Adherent-invasive Escherichia Coli Infection Research Articles

Intestinal Epithelial PTPN2 Limits Pathobiont Colonization by Immune-Directed Antimicrobial Responses.

Loss of activity of the inflammatory bowel disease (IBD) susceptibility gene, protein tyrosine phosphatase non-receptor type 2 (PTPN2), is associated with altered microbiome composition in both human subjects and mice. Further, expansion of the bacterial pathobiont, adherent-invasive E. coli (AIEC), is strongly linked to IBD pathogenesis. The mechanism by which intestinal epithelial cells (IEC) maintain equilibrium between commensal microbiota and immune cells to restrict invading pathobionts is poorly understood. Here, we investigated the role of IEC-specific PTPN2 in regulating AIEC colonization. Tamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice (Ptpn2 ΔIEC) and control Ptpn2 fl/fl mice were infected with either non-invasive E. coli K12, or fluorescent-tagged mAIEC (mAIECred) for four consecutive days or administered PBS. Subsequently, bacterial colonization in mouse tissues was quantified. mRNA and protein expression were assayed in intestinal epithelial cells (IECs) or whole tissue lysates by PCR and Western blot. Tissue cytokine expression was determined by ELISA. Intestinal barrier function was determined by in vivo administration of 4 kDa FITC-dextran (FD4) or 70kDa Rhodamine-B dextran (RD70) fluorescent probes. Confocal microscopy was used to determine the localization of tight-junction proteins. Ptpn2 ΔIEC mice exhibited increased mAIECred - but not K12 - bacterial load in the distal colon compared to infected Ptpn2 fl/fl mice. The higher susceptibility to mAIECred infection was associated with altered levels of antimicrobial peptide (AMPs). Ileal RNA expression of the alpha-defensin AMPs, Defa5 and Defa6, as well as MMP7, was significantly lower in Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice, after mAIECred but not K12 infection. Further, we observed increased tight junction-regulated permeability determined by elevated in vivo FD4 but not RD70 permeability in Ptpn2 ΔIEC-K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2 ΔIEC mAIEC-infected mice. Further, Ptpn2 ΔIEC mice displayed lower IL-22, IL-6, IL-17A cytokine expression post mAIEC infection compared to Ptpn2 fl/fl controls. Recombinant IL-22 reversed the FD4 permeability defect and reduced bacterial burden in Ptpn2 ΔIEC mice post mAIEC challenge. Our findings highlight that intestinal epithelial PTPN2 is crucial for mucosal immunity and gut homeostasis by promoting anti-bacterial defense mechanisms involving coordinated epithelial-immune responses to restrict pathobiont colonization.

Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection.

Creatine transporter (CrT1) mediates cellular uptake of creatine (Cr), a nutrient pivotal in maintaining energy homeostasis in various tissues including intestinal epithelial cells (IECs). The impact of CrT1 deficiency on the pathogenesis of various psychiatric and neurological disorders has been extensively investigated. However, there are no studies on its regulation in IECs in health and disease. Current studies have determined differential expression of CrT1 along the length of the mammalian intestine and its dysregulation in inflammatory bowel disease (IBD)-associated inflammation and Adherent Invasive E. coli (AIEC) infection. CrT1 mRNA and protein levels in normal intestines and their alterations in inflammation and following AIEC infection were determined in vitro in model IECs (Caco-2/IEC-6) and in vivo in SAMP1/YitFc mice, a model of spontaneous ileitis resembling human IBD. CrT1 is differentially expressed in different regions of mammalian intestines with its highest expression in jejunum. In vitro, CrT1 function (Na+-dependent 14C-Cr uptake), expression and promoter activity significantly decreased following TNFα/IL1β treatments and AIEC infection. SAMP1 mice and ileal organoids generated from SAMP1 mice also showed decreased CrT1 mRNA and protein compared to AKR controls. Our studies suggest that Cr deficiency in IECs secondary to CrT1 dysregulation could be a key factor contributing to IBD pathogenesis.

