Misagh Alipour

Mucosal Barrier Depletion And Loss Of Bacterial Diversity Are Primary Abnormalities In Paediatric Ulcerative Colitis

BACKGROUND AND AIMS Ulcerative colitis [UC] is associated with colonic mucosa barrier defects and bacterial dysbiosis, but these features may simply be the result of inflammation. Therefore, we sought to assess whether these features are inherently abrogated in the terminal ileum [TI] of UC patients, where inflammation is absent. METHODS TI biopsies from paediatric inflammatory bowel disease [IBD] subsets [Crohns disease [CD; n = 13] and UC [n = 10]], and non-IBD disease controls [n = 12] were histologically graded, and alcian blue/periodic acid-Schiff stained biopsies were quantified. The mucosal barrier was assessed for mucin [MUC2], immunoglobulin [Ig]A, IgG, and total bacteria (fluorescence in-situ hybridisation [FISH probe EUB338]) by immunofluorescence. The regulation of mucin secretion was investigated by NLRP6 gene expression and immunofluorescence. The composition of the active mucosa-associated microbiota was explored by sequencing the 16S rRNA amplicon generated from total RNA. RESULTS Despite the absence of ileitis, UC patients displayed ileal barrier depletion illustrated by reductions in mucin-containing goblet cells and mucin production and altered epithelial NLRP6 expression. In both CD patients with ileitis and UC patients with normal histology, bacteria coated with IgA and IgG penetrated the TI mucin layer. Biopsy 16S rRNA sequencing revealed a reduction in α-diversity by three methods [Shannon, Simpson, and Equitability indices] between UC and non-IBD paediatric patients. CONCLUSIONS These findings suggest an underlying defect in the UC-afflicted intestinal tract even in the absence of inflammation, implicating barrier and microbial changes as primary abnormalities in UC that may play a causative role in disease development.

A balanced IL-1β activity is required for host response to Citrobacter rodentium infection.

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3−/− mice but increased tissue damage when given to WT mice.

Epithelial cell extrusion leads to breaches in the intestinal epithelium.

Background:Two distinct forms of intestinal epithelial cell (IEC) extrusion are described: 1 with preserved epithelial integrity and 1 that introduced breaches in the epithelial lining. In this study, we sought to determine the mechanism underlying the IEC extrusion that alters the permeability of the gut epithelium. Methods:IEC extrusions in polarized T84 monolayer were induced with nigericin. Epithelial permeability was assessed with transepithelial electrical resistance and movements of latex microspheres and green fluorescent protein–transfected Escherichia coli across the monolayer. In vivo IEC extrusion was modulated in wild-type and a colitic (interleukin-10 knock-out) mouse model with caspase-1 activation and inhibition. Luminal aspirates and mucosal biopsies from control patients and patients with inflammatory bowel disease were analyzed for caspase-1 and caspase-3&7 activation. Results:Caspase-1–induced IEC extrusion in T84 monolayers resulted in dose-dependent and time-dependent barrier dysfunction, reversible with caspase-1 inhibition. Moreover, the movements of microspheres and microbes across the treated epithelial monolayers were observed. Increased caspase-1–mediated IEC extrusion in interleukin-10 knock-out mice corresponded to enhanced permeation of dextran, microspheres, and translocation of E. coli compared with wild type. Caspase-1 inhibition in interleukin-10 knock-out mice resulted in a time-dependent reduction in cell extrusion and normalization of permeability to microspheres. Increased IEC extrusion in wild-type mice was induced with caspase-1 activation. In human luminal aspirates, the ratio of positively stained caspase-1 to caspase-3&7 cells were 1:1 and 2:1 in control patients and patients with inflammatory bowel disease, respectively; these observations were confirmed by cytochemical analysis of mucosal biopsies. Conclusions:IEC extrusion mediated by caspase-1 activation contributes to altered intestinal permeability in vitro and in vivo.

