kinase C (PKC) at the brush border membranes (BBM) of mice jejunal mucosa. Results: We found that the raised glycemia in mice in response to oral glucose administration (OGTT) was decreased in presence of OMWW, being highly effective at 4 g/kg of body weight. Further, OMWW inhibited glucose induced short-circuit current (Isc) in mice jejunal, which is a marker of SGLT-1 activity. The effect was rapid, with maximal inhibition of 80 % versus control. Also, OMWW induced a strong decrease of SGLT-1 protein (60%) as well as PKC (30%), a key effector protein in BBM of mice jejunal mucosa in response to glucose. Conclusion: Our results suggest that OMWW may be beneficial for diabetes through its novel hypoglycemic effect by acting on the PKC-SGLT-1 axis.

Background & Aims To determine the pathogenesis of liver nodules, and lesions similar to obliterative portal venopathy, observed after portosystemic shunts or portal vein thrombosis in humans. Methods We conducted an experimental study comparing portacaval shunt (PCS), total portal vein ligation (PVL), and sham (S) operated rats. Each group were either sacrificed at 6 weeks (early) or 6 months (late). Arterial liver perfusion was studied in vivo using CT, and histopathological changes were noted. Liver mRNA levels were quantified by RT-QPCR for markers of inflammation (Il10, Tnfa), proliferation (Il6st, Mki67, Hgf, Hnf4a), angiogenesis: (Vegfa, Vegfr 1, 2 and 3; Pgf), oxidative stress (Nos2, and 3, Hif1a), and fibrosis (Tgfb). PCS and PVL were compared to the S group. Results Periportal fibrosis and arterial proliferation was observed in late PCS and PVL groups. CT imaging demonstrated increased arterial liver perfusion in the PCS group. RT-QPCR showed increased inflammatory markers in PCS and PVL early groups. Tnfa and Il10 were increased in PCS and PVL late groups respectively. All proliferative markers increased in the PCS, and Hnf4a in the PVL early groups. Mki67 and Hnf4a were increased in the PCS late group. Nos3 was increased in the early and late PCS groups, and Hif1a was decreased in the PVL groups. Markers of angiogenesis were all increased in the early PCS group, and Vegfr3 and Pgf in the late PCS group. Only Vegfr3 was increased in the PVL groups. Tgf was increased in the PCS groups. Conclusions Portal deprivation in rats induces a sustained increase in intrahepatic markers of inflammation, angiogenesis, proliferation, and fibrosis.

Whereas the remodeling of intestinal mucosa after bariatric surgeries has been the matter of numerous studies to our knowledge, very few reported on the remodeling of the residual gastric mucosa. In this study, we analyzed remodeling of gastric mucosa after Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) in rats. Diet-induced obese rats were subjected to RYGB, VSG or sham surgical procedures. All animals were assessed for food intake, body-weight, fasting blood, metabolites and hormones profiling, as well as insulin and glucose tolerance tests before and up to 5 weeks post-surgery. Remodeling of gastric tissues was analyzed by routine histology and immunohistochemistry studies, and qRT-PCR analyses of ghrelin and gastrin mRNA levels. In obese rats with impaired glucose tolerance, VSG and RYGB caused substantial weight loss and rats greatly improved their oral glucose tolerance. The remaining gastric mucosa after VSG and gastric pouch (GP) after RYGB revealed a hyperplasia of the mucous neck cells that displayed a strong immunoreactivity for parietal cell H+/K+-ATPase. Ghrelin mRNA levels were reduced by 2-fold in remaining fundic mucosa after VSG and 10-fold in GP after RYGB. In the antrum, gastrin mRNA levels were reduced after VSG in line with the reduced number of gastrin positive cells. This study reports novel and important observations dealing with the remaining gastric mucosa after RYGB and VSG. The data demonstrate, for the first time, a hyperplasia of the mucous neck cells, a transit cell population of the stomach bearing differentiating capacities into zymogenic and peptic cells.

Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.

Hepcidin, the key regulatory hormone of iron homeostasis, and iron carriers such as transferrin receptor1 (TFR1), divalent metal transporter1 (DMT1), and ferroportin (FPN) are expressed in kidney. Whether hepcidin plays an intrinsic role in the regulation of renal iron transport is unknown. Here, we analyzed the renal handling of iron in hemochromatosis Hepc(-/-) and Hjv(-/-) mouse models, as well as in phenylhydrazine (PHZ)-treated mice. We found a marked medullary iron deposition in the kidneys of Hepc(-/-) mice, and iron leak in the urine. The kidneys of Hepc(-/-) mice exhibited a concomitant decrease in TFR1 and increase in ferritin and FPN expression. Increased FPN abundance was restricted to the thick ascending limb (TAL). DMT1 protein remained unaffected despite a significant decrease of its mRNA level, suggesting that DMT1 protein is stabilized in the absence of hepcidin. Treatment of kidney sections from Hepc(-/-) mice with hepcidin decreased DMT1 protein, an effect confirmed in renal cell lines where hepcidin markedly decreased Fe-55 transport. In the kidneys of Hjv(-/-) mice exhibiting low hepcidin expression, the iron overload was similar to that in the kidneys of Hepc(-/-) mice. However, in PHZ mice, iron accumulation resulting from hemoglobin leak was detected in the proximal tubule. Thus, kidneys exhibit a tissue-specific handling of iron that depends on the extra iron source. Hepcidin may control the expression of iron transporters to prevent renal iron overload.