ZINC RESTRICTION BY INTESTINAL E. COLI - PRODUCED YERSINIABACTIN ACTIVATES HIF-1α IN MACROPHAGES TO PROMOTE CD-ASSOCIATED FIBROSIS

Abstract Intestinal fibrosis is a significant complication in Crohn’s disease (CD) patients, causing severe intestinal strictures and obstructions that are ultimately relieved by surgical bowel resection. Mechanisms driving CD-associated fibrosis are poorly understood but implicate the microbiome, especially adherent-invasive E. coli (AIEC). Previously, our work demonstrated that AIEC production of the metallophore yersiniabactin (Ybt) promotes fibrosis in the well-established gnotobiotic Il10-/- CD mouse model. These pro-fibrotic effects do not require bacterial uptake/utilization of Ybt, suggesting Ybt directly targets the host. Here we show that CD11b+ F4/80+ MHCIIlow macrophages are abundant in the submucosa and muscularis of fibrotic lesions in our mouse model. Similarly, CD68+ macrophages are abundant in strictures, not adjacent margins, of ileocolonic CD patients. We hypothesized that Ybt targets host macrophages to drive fibrosis. A critical metal-dependent pathway for immune cell function during inflammation is HIF-1α. We demonstrate that AIEC infection activates HIF-1α in macrophages, which is dependent upon Ybt production. Importantly, in fibrotic lesions of CD mouse models and resected strictures of ileocolonic CD patients, HIF-1α is activated and localized in nuclei of F4/80+ or CD68+ macrophages. We identified that zinc sequestration by Ybt is responsible for HIF-1α activation, a novel mechanism of HIF-1α stabilization. Furthermore, Ybt-producing AIEC induce potent pro-fibrotic gene expression in macrophage:fibroblast co-cultures, a process dependent on macrophage HIF-1α. This implies that Ybt induces pro-fibrotic macrophages, activating adjacent fibroblasts and fostering intestinal fibrosis. In summary, our findings suggest that as intestinal macrophages encounter AIEC, Ybt sequesters zinc, causes an accumulation of nuclear HIF-1α, and this imbalance of metal homeostasis drives pro-fibrotic macrophage and fibroblast activation. Ultimately, effective fibrotic remodeling fails and leads to intestinal fibrosis in CD patients.

PUFAs add fuel to Crohn’s disease-associated AIEC-induced enteritis by exacerbating intestinal epithelial lipid peroxidation

ABSTRACT Polyunsaturated fatty acids (PUFAs) have been shown to exacerbate Crohn’s disease (CD) by promoting lipid peroxidation (LPO) of intestinal epithelial cells (IECs). Dysbiosis of the gut microbiota may play a crucial role in this process. CD patients often exhibit an increased abundance of Escherichia coli (E. coli) in the gut, and the colonization of adherent-invasive E. coli (AIEC) is implicated in the initiation of intestinal inflammation in CD. However, the impact of AIEC on LPO remains unclear. In this study, we observed that AIEC colonization in the terminal ileum of CD patients was associated with decreased levels of glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH) in the intestinal epithelium, along with elevated levels of 4-Hydroxynonenal (4-HNE). In vitro experiments demonstrated that AIEC infection reduced the levels of GPX4 and FTH, increased LPO, and induced ferroptosis in IECs. Furthermore, arachidonic acid (AA) and docosahexaenoic acid (DHA) supplementation in AIEC-infected IECs significantly aggravated LPO and ferroptosis. However, overexpression of GPX4 rescued AIEC-induced LPO and ferroptosis in IECs. Our results further confirmed that AIEC with AA supplementation, associated with excessive LPO and cell death in IECs, worsened colitis in the DSS mouse model and induced enteritis in the antibiotic cocktail pre-treatment mouse model in vivo. Moreover, treatment with ferrostatin-1, a ferroptosis inhibitor, alleviated AIEC with AA supplementation-induced enteritis in mice, accompanied by reduced LPO and cell death in IECs. Our findings suggest that AIEC, in combination with PUFA supplementation, can induce and exacerbate intestinal inflammation, primarily through increased LPO and ferroptosis in IECs.