Inflammasome Activation by ATP Enhances Citrobacter rodentium Clearance through ROS Generation

Background: Nod-like receptor family, pyrin domain containing 3 (NLRP3) is an important cytosolic sensor of cellular stress and infection. Once activated, NLRP3 forms a multiprotein complex (inflammasome) that triggers the maturation and secretion of interleukin (IL)-1β and IL-18. We aimed to define the consequences of NLRP3 induction, utilizing exogenous adenosine triphosphate (ATP) as an inflammasome activator, to determine if inflammasome activation increases macrophage killing of Citrobacter rodentium and define mechanisms. Methods: Bacterial survival was measured using a gentamicin protection assay. Inflammasome activation or inhibition in mouse J774A.1 macrophages were assessed by measuring IL-1β; cytokines and reactive oxygen species (ROS) were measured by ELISA and DCFDA, respectively. Results: Activation of the inflammasome increased bacterial killing by macrophages and its inhibition attenuated this effect with no impact on phagocytosis or cell death. Furthermore, inflammasome activation suppressed pro-inflammatory cytokines during infection, possibly due to more effective bacterial killing. While the infection increased ROS production, this effect was reduced by inflammasome inhibitors, indicating that ROS is inflammasome-dependent. ROS inhibitors increased bacterial survival in the presence of ATP, suggesting that inflammasome-induced bacterial killing is mediated, at least in part, by ROS activity. Conclusion: Improving inflammasome activity during infection may increase bacterial clearance by macrophages and reduce subsequent microbe-induced inflammation.

P-323 Immunoglobulin G Selectively Identifies Pathosymbionts in Paediatric Inflammatory Bowel Diseases

Background: Inflammatory bowel diseases (IBD) are a group of complex and multifactorial disorders with unknown etiology. Both environmental and genetic factors contribute to disease pathogenesis. This complexity is further exacerbated with an uncontrolled immune response to the gut microbiota. Given that immunoglobulin (Ig) G is formed in response to invasive microbes, we anticipated that bacteria bound by IgG are more likely to be virulent; their isolation could help identify such invasive strains and define mechanisms of immune activation. We hypothesized that host IgG immune response can be used to identify bacteria that may be involved in the pathogenesis of IBD. Methods: Aspirate washes from the terminal ileum of pediatric IBD (including Crohn disease [CD] and ulcerative colitis [UC]) and non-IBD controls were collected during endsocopy, stringently washed, and then fixed in paraformaldehyde. An aliquot was stored (presort) and the remaining washes were labeled with propidium iodide and anti-IgG fluorescent antibody to allow for identification of bacteria bound by IgG and those that are not. Using fluorescence-activated cell sorting (FACS), the samples were then fractioned for IgG bound (IgG+) and non-IgG bound (IgG−) bacteria. Some samples were also imaged by image cytometry to validate the FACS procedure. After sorting, DNA was extracted using beads and purified by phenol/chloroform. DNA was then analyzed by 16S sequencing using the Ilumina MiSeq platform. Results: A total of 36 washes from children without IBD (n = 10), and with CD (n = 17), and UC (n = 9), were suitable for analysis with DNA meeting quality control criteria. The ileal mucosa-associated microbiome composition of non-IBD and IBD groups did not differ significantly, with predominance of Firmicutes and Bacteroidetes. When sorted for IgG coated bacteria, the ratio of IgG+/IgG−bacteria (indicating taxa over-represented by IgG binding) were increased in CD and UC patients by 2- and 1.5-fold, respectively. Notably, the IgG+/IgG−ratio of Bacteroidetes and Proteobacteria taxa were increased in CD, Actinobacteria was increased in UC patients, and there was no changes in Firmicutes, the major phyla of the ileal mucosal microbiome. Although there was considerable inter-individual variation, at the family level, IgG binding favored Porphyromonadaceae (Bacteroidetes) and Enterobacteriaceae (Proteobacteria) in children with CD; Bifidobacteriaceae (Actinobacteria) and Barnesiellaceae (Bacteroidetes) in UC; and Clostridiaceae and Veillonellaceae (both Firmicutes) in non-IBD. Conclusions: IgG coating of mucosa associated bacteria in the ileum of IBD patients is elevated and is selective for unique phyla compared to non-IBD controls. Taxa identified by IgG include bacteria that have been associated with disease and virulence. Identification of individual microbes using this method could facilitate development of targeted diagnostic and therapeutic approaches for IBD.

