Search

ObjectiveWhile appendectomy may reduce colorectal inflammation in patients with ulcerative colitis (UC), appendectomy has been suggested to be associated with an increased risk of colitis-associated cancer (CAC). The aim of this study was to explore the mechanism underlying the appendectomy-associated increased risk of CAC.DesignFive-week-old male BALB/c mice underwent appendectomy, appendicitis induction or sham laparotomy. They were then exposed to azoxymethane/dextran sodium sulfate (AOM/DSS) to induce CAC. Mice were sacrificed 12 weeks later, and colons were taken for pathological analysis and immunohistochemistry (CD3 and CD8 staining). Human colonic tumors from 21 UC patients who underwent surgical resection for CAC were immunophenotyped and stratified according to the appendectomy status.ResultsWhile appendectomy significantly reduced colitis severity and increased CAC number, appendicitis induction without appendectomy led to opposite results. Intra-tumor CD3+ and CD8+ T-cell densities were lower after appendectomy and higher after appendicitis induction compared to the sham laparotomy group. Blocking lymphocyte trafficking to the colon with the anti-α4β7 integrin antibody or a sphingosine-1-phosphate receptor agonist suppressed the inducing effect of the appendectomy on tumors’ number and on CD3+/CD8+ intra-tumoral density. CD8+ or CD3+ T cells isolated from inflammatory neo-appendix and intravenously injected into AOM/DSS-treated recipient mice increased CD3+/CD8+ T-cell tumor infiltration and decreased tumor number. In UC patients with a history of appendectomy, intra-tumor CD3+ and CD8+ T-cell densities were decreased compared to UC patients without history of appendectomy.ConclusionsIn UC, appendectomy could suppress a major site of T-cell priming resulting in a less efficient CAC immunosurveillance.Significance of this studyWhat is already known on this subject?The protective effect of preemptive appendectomy is currently investigated as a therapy for refractory ulcerative colitis (UC), with encouraging results.An increased risk of developing colitis-associated cancer (CAC) caused by this promising treatment has been identified.Since it is commonly accepted that CAC is related to colitis severity and extent, this finding is counterintuitive and the mechanisms of this paradoxical effect remain unknown.What are the new findings?In a mouse model of CAC, less extended colitis associated with an increased number of tumors was observed. Intra-tumor T-cell infiltration was significantly reduced after appendectomy. Blocking lymphocyte trafficking to the colon with current or experimental UC treatments mimicked the appendectomy-associated phenotype whereas neo-appendicitis or appendix-primed T-cell injection in recipient mice increased intra-tumor T-cell infiltration and strengthened protection against CAC.In UC patients with CAC, appendectomy was associated with a decreased intra-tumor T-cell infiltration.These findings suggest that, in UC, appendectomy could suppress a major site of T-cell priming in the colon, resulting in a reduced CAC immunosurveillance.How might it impact on clinical practice in the foreseeable future?This work emphasizes the fact that precautions will be necessary if appendectomy becomes an accepted therapeutic option for the treatment of refractory UC.Innovative cell-based therapies and immunotherapies, such as the administration of stimulated autologous appendicular T cells in patients with CAC are promising options.

Sexual dimorphism is an important factor in understanding various diseases, including inflammatory bowel disease (IBD). While females typically exhibit stronger immune responses, the role of sex in IBD remains unclear. This study aimed to explore the sex-dependent differences and inflammatory susceptibility in the most extensively used IBD mouse model as they developed colitis. We monitored IL10-deficient mice (IL-10−/−) up to 17 weeks of age and characterized their colonic and fecal inflammatory phenotype, as well as their microbiota changes. Here, we originally identified IL-10−/− female mice as more prone to developing intestinal inflammation, with an increase in fecal miR-21, and dysbiosis with more detrimental characteristics compared to males. Our findings provide valuable insights into the sex-based differences in the pathophysiology of colitis and emphasize the importance of considering sex in experimental designs. Moreover, this study paves the way for future investigations aiming at addressing sex-related differences for the development of adequate disease models and therapeutic strategies, ideally enabling personalized medicine.