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

Epigenetic master regulators HDAC1 and HDAC5 control pathobiont Enterobacteria colonization in ileal mucosa of Crohn’s disease patients

ABSTRACT Adherent-invasive Escherichia coli (AIEC), which abnormally colonize the ileal mucosa of Crohn’s disease (CD) patients, are known to contribute to the etiopathogenesis of CD. Molecular mechanisms favoring AIEC ileal colonization have not been completely characterized yet. The aim of this study was to investigate whether epigenetic regulators histone deacetylases (HDAC) expression in intestinal epithelial cells of CD patients regulate Enterobacteria and AIEC encroachment to intestinal mucosa. HDAC were inhibited in vitro and in CEABAC10 mice to decipher their involvement in the entry of AIEC within host cells. CD ileal samples from the REMIND cohort were used to study the relationship between HDAC expression level and Enterobacteria/AIEC colonization in patients. Mice were fed a westernized diet and orally challenged with AIEC to determine the impact of diet on HDAC expression. Global level of acetylated histone H3 is higher in patients colonized by AIEC bacteria compared to patients non-colonized by Enterobacteria and HDAC inhibition-mediated H3 hyperacetylation promotes the entry of AIEC bacteria within intestinal epithelial cells. HDAC1 and HDAC5 are central and antagonistic in the regulation of AIEC entry within host cells in vitro, in mouse models and in ileal mucosa of CD patients. In mice fed a western-type diet, AIEC infection decreases HDAC1 expression, inducing H3 hyperacetylation to favor their own colonization. CD patients under a western diet are more prone to be colonized by AIEC bacteria as such a diet affects intestinal homeostasis, enables AIEC access to intestinal mucosa where they then manipulate host-epigenome to their advantage. Abbreviations AIEC Adherent-Invasive Escherichia coli; BSA Bovine serum albumin; CD Crohn’s disease; CEABAC10 Carcinoembryonic antigen bacterial artificial chromosome 10; CEACAM Carcinoembryonic antigen-related cell adhesion molecule; FBS Fetal bovine serum; IBD Inflammatory Bowel Disease; HAT Histone acetyltransferase; HDAC Histone deacetylase; kDa KiloDalton; SAHA Suberoylanilide Hydroxamic Acid; Scr Scramble

Siderophore Immunization Restricted Colonization of Adherent-Invasive Escherichia coli and Ameliorated Experimental Colitis.

ABSTRACTInflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract and profound alterations to the gut microbiome. Adherent-invasive Escherichia coli (AIEC) is a mucosa-associated pathobiont that colonizes the gut of patients with Crohn’s disease, a form of IBD. Because AIEC exacerbates gut inflammation, strategies to reduce the AIEC bloom during colitis are highly desirable. To thrive in the inflamed gut, Enterobacteriaceae acquire the essential metal nutrient iron by producing and releasing siderophores. Here, we implemented an immunization-based strategy to target the siderophores enterobactin and its glucosylated derivative salmochelin to reduce the AIEC bloom in the inflamed gut. Using chemical (dextran sulfate sodium) and genetic (Il10−/− mice) IBD mouse models, we showed that immunization with enterobactin conjugated to the mucosal adjuvant cholera toxin subunit B potently elicited mucosal and serum antibodies against these siderophores. Siderophore-immunized mice exhibited lower AIEC gut colonization, diminished AIEC association with the gut mucosa, and reduced colitis severity. Moreover, Peyer’s patches and the colonic lamina propria harbored enterobactin-specific B cells that could be identified by flow cytometry. The beneficial effect of siderophore immunization was primarily B cell-dependent because immunized muMT−/− mice, which lack mature B lymphocytes, were not protected during AIEC infection. Collectively, our study identified siderophores as a potential therapeutic target to reduce AIEC colonization and its association with the gut mucosa, which ultimately may reduce colitis exacerbation. Moreover, this work provides the foundation for developing monoclonal antibodies against siderophores, which could provide a narrow-spectrum strategy to target the AIEC bloom in Crohn’s disease patients.