Background & Aims: Lipopolysaccharide (LPS)-expressing bacteria cause severe inflammation in cirrhotic patients. The global gene response to LPS is unknown in cirrhotic immune cells.Methods: Gene-expression profiling using Affymetrix Human Exon Array analyzed the expression of 14,851 genes in LPS-stimulated peripheral blood mononuclear cells (PBMCs) from 4 patients with cirrhosis and 4 healthy subjects. We performed validation studies using RT-qPCR in LPS-stimulated PBMCs from 52 patients and 9 healthy subjects and investigated the association of gene induction with mortality in 26 patients.Results: Gene-expression profiling of LPS-stimulated cirrhotic cells showed 509 upregulated genes and 1588 downregulated genes. In LPS-stimulated "healthy" cells, 952 genes were upregulated and 838 genes downregulated. The 741 LPS-regulated genes shared by cirrhotic and "healthy" cells were involved in cytokine production/activity and induction of "immune paralysis". Comparison of functions associated with the 1356 genes, specifically regulated by LPS in cirrhotic cells, to functions of the 1049 genes, specifically regulated in "healthy" cells, allowed to define a cirrhosis-specific phenotype. Unlike in "healthy" cells, LPS failed to induce an interferon-mediated program in cirrhotic cells. In cirrhotic PBMCs, LPS specifically induced certain molecules involved in apoptosis and downregulated molecules involved in endocytic trafficking. RT-qPCR experiments showed that LPS-stimulated cirrhotic PBMCs had an enhanced induction of certain proinflammatory cytokines and chemokines. In the prognosis study, higher ex vivo LPS-induction of the inflammatory genes IL6 and CXCL5 was a significant predictor of mortality.Conclusions: Our results show that LPS-stimulated cirrhotic PBMCs exhibit an extensive and often unexpected transcriptional response. (c) 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

BACKGROUND & AIMS:Circulating membrane-shed microparticles (MPs) participate in regulation of vascular tone. We investigated the cellular origins of MPs in plasma from patients with cirrhosis and assessed the contribution of MPs to arterial vasodilation, a mechanism that contributes to portal hypertension.METHODS:We analyzed MPs from blood samples of 91 patients with cirrhosis and 30 healthy individuals (controls) using flow cytometry; their effects on the vascular response to vasoconstrictors were examined in vitro and in vivo.RESULTS:Circulating levels of leuko-endothelial (CD31(+)/41(-)), pan-leukocyte (CD11a(+)), lymphocyte (CD4(+)), and erythrocyte (CD235a(+)) MPs were higher in patients with cirrhosis than in controls. Plasma of patients with cirrhosis contained hepatocyte-derived MPs (cytokeratin-18(+)), whereas plasma from controls did not. The severity of cirrhosis and systemic inflammation were major determinants of the levels of leuko-endothelial and hepatocyte MPs. MPs from patients with advanced cirrhosis significantly impaired contraction of vessels in response to phenylephrine, whereas MPs from healthy controls or from patients of Child-Pugh class A did not. This effect depended on cyclooxygenase type 1 and required phosphatidylserine on the surface of MPs. Intravenous injection of MPs from patients with cirrhosis into BALB/C mice decreased mean arterial blood pressure.CONCLUSIONS:Cirrhosis is associated with increases in circulating subpopulations of MPs, likely resulting from systemic inflammation and liver cell damage. The overall pool of circulating MPs from patients with advanced cirrhosis impairs vasoconstrictor responses and decreases blood pressure, contributing to the arterial vasodilation associated with portal hypertension.

Objective:Anemia is common in critically ill patients, due to inflammation and blood loss. Anemia can be associated with iron deficiency and low serum hepcidin levels. However, iron administration in this setting remains controversial because of its potential toxicity, including oxidative stress induction and sepsis facilitation. The objective of this work was to determine the efficacy and toxicity of iron administration using a mouse model mimicking critical care anemia as well as a model of acute septicemia. Design:Prospective, randomized, open label controlled animal study. Setting:University-based research laboratory. Subjects:C57BL/6 and OF1 mice. Interventions:Intraperitoneal injection of zymosan inducing generalized inflammation in C57BL/6 mice, followed in our full model by repeated phlebotomies. A dose equivalent to 15 mg/kg of ferric carboxymaltose was injected intravenously on day 5. To assess the toxicity of iron in a septicemia model, OF1 mice were simultaneously injected with iron and different Escherichia coli strains. Measurements and Main Results:To investigate the effect of iron on oxidative stress, we measured reactive oxygen species production in the blood using luminol-amplified chemiluminescence and superoxide dismutase 2 messenger RNA levels in the liver. These markers of oxidative stress were increased after iron administration in control mice but not in zymosan-treated mice. Liver catalase messenger RNA levels decreased in iron-treated control mice. Iron administration was not associated with increased mortality in the septicemia model or in the generalized inflammation model. Iron increased hemoglobin levels in mice fed with a low iron diet and subjected to phlebotomies and zymosan 2 wks after treatment administration. Conclusions:Adverse effects of intravenous iron supplementation by ferric carboxymaltose seem to be minimal in our animal models. Furthermore, iron appears to be effective in correcting anemia, despite inflammation. Studies of efficacy and safety of iron in critically ill patients are warranted.