Supplementary Data from Small-Molecule Drugs Mimicking DNA Damage: A New Strategy for Sensitizing Tumors to Radiotherapy

Intestinal barrier dysfunction may contribute to the development of chronic arthritis, but the underlying pathophysiological mechanisms are not fully understood. We took advantage of induced and spontaneous models of murine arthritis to study how translocation of peptidoglycan (PG) and its fractions impact the course of chronic arthritis. We experimentally increased the translocation of intestinal bacterial cell envelope moieties using oral gavage of muramyldipeptide (MDP) and lipopolysaccharide (LPS), as well as genetic (deletion of intestinal Hnf-4a) and nutritional (food additive carrageenan) perturbations of intestinal barrier function. Oral gavage with MDP and LPS increased arthritis without altering the gut barrier integrity. Translocated PG from the gut to blood and spleen was correlated with the severity of arthritis. Carrageenan treatment increased translocation of PG into the joints, and worsening of arthritis. Interestingly, MDP, LPS and dietary factors worsened the course of arthritis only when administered before, but not after, the onset of arthritis. In human patients, MDP was more abundant in the synovial fluids of children with Juvenile Idiopathic Arthritis than in those of children with transient forms of arthritis. In conclusion, there is a window during the early stages of arthritis, when translocation of gut bacterial components can modulate the progression of arthritis.

Background and Aims Nucleotide oligomerization domain 2 [NOD2] mutations are key risk factors for Crohn’s disease [CD]. NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the exact roles of NOD2 in CD and other NOD2-associated disorders remain poorly known. Methods We initially observed that NOD2 expression was increased in epithelial cells away from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation, and cytokines expression were examined in the small bowel of wild-type [WT], Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulphonic acid [TNBS]. Results In WT mice, Nod2 upregulation upstream to rectal injury was associated with pro-inflammatory cytokine expression but no overt histological inflammatory lesions. Conversely, in Nod2-deficient mice the inflammation spread from colitis to ileum and duodenum. Conclusions Nod2 protects the gut from colitis spreading to small intestine.

Background and Aims Nucleotide Oligomerisation Domain 2 [NOD2] is a key gene of innate immunity which participates in the host defence against pathogens. Several loss-of-function NOD2 mutations are associated with Crohn’s disease [CD]. Their high frequencies in populations of European ancestry suggest a model of balancing selection. Because NOD2 deficiency has been associated with a resistance to Yersinia pseudotuberculosis in mice, we hypothesised that NOD2 mutations have been selected during past plague outbreaks due to the closely related bacterium Yersinia pestis. Methods Contemporary frequencies of the main CD-associated NOD2 mutations [R702W, G908R, and 1007fs], measured in healthy people from European and Mediterranean countries, were collected from 60 studies via a PubMed search. Plague exposure was calculated from a dataset providing outbreaks from 1346 to 1860 in Europe and the Mediterranean Bassin. A plague index was built to capture the intensity of plague exposure in the studied geographical areas. Results NOD2 mutation frequencies were associated with the past exposure to plague. Statistical significance was obtained for the most frequent mutation [R702W, p = 0.03] and for the pooled three mutations [p = 0.023]. The association remained significant when putative demographic biases were considered. Conclusions This result argues for a selection of CD-associated NOD2 mutations by plague outbreaks and further questioned the role of exposure to enteropathogenic Yersinia species in CD.