Chitinase 3-like 1, Carcinoembryonic Antigen-related Cell Adhesion Molecule 6, and Ectopic Claudin-2 in the Carcinogenic Processes of Ulcerative Colitis.

The cumulative cancerous rate of colitis-associated cancer (CAC) has increased exponentially in patients with ulcerative colitis (UC). We have investigated the factors involved in the carcinogenic processes of CAC among UC patients. A total of 42 UC patients who underwent surgical treatments between January 2001 and December 2010 at Kurume University Hospital (Fukuoka, Japan) were enrolled. We conducted this study using 3 cases of CAC out of 42 UC cases and 1 case of colorectal cancer. cDNA microarray analyses were performed using normal, inflamed, and cancerous tissues from surgical CAC specimens and protein expression was confirmed by immunohistochemical analyses. cDNA microarray revealed 32 genes that were dominantly expressed in tumorous regions of CAC. Gene ontology analysis revealed that these genes were involved in inflammatory responses and cytokine-cytokine receptor interactions. Chitinase 3-like1 (CHI3L1), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), and Claudin-2 (CLND2) were selected from CAC-related genes as candidate molecules. Immunostaining revealed strong expression of each protein in cancerous regions. In this study, we identified CAC-related genes and found that CHI3L1, CEACAM6, and CLND2 were expressed in patient samples. All the above genes were associated with adherent invasive Escherichia coli (AIEC), which suggested that these molecules are likely involved in AIEC infection. Further analyses would be required to reveal unknown mechanisms of CAC-related genes in the tumor microenvironment.

Commensal Bacteria Derived Metabolite Butyrate Restores the Epithelial Mitochondrial Network Disrupted by the Crohn's Disease‐Associated Pathobiont Adherent Invasive E‐coli Infection

Background Mitochondrial dysfunction has been implicated in several autoimmune and inflammatory diseases. Adherent-invasive E. coli (AIEC) is a Crohn's disease-associated pathobiont that triggers pro-inflammatory responses. Previous work done by our group showed that AIEC (strain LF82) infection in gut epithelial cells (T84 human cell line) induces excessive mitochondrial network fragmentation with loss of mitochondrial membrane potential. Commensal bacteria in the gut produce useful metabolites such as short chain fatty acids (SCFA), which have a wide range of benefits including enhancing mitochondrial biogenesis. Aims (1) Determine the ability of sodium butyrate (Na-But) treatment to restore mitochondrial functions disrupted by AIEC (strain LF82) infection in T84 epithelial cells. (2) Identify the mechanism utilized by Na-But to impose its protective effect. Methods Mitochondrial network morphology was assessed via confocal microscopy live cell imaging of Mito-tracker-stained T84 epithelial cells, and images analyzed using ImageJ. We assessed the effect of Na-But (10mM) on the proliferation of bacteria and their invasion of T84-epithelia by growth curve analysis and gentamycin internalization assays. Mitochondrial membrane potential was measured by flow cytometry, and mRNA and protein expression of peroxisome proliferating activator receptor assessed by qPCR and immunoblotting. Results We confirmed that AIEC-infection (strain LF82, 108cfu, 4h) induces massive mitochondrial fragmentation in T84 cells (2x105). Co-treatment with Na-But and LF82 showed increased percentage of fused T84-cell mitochondrial networks compared to LF82-treated cells (Na-But also reduced epithelial mitochondrial fragmentation caused by dinitrophenol (0.1mM, 2h)). The protective effect of Na-But (3-20mM; 0-24hr) was neither due to inhibition of bacterial proliferation nor their invasion of the epithelia. Epithelia infected with E. coli-LF82 displayed reductions in mitochondrial membrane potential and mRNA expression of the master regulator of mitochondrial biogenesis, PGC1α: both effects were partially prevented by Na-But. Conclusions These data indicate that pathogen disruption of the epithelial mitochondrial network is a component of IBD that could identify novel targets to control enteric inflammation. These data underscore the complex bacterial-epithelium interaction: commensal organisms may preserve the mitochondrial network in the face of challenge from pathogens that seek to disrupt mitochondrial form and function.

