Manuela Costanzo

Characterization of adherent-invasive Escherichia coli isolated from pediatric patients with inflammatory bowel disease†

Background: Crohns disease (CD) and ulcerative colitis (UC), known as inflammatory bowel diseases (IBD), are characterized by an abnormal immunological response to commensal bacteria colonizing intestinal lumen and mucosa. Among the latter, strains of adherent‐invasive Escherichia coli (AIEC), capable of adhering to and invading epithelium, and to replicate in macrophages, have been described in CD adults. We aimed at identifying and characterizing AIEC strains in pediatric IBD. Methods: In all, 24 CD children, 10 UC, and 23 controls were investigated. Mucosal biopsies, taken during colonoscopy, were analyzed for the presence of AIEC strains by an adhesive‐invasive test. Protein expression of the specific AIEC receptor, the carcinoembryonic antigen‐related cell adhesion molecule 6 (CEACAM6), was evaluated by western blot and immunohistochemistry, while tumor necrosis factor alpha (TNF‐&agr;) and interleukin (IL)‐8 mRNA expression was detected by real‐time polymerase chain reaction (PCR), after bacterial infection. Transmission electron microscopy and trans‐epithelial electric resistance assays were performed on biopsies to assess bacteria‐induced morphological and functional epithelial alterations. Results: Two bacterial strains, EC15 and EC10, were found to adhere and invade the Caco2 cell line, similar to the well‐known AIEC strain LF82 (positive control): they upregulated CEACAM6, TNF‐&agr;, and IL‐8 gene/protein expression, in vitro and in cultured intestinal mucosa; they could also survive inside macrophages and damage the epithelial barrier integrity. Lesions in the inflamed tissues were associated with bacterial infection. Conclusions: This is the first study showing the presence of adhesive‐invasive bacteria strains in the inflamed tissues of children with IBD. Collective features of these strains indicate that they belong to the AIEC spectrum, suggesting their possible role in disease pathogenesis. (Inflamm Bowel Dis 2011)

Dipotassium Glycyrrhizate Inhibits HMGB1-Dependent Inflammation and Ameliorates Colitis in Mice

Background High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. Advances in targeting HMGB1 represent a major challenge to improve the treatment of acute/chronic inflammation. Aim This study is aimed at verifying whether the inhibition of HMGB1 through dipotassium glycyrrhizate (DPG) is a good strategy to reduce intestinal inflammation. Methods Human colon adenocarcinoma cell line, HT29, human epithelial colorectal adenocarcinoma, Caco2, and murine macrophage cell line, RAW 264.7, were cultured to investigate the effect of DPG on the secretion of HMGB1. Acute colitis was induced in C57BL/6 mice through administration of 3% dextran sodium sulphate (DSS); a combined treatment with DSS and 3 or 8 mg/kg/day DPG was used to investigate the effects of DPG on intestinal inflammation. Animals were euthanized at seventh day and colonic samples underwent molecular and histological analyses. Results DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1. Moreover, DPG significantly decreases the severity of DSS-induced colitis in mice. Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4. Finally, HMGB1, abundantly present in the feces of mice with DSS-induced colitis, is strongly reduced by DPG. Conclusions HMGB1 is an early pro-inflammatory cytokine and an active protagonist of mucosal gut inflammation. DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation. Thus, we reason that DPG could represent an innovative tool for the management of human intestinal inflammation.

NOD2 Is Regulated by MIR-320 in Physiological Conditions but this Control Is Altered in Inflamed Tissues of Patients with Inflammatory Bowel Disease