Introduction L’arthrite juvenile idiopathique (AJI) est la pathologie rhumatologique a debut pediatrique la plus frequente. Ses causes restent inconnues. De nombreuses donnees suggerent que le microbiote intestinal et une dysfonction de la barriere intestinale pourraient jouer un role dans la physiopathologie de l’AJI. De plus, des publications suggerent un role des Microbe-Associated Molecular Patterns (MAMPs) dans le developpement de l’arthrite dans un modele murin. Afin de developper de nouvelles approches therapeutiques dans l’arthrite inflammatoire, il est necessaire de mieux comprendre le lien entre une dysfonction de la barriere intestinale et le developpement de l’arthrite inflammatoire. L’objectif est ici d’etudier le lien entre la permeabilite intestinale, la translocation systemique de composants bacteriens et le developpement de l’arthrite dans un modele murin. Materiels et methodes Nous avons utilise le modele d’arthrite induite par le collagene (CIA) et un modele d’arthrite spontanee les souris IL1RaKO. La severite de l’arthrite etait mesuree a l’aide d’un score clinique. La permeabilite intestinale a ete etudiee ex vivo en utilisant la translocation de composants marques dans des chambres de Ussing. Resultats Le traitement oral par carraghenanes (CGN) augmentait la permeabilite intestinale (moy. 859 vs 313 pmol/cm2/h, p Conclusion Des modifications de la permeabilite intestinale impactent la severite de l’arthrite. Le MDP et le LPS biodisponibles contribuent au developpement de l’arthrite. D’autres experiences sont necessaires pour mieux comprendre exactement comment les MAMPS systemiques conduisent a une aggravation de l’arthrite.

Background: Chronic Inflammatory Bowel Diseases (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC) are gastrointestinal disorders under the influence of a complex genetic basis. One hundred sixty-three predisposition loci were identified by genome-wide association (GWAS) studies, refocusing the pathogenesis of IBD on immunity genes. Autophagy is a fundamental mechanism in the maintenance of intestinal homeostasis. Failures of this mechanism appear to be a major risk factor in the setting up of chronic intestinal inflammation. Two single nucleotide polymorphisms (SNPs) within two genes, were selected namely (rs2241880 AaG) for ATG16L1 and (rs10065172 CaT) for IRGM.These two genes are both involved in the autophagy mechanism. We investigated these two SNP in an Algerian cohort for the first time since no previous association studies between IBD and the two studied genes were available for the Algerian population. Methods: A case-control study was performed on a cohort including 95 Algerian patients with Crohn's disease and ulcerative colitis versus 116 Controls. Genotyping of the cohort involved allelic discrimination by TaqMan SNP Genotyping Assay. Results: A statistically significant association of the rs2241880 AaGmutation of the ATG16L1 gene was demonstrated in IBD (p = 0.04) and, more particularly in Crohn's disease (p = 0.03).The presence of this mutation would increase the risk of Crohn's disease by a factor of 2 in its GG homozygous mutated form (OR = 2.11 95% CI (1.07-4.16)). No significant associations were found for the rs10065172 CaT mutation of the IRGM gene. Conclusion: Our data highlight the involvement of the ATG16L1 gene in the genesis and/or evolution of IBD cases, notably Crohn's disease, indicating the impact that deregulation of the autophagy mechanism could have on the Algerian population. Nevertheless, it would be interesting to expand the sampling and carry out the functional studies to elucidate the impact of the deregulation of the ATG16L1 gene in IBD.

NOD2 mutations are key risk factors for Crohn’s disease (CD). NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the roles of NOD2 during gut inflammation is not known. We initially observed that NOD2 expression was increased in epithelial cells remote from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation and gut permeability were examined in the small bowel of wild-type (WT), Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). In WT mice, Nod2 upregulation remote to rectal injury was associated with pro-inflammatory cytokine expression, recirculating CD4+ T-cells, increased paracellular permeability and myosin like chain kinase activity. Nod2 knockout or mutation led to duodenitis and ileitis demonstrating the remote protective role of Nod2. Bone morrow stem cell (BMSC) transplantations indicated that the small intestinal inflammation was due to NOD2 loss in both hematopoietic and non-hematopoietic compartments. As a whole, WT but not mutant NOD2 prevents disease extension at sites remote from the initial intestinal injury.