Tu1702 A Balanced IL-1β Activity Is Required for Host Response to Citrobacter Rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1b. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1b to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3 2/2 and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1b (0.5 mg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3 2/2 mice developed severe colitis; IL-1b treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3 2/2 mice. In contrast, IL-1b treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3 2/2 mice, and increased severity of disease in WT mice with IL-1b treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3 2/2 compared to WT mice. IL-1b treatments elevated macrophage infiltration into infected crypts in Nlrp3 2/2 mice, suggesting that IL-1b may improve macrophage function, as exogenous administration of IL-1b increased phagocytosis of C. rodentium by peritoneal Nlrp3 2/2 macrophages in vitro .A s well, the exogenous administration of IL-1b to WT peritoneal macrophages damaged the epithelial barrier of C. rodentiuminfected polarized CMT-93 cells. Treatment of Nlrp3 2/2 mice with IL-1b seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL1b appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1b improved bacterial clearance in Nlrp3 2/2 mice but increased tissue damage when given to WT mice.

Tu1952 Title: Increased Epithelial Gap Density in the Small Bowel Is Mediated by Caspase-1 Activation of Intestinal Epithelial Cells: A Quantitative Analysis of Confocal Laser Endomicroscopy Images and Mucosal Biopsy Samples

BACKGROUND AND AIMS: There are only few case reports describing the frequency and the clinical features of pancreatic involvement (PI) in inflammatory bowel disease (IBD). We aimed to estimate the prevalence and to characterize IBD pediatric patients with PI. METHODS: We retrospectively reviewed data collected in the IBD web-registry of the Italian Society for Pediatric Gastroenterology, Hepatology and Nutrition. All children presenting with hyperamylasemia and/or hyperlipasemia were identified. Demographic and clinical data, IBD type, disease exstension and activity, laboratory data, IBD therapy, imaging findings and therapeutic interventions were evaluated. Acute pancreatitis (AP) was defined by the occurrence of at least two of the following findings: acute epigastric abdominal pain, elevated serum amylase and/or lipase ≥ 3 times the upper level of normal, and characteristic radiological changes. RESULTS: We found 27 children out of 649 (4.1%) with an increased value of amylase and/or lipase [Median Age: 148, range 65-191, mths; F/M: 14/13; Ulcerative colitis (UC): 12; Crohns disease (CD): 13; Unclassified Colitis (IBDU): 2)]. Mean serum amylase level was 205.3 ± 91.5 (range 61-413 IU/L) and mean serum lipase level was 526.8 ± 598.8 (range 37-2565 IU/L). Eleven patients (1.6%) fulfilled diagnostic criteria for AP (6 CD, 4 UC, 1 IBDU). Ten (37%) patients underwent imaging ultrasound scan, 8 (29.6%) patients had magnetic resonance cholangiopancreatography and 2 (7.4%) patients had a computed tomography scan. Pancreatic pathological findings were found in all subjects with AP. The mean lag time period between the diagnosis of IBD and the PI was 12.1 mths (range 0-65). Five patients out of 27 (18.5%) showed PI at diagnosis of IBD. Twenty-four out of 27 (88.8%) had colonic disease [10 CD, 12 UC (8 pancolitis) and 2 IBDU]. Ten patients were receiving mesalamine (ASA) and 16 patients were under immunosuppressive therapy [3 streoids, 6 azathioprine (AZT), 5 AZT + ASA, 1 methotrexate, 1 infliximab (IFX), and 1 IFX+ASA+AZT], while 1 patient was not treated. Comparing with the patients with an exclusive hyperamylasemia and/or hyperlipasemia, AP was significantly associated with female gender (p=0.018), whereas type of IBD, ongoing treatments, activity and extension of disease did not result significant risk factors. Seven out of 11 (63.6%) patients with AP needed therapeutic measures including AZT and/or ASA withdrawal, compared to only 4 (25%) patients out of 16 with serum hyperamylasemia and/or hyperlipasemia (p=0.06). CONCLUSIONS: Our study suggests that prevalence of AP in children is similar to that reported in adults. PI was more common in colonic disease. Female gender seems to be a risk factor for developing AP in children. In contrast to previous study, PI was not associated with any type of IBD.