Background:Large evidence supports the role of microRNAs as new important inflammatory mediators by regulating both the adaptive and innate immunity. In the present study, we speculated that miR-320 controls NOD2 (nucleotide-binding oligomerization domain) expression, because it contains multiple binding sites in the 3′-untranslated region of the gene. NOD2, the first gene associated to increased susceptibility to Crohns disease, is a cytosolic receptor that senses wall peptides of bacteria and promotes their clearance through initiation of a proinflammatory transcriptional program. This study aims at demonstrating that NOD2 is a target of miR-320 as well as investigating the role of inflammation in modulating the miR-320 control on NOD2 expression and analyzing miR-320 expression in intestinal biopsies of children with inflammatory bowel disease. Methods:The colonic adenocarcinoma cell line HT29 was used to assess the miR-320-mediated regulation of NOD2 expression. MiR-320 and NOD2 expression were analyzed in mucosal samples of 40 children with inflammatory bowel disease. Results:During inflammation, NOD2 expression is inversely correlated with miR-320 expression in vitro and ex vivo. Exogenous miR-320 transfection in HT29 cells leads to a significant decrease of NOD2 expression, whereas the miR-320 inhibitor transfection leads to increase of NOD2 expression, nuclear translocation of nuclear factor &kgr;B, and activation of downstream cytokines. Conclusions:We show for the first time that NOD2 expression is under the control of miR-320. We also show in vitro and ex vivo that inflammation induces a decrease of miR-320 and the latter correlates negatively with NOD2 expression.

Lactoferrin prevents invasion and inflammatory response following E. coli strain LF82 infection in experimental model of Crohn's disease

BACKGROUND Crohns disease is a multifactorial disease in which an aberrant immune response to commensal intestinal microbiota leads to chronic inflammation. The small intestine of patients with Crohns disease is colonized by a group of adherent-invasive Escherichia coli strongly able to adhere and invade intestinal epithelial cells lactoferrin is an iron-binding glycoprotein known to have anti-bacterial and anti-inflammatory activities. AIMS We explore the ability of bovine lactoferrin to modulate the interactions between the adherent-invasive E. coli strain LF82 and intestinal epithelial cells as well as the inflammatory response. METHODS Bacterial adhesion and invasion assays were used to assess the antimicrobial activity of lactoferrin. Electron microscopy was used to characterize bacteria-cell interactions. The mRNA expression of pro-inflammatory cytokines was measured both in cultured cells and in biopsies taken from intestine of patients affected by Crohns disease. RESULTS Lactoferrin inhibited bacterial invasion through minimally affecting adhesion. This divergence was due to a mannose-dependent lactoferrin binding to the bacterial type 1 pili and consequent bacterial aggregation on the intestinal epithelial cell surface. Expression of pro-inflammatory cytokines, such as TNF-alpha, IL-8, and IL-6, was markedly inhibited by lactoferrin both in cultured and Crohn-derived intestinal cells. CONCLUSIONS Bovine lactoferrin might function via an antibacterial and/or anti-inflammatory mechanism in the treatment of Crohns disease.

Krill oil reduces intestinal inflammation by improving epithelial integrity and impairing adherent-invasive Escherichia coli pathogenicity

BACKGROUND Krill oil is a marine derived oil rich in phospholipids, astaxanthin and omega-3 fatty acids. Several studies have found benefits of krill oil against oxidative and inflammatory damage. AIMS We aimed at assessing the ability of krill oil to reduce intestinal inflammation by improving epithelial barrier integrity, increasing cell survival and reducing pathogenicity of adherent-invasive Escherichia coli. METHODS CACO2 and HT29 cells were exposed to cytomix (TNFα and IFNγ) to induce inflammation and co-exposed to cytomix and krill oil. E-cadherin, ZO-1 and F-actin levels were analyzed by immunofluorescence to assess barrier integrity. Scratch test was performed to measure wound healing. Cell survival was analyzed by flow cytometry. Adherent-invasive Escherichia coli LF82 was used for adhesion/invasion assay. RESULTS In inflamed cells E-cadherin and ZO-1 decreased, with loss of cell-cell adhesion, and F-actin polymerization increased stress fibres; krill oil restored initial conditions and improved wound healing, reduced bacterial adhesion/invasion in epithelial cells and survival within macrophages; krill oil reduced LF82-induced mRNA expression of pro-inflammatory cytokines. CONCLUSIONS Krill oil improves intestinal barrier integrity and epithelial restitution during inflammation and controls bacterial adhesion and invasion to epithelial cells. Thus, krill oil may represent an innovative tool to reduce intestinal inflammation.