Background and Aims: Crohn’s disease [CD] is a complex disorder characterised by an inappropriate immune response, impaired barrier function and microbial dysbiosis. Mutations in nucleotide oligomeriation domain 2 [ NOD2 ] are CD risk factors. Increase of intestinal permeability, CD4+ T cell infiltration, and bacterial dysbiosis are also seen in Nod2 -knockout [ Nod2 KO] mice. However, the specificity and relationship between these Nod2 -associated abnormalities remain largely unexplored. Methods: Wild-type [WT], Nod1 -knockout [ Nod1 KO] and Nod2 KO mice were analysed in parallel. Microbial composition was defined by 454-pyrosequencing of bacterial 16S rRNA genes. Mucin and antimicrobial peptide expression was assessed by RT-PCR. Cell populations from Peyer’s patches were determined by flow cytometry. Ussing chambers were used to measure intestinal permeability and bacterial translocation. Finally, to explore the impact of colonisation with mother’s microbiota at birth, analyses were also performed in Nod2 KO and WT mice born from WT surrogate mothers after embryo transfer. Results: Nod2 KO mice exhibited colonic bacterial dysbiosis different from WT and Nod1 KO mice. Altered expression of antimicrobial peptides and mucins in ileum and colon was associated with the microbial composition. Bacterial composition of Nod2 KO and WT mice obtained by embryo transfer was similar to that observed in Nod2 KO mice, arguing for a dominant effect of Nod2 KO-associated dysbiosis. In contrast, increased levels of CD4+ T cells and gut barrier defects across Peyer’s patches were specific to Nod2 deficiency and independent of Microbial dysbiosis. Conclusions: Nod2 deficiency is associated with a specific dominant dysbiosis which does not drive mucosal tissue and immune alterations.

Yersinia are common contaminants of food products, but their prevalence in the human gut is poorly documented. Yersinia have been implicated in Crohn's Disease (CD, an inflammatory bowel disease) however their role in CD is controversial. We performed highly sensitive PCR assays of specific sequences for the gyrB gene of Y. aldovae, Y. bercovieri, Y. enterocolitica, Y. intermedia, Y. mollaretii and the inv gene of Y. pseudotuberculosis. We analyzed a total of 470 ileal samples taken from 338 participants (262 CD patients and 76 controls) belonging to three independent cohorts. All patients and controls were phenotyped and genotyped for the main CD susceptibility variants: NOD2, ATG16L1, and IRGM. Yersinia were found in 7.7% of ileal samples (respectively 7.9 and 7.6% in controls and CD patients) corresponding to 10% of participants (respectively 11.8 and 9.5% in controls and CD patients). Y. enterocolitica, Y. pseudotuberculosis and Y. intermedia were the most frequently identified species. The bacteria were more frequent in resected specimens, lymph nodes and Peyer's patches. Yersinia were no more likely to be detected in CD tissues than tissues from inflammatory and non-inflammatory controls. CD patients treated with immunosuppressants were less likely to be Yersinia carriers. In conclusion, this work shows that Yersinia species are frequently found at low levels in the human ileum in health and disease. The role of Yersinia species in this ecosystem should now be explored.

Background Ileal Crohn's disease is related to NOD2 mutations and to a gut barrier dysfunction. Pseudomonas fluorescens has also been associated with ileal Crohn's disease. The aim of this study was to determine the impact of P. fluorescens on the paracellular permeability in ileum and Peyer's patches. Methods To explore this question, in vivo and ex vivo experiments were performed in wild-type, Nod2, Nod2, and IL-1R mice together with in vitro analyses using the Caco-2 (epithelial) and the THP-1 (monocyte) human cell lines. Results Pseudomonas fluorescens increased the paracellular permeability of the intestinal mucosa through the secretion of IL-1β by the immune cell populations and the activation of myosin light chain kinase in the epithelial cells. Induction of the IL-1β pathway required the expression of Nod2 in the hematopoietic compartment, and muramyl dipeptide (a Nod2 ligand) had an inhibitory effect. Conclusions Pseudomonas fluorescens thus alters the homeostasis of the epithelial barrier function by a mechanism similar to that previously observed for Yersinia pseudotuberculosis. This work further documents a putative role of psychrotrophic bacteria in Crohn's disease.

