Anne Jarry

Proprotein Convertase Subtilisin Kexin Type 9 Null Mice Are Protected From Postprandial Triglyceridemia

Objectives—Proprotein convertase subtilisin kexin type 9 (PCSK9) is a natural inhibitor of the low-density lipoprotein receptor, and its deficiency in humans results in low plasma LDL-cholesterol and protection against cardiovascular disease. We explored whether PCSK9 expression impacts postprandial triglyceridemia, another important cardiovascular risk factor. Methods and Results—Real-time PCR and confocal microscopy were used to show that PCSK9 is expressed throughout the entire small intestine and in human enterocytes. On olive oil gavage, PCSK9-deficient mice showed a dramatically decreased postprandial triglyceridemia compared with their wild-type littermates. Lymph analysis revealed that intestinal TG output is not quantitatively modified by PCSK9 deletion. However, PCSK9−/− mice present with a significant reduction of lymphatic apoB secretion compared to PCSK9+/+ mice. Modulating PCSK9 expression in polarized CaCo-2 cells confirmed the relationship between PCSK9 and apoB secretion; PCSK9−/− mice consistently secrete larger TG-rich lipoprotein than wild-type littermates. Finally, kinetic studies showed that PCSK9-deficient mice have an increased ability to clear chylomicrons compared to wild-type littermates. Conclusion—These findings indicate that in addition to its effect on LDL-cholesterol, PCSK9 deficiency might protect against cardiovascular disease by reducing postprandial triglyceridemia.

CD4CD8αα Lymphocytes, A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease

Gut bacterium Faecalibacterium prausnitzii activates a newly identified set of human IL-10-producing Treg cells (CD4CD8αα lymphocytes), revealing a mechanism by which commensal microbes contribute to host immunity.

N-cadherin serves as diagnostic biomarker in intrahepatic and perihilar cholangiocarcinomas

As a definite immunoprofile of this tumor is missing, the histopathologic diagnosis of intrahepatic cholangiocarcinoma is difficult. The aim of this study was to explore E- and N-cadherin expressions in intrahepatic bile duct tumors, and to determine their potential interest in differential diagnosis. Normal liver tissue, 5 cirrhosis with ductular reaction, 5 focal nodular hyperplasia, 5 bile duct hamartomas, 5 bile duct adenomas, and 45 intrahepatic cholangiocarcinomas from Causasian patients were studied. Tissue-microarrays including 20 esophageal, 86 gastric, 8 small bowel, 64 colonic, 18 pancreatic, 6 gallbladder, and 7 extrahepatic biliary tract adenocarcinomas, 22 hepatocellular carcinomas, and normal tissues were constructed. Immunohistochemistry was performed using E-cadherin, N-cadherin, NCAM, Hep Par1, and cytokeratins 7, 19 and 20. Immunoblot analysis of frozen liver tissues was performed to control the specificity of E- and N-cadherin antibodies used. In normal liver, epithelial cells of intrahepatic bile ducts, whatever their caliber, as well as hepatocytes, coexpressed E- and N-cadherins at their plasma membranes. In cirrhosis, ductular reactions completely expressed E- and N-cadherins. All the benign lesions and 30 of the 45 intrahepatic cholangiocarcinomas (23/29 peripheral and 7/16 hilar) also expressed N-cadherin. E-cadherin was detected in all the lesions. The expression of N-cadherin at the plasma membrane of tumor cells was significantly more frequent in peripheral than in hilar intrahepatic cholangiocarcinomas (P=0.003). Among noncholangiocarcinomas, only 1% gastric and 66% gallbladder adenocarcinomas and all the hepatocellular carcinomas expressed N-cadherin at the membrane of tumor cells. Finally, for the diagnosis of intrahepatic cholangiocarcinomas, the specificity value of membranous expression of N-cadherin was 88%, whereas that of the combination cytokeratin 7/membranous N-cadherin was 98%. In the gastrointestinal and liver tract, membranous N-cadherin is restricted to the hepatocytes and intrahepatic biliary cells. In combination with cytokeratin 7 and Hep Par1, N-cadherin is a reliable tool for the histopathological diagnosis of primary hepatic tumors.