New insights into the pathogenesis of inflammatory bowel disease: Transcription factors analysis in bioptic tissues from pediatric patients

Objectives: Our work is aimed at identifying ex vivo new transcription factors, potentially involved in the pathogenesis of pediatric inflammatory bowel disease (IBD), by using a microarray approach. Patients and Methods: Microarray, including 84 transcription factors, was performed in inflamed and uninflamed mucosal tissues of pediatric patients with Crohn disease (CD) and in healthy controls. Real-time polymerase chain reaction was used to confirm microarray results on a larger size of CD and patients with ulcerative colitis (UC). Protein expression was evaluated by Western blot assay. Results: Microarray assay showed 40 genes differentially regulated in the inflamed mucosa and 17 in the uninflamed mucosa of patients with CD as compared with controls. Real-time polymerase chain reaction analysis revealed 10 transcripts in CD and 4 in UC, selected among those with higher differences as compared with healthy controls, significantly overexpressed in the inflamed tissues of patients. Moreover, 4 transcripts in CD and 2 in UC were found significantly upregulated in the uninvolved tissue. A further investigation evidenced an increased protein expression of activating transcription factor 3 and hypoxia-inducible transcription factor-1α in patients with CD as well as in Caco2 cell line stimulated by cytokines and hypoxia. Conclusions: The present study shows an evident upregulation of several transcription factors in the inflamed and uninflamed mucosa of children with IBD, suggesting that the inflammatory process is somehow activated at molecular levels even in the macroscopically normal mucosa of patients. A differential pattern of gene expression between CD and UC indicates distinct molecular mechanisms underlying the pathogenesis of 2 diseases. Finally, activating transcription factor 3 and hypoxia-inducible transcription factor-1α are proposed as new transcription factors potentially involved in the onset and maintenance of IBD.

RIP3 AND pMLKL promote necroptosis-induced inflammation and alter membrane permeability in intestinal epithelial cells

BACKGROUND Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). AIMS The aim of this study is to examine in depth in vitro and ex vivo the contribution of necroptosis to intestinal inflammation. METHODS In vitro: we used an intestinal cell line, HCT116RIP3, produced in our laboratory and overexpressing RIP3. Ex vivo: intestinal mucosal biopsies were taken from patients with inflammatory bowel disease (IBD) (20 with Crohns disease; 20 with ulcerative colitis) and from 20 controls. RESULTS RIP3-induced necroptosis triggers MLKL activation, increases cytokine/alarmin expression (IL-8, IL-1β, IL-33, HMGB1), NF-kBp65 translocation and NALP3 inflammasome assembly. It also affects membrane permeability by altering cell-cell junctional proteins (E-cadherin, Occludin, Zonulin-1). Targeting necroptosis through Necrostatin-1 significantly reduces intestinal inflammation in vitro and in cultured intestinal explants from IBD. CONCLUSION We show for the first time in vitro and ex vivo that RIP3-driven necroptosis seriously affects intestinal inflammation by increasing pMLKL, activating different cytokines and alarmins, and altering epithelial permeability. The inhibition of necroptosis causes a significant decrease of all these effects. These data strongly support the view that targeting necroptosis may represent a promising new option for the treatment of inflammatory enteropathies.

The Nuclear Protein HMGB1 is Increased in the Stools of IBD Patients and Appears to Be a Novel Marker of Intestinal Inflammation