Background and aims Digestive perianastomotic ulcerations (DPAU) have been occasionally reported as late complications of neonatal or childhood surgery. Methods We report here a series of 14 new cases. Results Cases were revealed by severe anemia, diarrhea, abdominal pain and growth failure in average 11.5 years after surgery. Ulcerations were most often multiple (n = 11), located on the upper part of ileocolonic anastomoses (n = 12) and difficult to treat. No granulomas were seen but lymphoid follicles were frequent. In addition, either ASCA or ANCA were positive in 4/9 tested patients and 8/11 genotyped patients exhibited a NOD2 mutation (P Conclusion Altogether, these findings argue for common physiopathological features between DPAU and Crohn's disease and for a prospective follow-up of selected operated children to explore the early events involved in gut inflammatory lesions.

Background: Crohn's disease (CD) pathogenesis is multifactorial involving genetic and environmental factors. Loss of function mutations in the nucleotide oligomerization domain 2 (NOD2) gene are the main genetic risk factor for CD. Like patients with CD, Nod2(KO) mice are characterized by an enhanced Th1 immune response and a defective mucosal barrier function evidenced by increased intestinal permeability. We previously showed that the latter is related to hematopoietic Nod2 deficiency. Our aim was to explore the mechanisms by which Nod2 expressed in the hematopoietic and in the nonhematopoietic compartments interplay to control epithelial paracellular permeability. Methods: Depletion of CD4(+) T cells in Nod2(KO) mice and treatments with inhibitors were conducted in chimeric mice transplanted with bone marrow cells from Nod2-deficient donors into Nod2-sufficient recipients or vice versa. Caco-2 cells overexpressing a NOD2 gene which did or did not include a CD-associated polymorphism were treated with inhibitors or siRNAs and cocultured with hematopoietic cells from Peyer's patches. Results: In vivo and in vitro Nod2 in hematopoietic cells regulates epithelial paracellular permeability through cytokine production influencing myosin light chain kinase (MLCK) activity. Indeed, tumor necrosis factor-a and interferon-g secretion by CD4(+) T cells upregulated expression and activity of epithelial MLCK leading to increased epithelial tight junction opening. When stimulated by muramyl dipeptide, Nod2 in the nonhematopoietic compartment normalized the permeability and T-cell cytokine secretion and regulated MLCK activity. This MLCK regulation is mediated by TAK1 and RICK-dependent mechanisms. Conclusions: Our study demonstrates how hematopoietic and nonhematopoietic Nod2 regulate intestinal barrier function, improving our knowledge on the mechanisms involved in CD pathogenesis.

International audience; Background: NOD2 mutations are associated with Crohn's disease (CD). Both CD (in human) and Nod2 deficiency (in mice) are characterized by increased mucosal CD4(+) T-cells, an altered permeability and a microbial dysbiosis. However, the respective roles of the gut epithelial and immune compartments on the phenotype are not known. Methods: Microbial composition, epithelial peptide secretion, intestinal permeability, and immune cell composition of Peyer patches were studied in Nod2 knock-out mice transplanted with wild-type bone marrow cells and vice versa. Results: The nonhematopoietic cells control the microbiota composition and epithelial secretion of mucins and antimicrobial peptides. These parameters are correlated with recurrent associations between bacterial species and luminal products. In contrast, Nod2 in the hematopoietic compartment regulates the epithelial permeability and the gut-associated lymphoid tissue independently of the bacterial composition. Conclusions: The immune system and the gut permeability in one hand and the microbial and epithelial peptide compositions in the other hand are separate couples of interdependent parameters, both controlled by Nod2 in either the hematopoietic or nonhematopoietic lineages.