Yersiniabactin Siderophore of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Is Involved in Autophagy Activation in Host Cells.

Background: Adherent-invasive Escherichia coli (AIEC) have been implicated in the etiology of Crohn’s disease. The AIEC reference strain LF82 possesses a pathogenicity island similar to the high pathogenicity island of Yersinia spp., which encodes the yersiniabactin siderophore required for iron uptake and growth of the bacteria in iron-restricted environment. Here, we investigated the role of yersiniabactin during AIEC infection. Methods: Intestinal epithelial T84 cells and CEABAC10 transgenic mice were infected with LF82 or its mutants deficient in yersiniabactin expression. Autophagy was assessed by Western blot analysis for p62 and LC3-II expression. Results: Loss of yersiniabactin decreased the growth of LF82 in competitive conditions, reducing the ability of LF82 to adhere to and invade T84 cells and to colonize the intestinal tract of CEABAC10 mice. However, yersiniabactin deficiency increased LF82 intracellular replication. Mechanistically, a functional yersiniabactin is necessary for LF82-induced expression of HIF-1α, which is implicated in autophagy activation in infected cells. Conclusion: Our study highlights a novel role for yersiniabactin siderophore in AIEC–host interaction. Indeed, yersiniabactin, which is an advantage for AIEC to growth in a competitive environment, could be a disadvantage for the bacteria as it activates autophagy, a key host defense mechanism, leading to bacterial clearance.

A49 BUTYRATE PRESERVES THE EPITHELIAL MITOCHONDRIAL NETWORK DISRUPTED BY THE CROHN’S DISEASE PATHOBIONT ADHERENT-INVASIVE E. COLI

Abstract Background Mitochondria exist in a dynamic network that undergoes continuous cycles of fission and fusion that is tightly controlled. Mitochondrial dysfunction is implicated in several autoimmune and inflammatory diseases. Adherent-invasive E. coli (AIEC) is a pathogenic strain of bacteria associated with Crohn’s disease (CD), that can evoke pro-inflammatory responses. Data from our lab showed that AIEC (strain LF82) infection in gut epithelial cells (T84 human cell line) caused dramatic mitochondrial network fragmentation and loss of mitochondrial membrane potential. Short chain fatty acids (SCFA) produced by commensal bacteria in the gut have a wide range of benefits including enhancing mitochondrial biogenesis. Aims To determine (1) if sodium butyrate (NaB) treatment can protect against mitochondrial dysfunction induced by AIEC (strain LF82) infection in T84 epithelial cells and then (2) to identify the mechanism by which NaB restores mitochondrial functions. Methods We assessed changes in mitochondrial network morphology through confocal microscopy live cell imaging of mitotracker-stained T84 epithelial cells based on unbiased Hoechst-stained nuclei to select the field of view. The effect of NaB on the proliferation of bacteria and their invasion of T84-epithelia was assessed by growth curve analysis and bacterial internalisation assays. Finally, mRNA and protein expression of peroxisome proliferating activator receptor gamma co-activator 1 alpha (PGC1α), a regulator of mitochondrial biogenesis, were assessed by qPCR and Western Blot. Results We confirmed that AIEC infection (strain LF82, 108cfu, 4h) induces massive mitochondrial fragmentation in T84 cells (2x105). We also found that cotreatment of T84 cells with NaB (10mM) and LF82 showed increased percentage of fused mitochondrial networks compared to LF82-treated cells. This result was also seen in cells treated with mitochondrial uncoupler dinitrophenol (DNP; 0.1 mM, 2h) and NaB. Moreover, the protective effect of NaB was not related to inhibition of proliferation of the bacteria as we demonstrated that LF82 growth and its invasive phenotype was not compromised by NaB (3-20mM; 0-24hr). In agreement with the effect of LF82 on mitochondrial functions, LF82 significantly reduced PGC1α mRNA expression in T84 cells, that was prevented by co-treatment with NaB. Conclusions These data suggest the pathogen disruption of the epithelial mitochondrial network is a component of IBD; thus, identifying mitochondrial fission and fusion pathways as novel therapeutic targets to control enteric inflammation. The data underscore the complex interplay between bacteria and the epithelium, such that commensal organisms may preserve the mitochondrial network in the face of challenge from pathogens that seek to disrupt mitochondrial form and function Funding Agencies CIHR