Background The balance between T regulatory cells (Treg) and T helper type 17 (Th17) cells appears important in modulating immune responses in inflammatory bowel disease. CD39 is an immune cell ectonucleotidase, expressed in lipid rafts, which generates immune suppressive adenosine. Genetic polymorphisms of CD39 are noted in IBD and the ectoenzyme has been validated as a marker of memory type Treg. CD161 (killer cell lectin subfamily B, member 1 or NKR-P1A) is a relatively specific surface marker for human Th17 cells and is linked to activation of acid sphingomyelinase that might in turn impact CD39 bioactivity. It is unknown whether there are T cell populations in IBD that express both markers and the putative relevance is unexplored. Aims: To identify dual expression of CD39 and CD161 in blood T cells from patients with IBD, and explore the role of CD161 in regulating immune deviation of CD4+CD39+ T cell populations. Methods The percentage of CD39+CD161+ CD4 T cells in peripheral blood of patients with active or inactive Crohns disease was determined by FACS. Four phenotypic subtypes of peripheral blood CD4 T cells including CD39+CD161+, CD39+CD161-, CD39-CD161+, and CD39-CD161from healthy donors were sorted by FACS, and stimulated respectively by anti-CD3/CD28 antibodies with with TGF-beta and IL-2 (for Treg cell expansion) or IL-6, IL-23, IL-1beta and TGFbeta (for Th17 cell expansion) for 3 days, and intracellular IL-17 and INF-gamma or FOXP3 were detected by FACS. These four subgroups were treated by ATP or NAD+ for 1 hour, and stained by Annexin V to detect early events of P2X7-mediated apoptosis. Result The percentage of CD39+CD161+ CD4 T cells in peripheral blood of normal controls, active and inactive Crohns disease patients was 1.26±0.73, 4.55±2.39 and 0.95±0.52%, respectively (p=0.01, for active disease vs. both controls and inactive Crohns disease). After 3 days, Treg expansion, of FOXP3-positive cells within the CD39+CD161+ group, was less than from the CD39+CD161group (p=0.01). After 3 days In Vitro expansion, the percentages of intracellular IL-17-positive and/or IFN-gamma-positive cells in CD4+CD39+CD161+ populations were higher than other three groups (p=0.01). However, CD39+CD161+ CD4 T cells also exhibited highest levels of NAD+-induced apoptosis (p=0.05 vs. other groups). Conclusions Peripheral blood CD4+CD39+CD161+ T cells are increased in active Crohns disease. CD161 expression impacts the reciprocal balance of Treg and Th17 cells generated from CD4+CD39+ cells.

Krill oil, vitamin D and Lactobacillus reuteri cooperate to reduce gut inflammation

Current research into original therapies to treat intestinal inflammation is focusing on no-drug therapies. KLD is a mixture of krill oil (KO), probiotic Lactobacillus reuteri (LR), and vitamin D (VitD3). The aim of this study was to assess in vitro and in vivo the potential cooperative effects of KLD in reducing gut inflammation. Colorectal adenocarcinoma cell lines, CACO2 and HT29, and C57BL/6 mice were used for in vitro and in vivo analyses, respectively. Cells were exposed to cytomix (interferon gamma + tumour necrosis factor alpha (TNF-α)) to induce inflammation or co-exposed to cytomix and KO, LR and VitD3 alone or to cytomix and KLD. Animals were treated for 7 days with dextran sodium sulphate (DSS) to induce colitis or with DSS and KLD. In vitro assays: F-actin expression was analysed by immunofluorescence; scratch test and trans-epithelial electric resistance test were performed to measure wound healing; adhesion/invasion assays of adhesive and invasive Escherichia coli (AIEC) bacteria were made; mRNA expression of TNF-α, interleukin (IL)-8 and vitamin D receptor (VDR) was detected by quantitative PCR. In vivo assays: body weight, clinical score, histological score and large intestine weight and length were estimated; mRNA expression of TNF-α, IL-1β, IL-6, IL-10 by quantitative PCR; VDR expression was detected by quantitative PCR and immunohistochemistry. In vitro: KLD restores epithelial cell-cell adhesion and mucosal healing during inflammation, while decreases the adhesiveness and invasiveness of AIEC bacteria and TNF-α and IL-8 mRNA expression and increases VDR expression. In vivo: KLD significantly improves body weight, clinical score, histological score and large intestine length of mice with DSS-induced colitis and reduces TNF-α, IL-1β and IL-6 mRNA levels, while increases IL-10 mRNA and VDR levels. KLD has significant effects on the intestinal mucosa, strongly decreasing inflammation, increasing epithelial restitution and reducing pathogenicity of harmful commensal bacteria.