Purpose: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage. Experimental Design: A novel strategy for inhibiting DNA repair was tested. We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait) and act by disorganizing damage signaling and DNA repair. We analyzed the effects of Dbait in cultured cells and on xenografted tumors growth and performed preliminary studies of their mechanism(s) of action. Results: The selected Dbait molecules activate H2AX phosphorylation in cell culture and in xenografted tumors. In vitro, this activation correlates with the reduction of Nijmegen breakage syndrome 1 and p53-binding protein 1 repair foci formation after irradiation. Cells are sensitized to irradiation and do not efficiently repair DNA damage. In vivo, Dbait induces regression of radioresistant head and neck squamous cell carcinoma (Hep2) and melanoma (SK28 and LU1205) tumors. The combination of Dbait32Hc treatment and fractionated radiotherapy significantly enhanced the therapeutic effect. Tumor growth control by Dbait molecules depended directly on the dose and was observed with various irradiation protocols. The induction of H2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of “false” DNA damage signaling and repair inhibition. Conclusions: These data validate the concept of introducing small DNA molecules, which mimic DNA damage, to trigger “false” signaling of DNA damage and impair DNA repair of damaged chromosomes. This new strategy could provide a new method for enhancing radiotherapy efficiency in radioresistant tumors.

AIM: To analyse allelic frequency of NOD2 gene variants and to assess their correlation with inflammatory bowel disease (IBD) in Algeria. METHODS: We studied 132 unrelated patients diagnosed with IBD, 86 with Crohn’s disease (CD) and 46 with ulcerative colitis (UC). Data was prospectively collected between January 2011 and December 2013. The demographic and clinical characteristics were recorded for all the patients. A group of 114 healthy unrelated individuals were selected as controls. All groups studied originated from different regions of North Algeria and confirmed the Algerian origin of their parents and grandparents. Informed and written consent was obtained from each of the participants. All individuals were genotyped for the three CD-associated NOD2 variants (p.Arg702Trp, p.Gly908Arg and p.Leu1007fsinsC mutations) using the polymerase chain reaction-restriction fragment length polymorphism method. Allele and genotype frequencies in patients and control subjects were compared by χ2 test and Fisher’s exact test where appropriate. Odds ratios (OR) and 95% confidence intervals (95%CI) were also estimated. Association analyses were performed to study the influence of these variants on IBD and on clinical phenotypes. RESULTS: The p.Arg702Trp mutation showed the highest frequency in CD patients (8%) compared to UC patients (2%) (P = 0.09, OR = 3.67, 95%CI: 0.48-4.87) and controls (5%) (P = 0.4, OR = 1.47, 95%CI: 0.65-3.31). In CD patients allelic frequencies of p.Gly908Arg and p.Leu1007fsinsC variants compared to HC were 3% vs 2% (P = 0.5, OR = 1.67, 95%CI: 0.44-6.34); 2% vs 1% (P = 0.4 OR = 2.69 95%CI: 0.48-14.87 respectively). In UC patients, allelic frequencies of p.Gly908Arg and p.Leu1007fsinsC variants compared to HC were 1% vs 2% (P = 1, OR = 1.62, 95%CI: 0.17-4.74) and 2% vs 1% (P = 0.32, OR = 0.39, 95%CI: 0.05-2.87). The total frequency of the mutated NOD2 chromosomes was higher in CD (13%), than in HC (8%) and UC (5%). In addition, NOD2 variants were linked to a particular clinical sub-phenotype in CD in this Algerian cohort. As expected, the three NOD2 variants showed a significant association with CD but did not reach statistical significance, despite the fact that the allele frequency of NOD2 variants was in the range found in most of the European populations. This might be due to the non-exposure of the NOD2 carriers to environmental factors, required for the expression of the disease. CONCLUSION: Further analyses are necessary to study genetic and environmental factors in IBD in the Algerian population, using larger patient groups.