TARGETING SIDEROPHORES TO REDUCE COLONIZATION OF ADHERENT INVASIVE E. COLI IN CROHN’S DISEASE

Abstract The pathovar adherent-invasive Escherichia coli (AIEC) is a mucosa-associated bacterium that is frequently found in patients with Crohn’s disease (CD), but targeted treatment options are currently lacking. During intestinal inflammation, host-mediated iron limitation is a key strategy to restrict the outgrowth of pathogens. Various bacterial pathogens evade this nutrient limitation by secreting small, iron-chelating molecules known as siderophores. The siderophore enterobactin (Ent) is an important virulence factor for Gram-negative pathogens such as AIEC by promoting their expansion and competition over commensal microbes during colitis. Here, we developed an immunization-based approach to target bacterial siderophores during colitis and to reduce AIEC colonization in mice. We demonstrate that immunization with Ent (conjugated to the mucosal adjuvant cholera toxin subunit B, CTB) potently elicits fecal antibodies against Ent. Immunized animals show reduced AIEC colonization along with milder weight loss, less colonic shortening, and decreased histological severity scores. This phenotype is largely B cell-dependent, as immunized mice lacking mature B lymphocytes do not show protection during AIEC infection. Ent-specific B cells are primarily found in small intestinal Peyer’s patches and can be isolated by a flow cytometry-based sorting approach. This project paves the way to develop monoclonal antibodies against siderophores, which could provide a more targeted and narrow-spectrum strategy to reduce AIEC colonization in patients with CD.

Characterising the role of Proline Tyrosine Kinase 2 during Adherent-invasive Escherichia coli infection of macrophages

Studies have implicated that Adherent-invasive Escherichia coli (AIEC) in the aetiology of Crohn’s Disease (CD), a chronic inflammatory bowel disease (IBD). CD-associated AIEC are characterised by an ability to adhere to and invade intestinal epithelial cells and replicate intracellularly in macrophages. This occurs as AIEC form vacuoles within phagolysosomes, preventing macrophage-mediated killing. However, little is known about the interaction of macrophage proteins with AIEC. In this study, we used an in vitro infection model to identify macrophage proteins associated with AIEC intracellular replication. We identified the importance of proline-rich tyrosine kinase 2 (PYK2) in AIEC infection. PYK2 is widely expressed in epithelial cells and hematopoietic cells and plays essential roles in tumorigenesis and tumour progression. PYK2 is also overexpressed in patients with intestinal and colorectal cancer (CRC). CRC is one of the major long-term complications of IBD, especially in patients with long disease duration, a large extent of colitis, and uncontrolled inflammation. Moreover, PYK2 has been identified as an IBD risk loci via large scale genome-wide association studies. Our in vitro models have demonstrated that the addition of PYK2 inhibitor PF-431396 during infection results in a significant decrease in intracellular replication of AIEC. This has been quantified using fluorescence immunostaining and imaging flow cytometry. These results suggest that PYK2 may play a role in intracellular bacteria survive and replication. Our future work aims to further understand the relationship between PYK2, AIEC and CD.

Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

ABSTRACT Adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of Crohn’s disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic E. coli K12 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred via exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.

Adherent-Invasive E. coli: Update on the Lifestyle of a Troublemaker in Crohn's Disease.

Besides genetic polymorphisms and environmental factors, the intestinal microbiota is an important factor in the etiology of Crohn’s disease (CD). Among microbiota alterations, a particular pathotype of Escherichia coli involved in the pathogenesis of CD abnormally colonizes the intestinal mucosa of patients: the adherent-invasive Escherichia coli (AIEC) pathobiont bacteria, which have the abilities to adhere to and to invade intestinal epithelial cells (IECs), as well as to survive and replicate within macrophages. AIEC have been the subject of many studies in recent years to unveil some genes linked to AIEC virulence and to understand the impact of AIEC infection on the gut and consequently their involvement in CD. In this review, we describe the lifestyle of AIEC bacteria within the intestine, from the interaction with intestinal epithelial and immune cells with an emphasis on environmental and genetic factors favoring their implantation, to their lifestyle in the intestinal lumen. Finally, we discuss AIEC-targeting strategies such as the use of FimH antagonists, bacteriophages, or antibiotics, which could constitute therapeutic options to prevent and limit AIEC colonization in CD patients.