KLF4‐dependent, PPARγ‐induced expression of GPA33 in colon cancer cell lines

The glycoprotein A33 (GPA33) is a colon cancer antigen. Phase I trials with 131I and 125I monoclonal antibody A33 in colon carcinoma patients showed excellent localization to colorectal cancer and some evidence of tumor response. Using DNA microarrays, we have identified the GPA33 gene as a target of PPARγ in HT29‐Cl.16E colon cancer cells. Treatment of HT29‐Cl.16E, Caco2, SW1116 and LS174T colon cancer cells with the PPARγ agonist GW7845 induced a 2‐ to 6‐fold increase in GPA33 mRNA as determined by real‐time PCR. This induction was also found in HT29‐Cl.16E cells treated with rosiglitazone and ciglitazone and was prevented by cotreatment with the PPARγ antagonist GW9662, indicating that this regulation was PPARγ dependent. No canonical PPAR responsive element was found in the GPA33 promoter. We therefore analyzed the expression of transcription factors involved in GPA33 expression. CDXl, CDX2 and KLF5 expression was not modified by PPARγ activation. By contrast, a significant increase in KLF4 was seen, both at mRNA and protein levels. Furthermore, chromatin immunoprecipitation studies demonstrated that an increased amount of KLF4 protein was bound to the GPA33 promoter in cells treated with rosiglitazone. Finally, downregulation of KLF4 expression by siRNA reduced rosiglitazone‐induced GPA33 expression. This indicates that PPARγ activation induces KLF4 expression, which in turn increases GPA33 expression. We also demonstrate that PPARγ activation leads to increased (p21WAF1/Cip1 and keratin 19) or decreased (cyclin D1) expression of known KLF4 targets, suggesting that KLF4 is a nodal player in a network of PPARγ‐regulated genes.

Loss of Interleukin-10 or Transforming Growth Factor β Signaling in the Human Colon Initiates a T-Helper 1 Response Via Distinct Pathways

BACKGROUND & AIMS Signaling via interleukin (IL)-10 or transforming growth factor (TGF)-β is disrupted in subpopulations of patients with inflammatory bowel disease, but it is not clear how a T-helper (Th) 1 cell response is induced. We studied conversion of human mucosal innate immune cells into inflammatory cells and the initiation of a Th1 cell response following loss of IL-10 or TGF-β signaling. METHODS We depleted IL-10 or TGF-β from explant cultures of human normal colonic mucosa using immunoneutralization. Pharmacologic inhibitors and antibodies were used to determine the factors involved in the initiation of an interferon (IFN)-γ response following loss of TGF-β or IL-10 signaling. Cytokines produced by mucosal cells were assessed by enzyme-linked immunosorbent assay and quantitative reverse-transcriptase polymerase chain reaction. The subsets of cells involved in cytokine production were determined by in situ immunofluorescence analysis and flow cytometry after digestion of the explants with collagenase. RESULTS Depletion of IL-10 from human normal colonic mucosa resulted in an IFN-γ response, characterized by early-stage secretion of mature IL-18 and production of the active form of caspase-1 by macrophages and some epithelial cells. A caspase-1 inhibitor or the IL-18 antagonist IL-18-binding protein blocked this response. By contrast, depletion of TGF-β resulted in an IFN-γ response that was preceded by and required secretion of IL-12 from macrophages, dendritic cells, and epithelial cells. CONCLUSIONS Innate immune cells (macrophages and epithelial cells) activate a Th1 cell response in explant cultures of human normal colonic mucosa depleted in IL-10 or TGF-β via distinct, nonredundant pathways. These pathways might contribute to the pathogenesis of inflammatory bowel disease.

Acute cytotoxicity of MIRA-1/NSC19630, a mutant p53-reactivating small molecule, against human normal and cancer cells via a caspase-9-dependent apoptosis

Although numerous studies have focused on the mechanisms of action of the candidate chemotherapeutic drug MIRA-1/NSC19630, initially described as a mutant p53-reactivating small molecule, the issue of its toxicological evaluation remains open. Here, we devised a strategy to examine the effects of MIRA-1 on a variety of human normal cells and cancer cell lines. First, we demonstrated a massive and rapid (within 2 hours) MIRA-1 apoptotic effect on human normal primary epithelial cells as shown using an intestinal mucosa explant assay. MIRA-1 was also cytotoxic to primary and subcultured human mesenchymal cells. Interestingly these effects were restricted to actively proliferating cells. Second, MIRA-1 acute toxicity was independent of p53, since it occurred in human normal cells with increased or silenced p53 expression level, in cancer cells derived from solid or liquid tumors, with either mutated or wt TP53, and in cancer cells devoid of p53. Third, combined pharmacological and genetic approaches showed that MIRA-1 acute cytotoxicity was mediated by a caspase-9-dependent apoptosis. In conclusion, our strategy unveils the limitations of the targeted action of a small molecule designed to reactivate mutant p53.

Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli

Adherent-invasive Escherichia coli (AIEC), associated with Crohn’s disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

Interferon-Alpha Promotes Th1 Response and Epithelial Apoptosis via Inflammasome Activation in Human Intestinal Mucosa

Backgound & Aims Several lines of investigation suggest that interferon (IFN) alpha can alter human intestinal mucosa homeostasis. These include the endogenous production of IFN alpha in celiac disease or inflammatory bowel diseases, as well as the occurrence of intestinal side effects of exogenous IFN alpha used as a therapeutic tool. Here, we present an ex vivo translational approach to investigate the effects of IFN alpha on the human normal intestinal mucosa, as well as its underlying mechanisms. Methods Human normal colonic mucosa explants were cultured in the presence or absence of IFN alpha 2a. Epithelial homeostasis was assessed using the immunohistochemical marker of apoptosis M30. The Wnt inhibitor Dickkopf-Homolog-1 (DKK1) was assayed in the supernatants by enzyme-linked immunosorbent assay. Activation of the inflammasome (caspase-1/interleukin [IL]18) and of a Th1 response was determined by in situ detection of active caspase-1, as well as by measurement of mature IL18 production and the prototype Th1 cytokine IFN gamma by enzyme-linked immunosorbent assay. In addition, mechanistic studies were performed using the specific caspase-1 inhibitor Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (YVAD-FMK), IL18-binding protein, neutralizing anti–IFN gamma, and anti-DKK1 antibodies. Results IFN alpha 2a elicited a rapid (24 hours) disruption of surface and crypt colonic epithelial cells via apoptosis that was variable in intensity among the 20 individuals studied. This apoptotic effect was dependent on the initiation of an IFN gamma response elicited by resident T box expressed in T cells–positive lamina propria cells. Both apoptosis and Th1 response were subordinated to active caspase-1 and IL18 production. Finally, neutralization of IFN gamma–induced DKK1 partially protected against IFN alpha–induced epithelial apoptosis. Conclusions By using an ex vivo model, we show an interindividual heterogeneity of IFN alpha effects. We show that IFN alpha is able to disrupt both epithelial and immune homeostasis in the human intestine, by activation of an innate immunity platform, the inflammasome, which drives a Th1 response and leads to epithelial barrier disruption.

In situ evidence of involvement of Schwann cells in ulcerative colitis: autocrine and paracrine signaling by A disintegrin and metalloprotease-17–mediated tumor necrosis factor α production

An increase in S100 protein-positive cells has been reported in inflammatory bowel diseases, mainly Crohn disease. These cells were interpreted as myeloid-derived dendritic cells, chiefly in follicular areas. We were prompted to assess the nature of these cells in interfollicular areas of inflamed colonic mucosa in ulcerative colitis and study their involvement in tumor necrosis factor alpha production, the main inflammatory cytokine in ulcerative colitis. The number and distribution of cells expressing S100 protein, nerve growth factor receptor, CD68, CD1a, CD83, and calretinin were studied in samples from 16 patients with active ulcerative colitis using simple and double immunohistochemistry. Then, the localization in S100 protein-positive cells of (1) tumor necrosis factor alpha, (2) its sheddase A disintegrin and metalloprotease-17, and (3) the receptors TNFR1 was assessed using double immunofluorescence followed by confocal microscopy. In active ulcerative colitis, there was an increased number in S100 protein-positive cells in interfollicular areas of colonic mucosa compared with quiescent ulcerative colitis, nonulcerative colitis, or normal mucosa. All S100 protein-positive cells ensheathed calretinin-positive axons, indicating their Schwann cell origin. No CD1a- or CD83-positive dendritic cells were detected. Double immunofluorescence studies showed that in normal colon, Schwann cells of the mucosa and submucosal plexuses weakly expressed A disintegrin and metalloprotease-17 but did not express tumor necrosis factor alpha. By contrast, in active ulcerative colitis, they expressed both A disintegrin and metalloprotease-17 and tumor necrosis factor alpha. Schwann cells as well as calretinin-positive axons strongly expressed TNFR1. This study shows (1) a Schwann cell proliferation in the inflamed colonic mucosa during active ulcerative colitis and (2) that Schwann cells produce tumor necrosis factor alpha. Tumor necrosis factor alpha is thus likely to stimulate Schwann cell proliferation through an autocrine/paracrine mechanism.