Intestinal barrier function requires intricate cooperation between intestinal epithelial cells and immune cells. Enteropathogens are able to invade the intestinal lymphoid tissue known as Peyer’s patches (PPs) and disrupt the integrity of the intestinal barrier. However, the underlying molecular mechanisms of this process are poorly understood. In mice infected with Yersinia pseudotuberculosis, we found that PP barrier dysfunction is dependent on the Yersinia virulence plasmid and the expression of TLR-2 by hematopoietic cells, but not by intestinal epithelial cells. Upon TLR-2 stimulation, Y. pseudotuberculosis–infected monocytes activated caspase-1 and produced IL-1β. In turn, IL-1β increased NF-κB and myosin light chain kinase activation in intestinal epithelial cells, thus disrupting the intestinal barrier by opening the tight junctions. Therefore, Y. pseudotuberculosis subverts intestinal barrier function by altering the interplay between immune and epithelial cells during infection.

International audience; Yersinia pseudotuberculosis is an enteropathogenic bacteria that disrupts the intestinal barrier and invades its host through gut-associated lymphoid tissue and Peyer's patches (PP). We show that the Y. pseudotuberculosis effector YopJ induces intestinal barrier dysfunction by subverting signaling of the innate immune receptor Nod2, a phenotype that can be reversed by pretreating with the Nod2 ligand muramyl-dipeptide. YopJ, but not the catalytically inactive mutant YopJ(C172A), acetylates critical sites in the activation loops of the RICK and TAK1 kinases, which are central mediators of Nod2 signaling, and decreases the affinity of Nod2 for RICK. Concomitantly, Nod2 interacts with and activates caspase-1, resulting in increased levels of IL-1β. Finally, IL-1β within PP plays an essential role in inducing intestinal barrier dysfunction. Thus, YopJ alters intestinal permeability and promotes the dissemination of Yersinia as well as commensal bacteria by exploiting the mucosal inflammatory response.

Cell adhesion is one function regulated by cellular prion protein (PrP(c)), a ubiquitous, glycosylphosphatidylinositol-anchored glycoprotein. PrP(c) is located in cell-cell junctions and interacts with desmosome proteins in the intestinal epithelium. We investigated its role in intestinal barrier function.We analyzed permeability and structure of cell-cell junctions in intestine tissues from PrP(c) knockout (PrP(c-/-)) and wild-type mice. PrP(c) expression was knocked down in cultured human Caco-2/TC7 enterocytes using small hairpin RNAs. We analyzed colon samples from 24 patients with inflammatory bowel disease (IBD).Intestine tissues from PrP(c-/-) mice had greater paracellular permeability than from wild-type mice (105.9 ± 13.4 vs 59.6 ± 10.1 mg/mL fluorescein isothiocyanate-dextran flux; P.05) and impaired intercellular junctions. PrP(c-/-) mice did not develop spontaneous disease but were more sensitive than wild-type mice to induction of colitis with dextran sulfate (32% mortality vs 4%, respectively; P = .0033). Such barrier defects were observed also in Caco-2/TC7 enterocytes following PrP(c) knockdown; the cells had increased paracellular permeability (1.5-fold over 48 hours; P.001) and reduced transepithelial electrical resistance (281.1 ± 4.9 vs 370.6 ± 5.7 Ω.cm(2); P.001). Monolayer shape and cell-cell junctions were altered in cultures of PrP(c) knockdown cells; levels of E-cadherin, desmoplakin, plakoglobin, claudin-4, occludin, zonula occludens 1, and tricellulin were decreased at cell contacts. Cell shape and junctions were restored on PrP(c) re-expression. Levels of PrP(c) were decreased at cell-cell junctions in colonic epithelia from patients with Crohn's disease or ulcerative colitis.PrP(c) regulates intestinal epithelial cell-cell junctions and barrier function. Its localization is altered in colonic epithelia from patients with IBD, supporting the concept that disrupted barrier function contributes to this disorder.