DOP49 Quantitative proteomics analysis of macrophages from Crohn’s disease patients and infected with adherent-invasive Escherichia coli

Abstract Background Crohn’s disease (CD) is a disabling inflammatory bowel disease. Currently, the available treatments only alleviate the symptoms. The anti-TNFα represent the most efficient therapeutic class, but its mechanisms are poorly known. Experimental data have highlighted the role of intestinal macrophages in the pathogenesis of CD. These macrophages present a defect in the control of CD-associated adherent-invasive E. coli (AIEC) replication, which is linked to altered autophagy. The main aim of this study was to compare the proteomic profile of macrophages from CD patients with and without anti-TNFα treatment, to those from ulcerative colitis (UC) patients or healthy subjects, both at the basal state and after AIEC infection. Methods Peripheral blood monocyte-derived macrophages (MDM) were obtained from 44 CD patients including 22 with and 22 without anti-TNFα treatment, 22 UC patients and 22 healthy subjects. MDM was infected or not with AIEC LF82 reference strain. The numbers of intracellular bacteria were determined at 1h and 6h post-infection, using gentamicin protection assay. The ‘bottom-up’ proteomic analysis of macrophages after 6h of AIEC LF82 infection or not, was assessed by a mass spectrometer, using the label-free quantification. Results AIEC survival was reduced within MDM from CD patients with anti-TNFα treatment compared with those from CD without TNFα treatment (p = 0.0235). The proteomic analysis identified 1359 proteins, of which, 71 were differentially expressed between MDM at the basal state and infected by AIEC LF82 whatever the origin, 30 proteins were specific in CD patients with anti-TNFα treatment and 16 in CD patients without anti-TNFα treatment (fold change >1.3). The bioinformatics analysis shows an impact of several pathways involved in cellular activation, immune process and lysosomal activity. Interestingly, CD macrophages expression levels of β3 integrin (ITGB3), glutathione peroxidase-1 (GPx-1) and peroxiredoxin 3 (PRDX3) are significantly correlated with AIEC survival at 6h post-infection and faecal calprotectin. The stratification of MDM from CD patients according to the level of faecal calprotectin highlighted that levels of calprotectin and α-L-Fucosidase 1 (FUCA-1) increased in a similar way, both at the basal state and after AIEC LF82 infection. Conclusion Our study highlighted that the anti-TNFα treatment limits bacteria replication and acts on macrophages function. For the first time, global proteomic analysis suggests that CD macrophages-AIEC interaction could be orchestrated by ITGB3, GPx-1, PRDX3 and FUCA-1. These proteins could represent potential therapeutic targets in CD patients that need to be further investigated.

Macrophages Inability to Mediate Adherent-Invasive E. coli Replication is Linked to Autophagy in Crohn's Disease Patients.

The macrophages from Crohn’s Disease (CD) patients are defective to control the replication of CD-associated adherent-invasive E. coli (AIEC). We aimed to identify the host factors associated with AIEC replication focusing on polymorphisms related to autophagy. Peripheral blood monocyte-derived macrophages (MDM), obtained from 95 CD patient, 30 ulcerative colitis (UC) patients and 15 healthy subjects, were genotyped for several CD-associated polymorphisms. AIEC bacteria survival increased within MDM from CD patients compared to UC (p = 0.0019). AIEC bacteria survival increased in patients with CD-associated polymorphism IRGM (p = 0.05) and reduced in those with CD-associated polymorphisms XBP-1 (p = 0.026) and ULK-1 (p = 0.033). AIEC infection led to an increase of pro-inflammatory cytokines IL-1β (p < 0.0001) and TNF-α (p < 0.0001) in CD macrophages. ULK-1 expression increased in AIEC-infected MDM from CD patients compared to MDM from UC patients or healthy subjects (p = 0.0056) and correlated with AIEC survival (p = 0.0013). Moreover, the expression of ULK-1 phosphorylation on Serine 757 decreased following to AIEC infection (p < 0.0001). Short-term silencing of ULK-1 and IRGM genes restricted and promote, respectively, AIEC survival within MDM (p = 0.0018 and p = 0.0291). In conclusion, the macrophage defect to mediate AIEC clearance in CD patients is linked to polymorphisms related to autophagy such as IRGM and ULK-1.