Background: Mounting evidence indicates that aberrant immune responses to commensal intestinal microbes contribute to the development of inflammatory bowel diseases (IBDs). However, little is known about the effects of inflammation on gut microbial function. We previously reported that colitis induces commensal luminal Escherichia coli to upregulate stress-response genes in monoassociated Il-10-/(KO) mice, but colitis-associated microbial transcription in complex luminal bacterial communities has not been explored. Understanding how inflammation affects gut microbes in a manner that perpetuates colitis could identify novelmicrobial pathways that can be targeted for therapeutic purposes. Hypothesis:Mixtures of commensal bacterial isolates induce colitis in KO mice and respond to inflammation by upregulating genes that promote microbial survival. Methods: Germ-free wild-type (WT) and KO 129 SvEv mice were colonized for up to 10 weeks with 8 commensal, predominantly human-derived bacterial isolates (Table) representing each major phylum of the gut microbiome. Intestinal inflammation was quantified by blinded histological scores and spontaneous secretion of Il-12/23p40 by colon explant cultures. Fecal bacterial composition at 5 wks was measured by real-time PCR. Cecal microbial metatranscriptomes at 10 wks were characterized using Illumina-based microbial RNA-Seq and Gene Set Enrichment Analysis of conserved domains. Results: At 5 and 10 weeks, KO mice had increased histological inflammation and Il-12/23p40 secretion by colon explant cultures compared to WT mice. At 5 weeks, fecal bacterial composition in WT and KOmice was similar and was dominated by Bacteroides thetaiotaomicron and Ruminococcus gnavus. Gut microbial metatranscriptomes in KO and WT mice were significantly different at 10 weeks. While relatively few E. faecalis mRNA's were detected in cecal contents, they comprise the majority of differentially expressed microbial genes (Table). Overall, 76 conserved domains were significantly enriched with genes that are upregulated in bacteria from KO mice including tRNA synthetase (false discovery rate/ FDR=0.001), translation factors (FDR=0.002), cell wall biosynthesis (FDR=0.011), nucleotide salvage pathways (FDR=0.013), bacterial sugar transferase (FDR=0.015), and DNA repair (FDR=0.039). 7 conserved domains were enriched with genes that are downregulated in bacteria from KO mice including thioredoxin (FDR=0.052) and cation transporting ATPase (FDR=0.155). Conclusions: A representative mixture of predominantly human commensal bacterial isolates induces colitis in gnotobiotic Il-10-/mice and responds to inflammation by upregulating genes in metabolic, synthetic and DNA repair pathways. Mechanistic studies of differentially expressed bacterial genes may reveal novel pathways that initiate and/or perpetuate IBDs.

Backgrounds/Aims: Macroautophagy is a bulk degradation system that enables recycling of long-lived proteins and damaged organelles. It is at the crossroads of innate and adaptive immunity, inflammation and apoptosis. Previous studies have associated the NOD2 frameshift 1007fs mutation with a defect of autophagy induction and an increased intestinal permeability. The aim of this study was to investigate the role of autophagy in the intestinal permeability and to define the involvement of NOD2 and its variant 1007fs. Methods: Paracellular permeability was assessed by FITC 4kD Dextran flux and transepithelial resistance in Caco2 monolayers. Stably transfected cells with the wild-type or 1007fs NOD2 variant were also used. Activation of autophagy was induced by 24H of nutrient starvation and rapamycin treatment. Results: Nutrient starvation of Caco-2 monolayers increased significantly the paracellular permeability compared to basal conditions (respectively, 10.84% vs 0.84% of 4kD FITC dextran; P