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Manipulate Host Autophagy by Impairing SUMOylation.

The intestinal mucosa of Crohn’s disease (CD) patients is abnormally colonized with adherent-invasive Escherichia coli (AIEC) that are able to adhere to and to invade intestinal epithelial cells (IECs), to survive in macrophages, and to induce a pro-inflammatory response. AIEC persist in the intestine, and induce inflammation in CEABAC10 transgenic mice expressing human CAECAM6, the receptor for AIEC. SUMOylation is a eukaryotic-reversible post-translational modification, in which SUMO, an ubiquitin-like polypeptide, is covalently linked to target proteins. Here, we investigated the role of SUMOylation in host responses to AIEC infection. We found that infection with the AIEC LF82 reference strain markedly decreased the levels of SUMO-conjugated proteins in human intestinal epithelial T84 cells. This was also observed in IECs from LF82-infected CEABAC10 transgenic mice. LF82-induced deSUMOylation in IECs was due in part to increased level of microRNA (miR)-18, which targets PIAS3 mRNA encoding a protein involved in SUMOylation. Over-expression of SUMOs in T84 cells induced autophagy, leading to a significant decrease in the number of intracellular LF82. Consistently, a decreased expression of UBC9, a protein necessary for SUMOylation, was accompanied with a decrease of LF82-induced autophagy, increasing bacterial intracellular proliferation and inflammation. Finally, the inhibition of miR-18 significantly decreased the number of intracellular LF82. In conclusion, our results suggest that AIEC inhibits the autophagy response to replicate intracellularly by manipulating host SUMOylation.

Antibiotics Potentiate Adherent-InvasiveE. coliInfection and Expansion

Crohn's disease (CD) is an inflammatory bowel disease with a complex etiology. Paradoxically, CD is associated with the use of antibiotics and with an increased abundance of an unusual phenotypic group of Escherichia coli known as adherent-invasive E. coli (AIEC). However, the impact of antibiotics on AIEC infection has not been well studied in controlled models of infection. We infected mice with AIEC before or after treatment with a variety of different classes of antibiotics. We assessed levels of AIEC in the feces and tissues, AIEC localization by immunofluorescence microscopy, and tissue pathology. We found that a wide range of antibiotic classes strongly potentiated initial AIEC infection and expanded AIEC in chronically infected mice. We found that the ability of antibiotics to potentiate AIEC infection did not correlate with a stereotyped shift in the gut bacterial community but was correlated with a decrease in overall diversity and a divergence from the pre-antibiotic state. We found that antibiotic-induced inflammation provided a fitness advantage for AIEC expansion through their use of oxidized metabolites in the postantibiotic period. Our results show that antibiotics can render hosts more susceptible to initial AIEC infection and can worsen infection in previously colonized hosts. AIEC appears to exploit host inflammatory responses that arise in the postantibiotic period, highlighting a previously unknown interaction between CD risk factors.

AIEC infection triggers modification of gut microbiota composition in genetically predisposed mice, contributing to intestinal inflammation

A high prevalence of adherent-invasive E. coli (AIEC) in the intestinal mucosa of Crohn’s disease patients has been shown. AIEC colonize the intestine and induce inflammation in genetically predisposed mouse models including CEABAC10 transgenic (Tg) mice expressing human CEACAM6-receptor for AIEC and eif2ak4−/− mice exhibiting autophagy defect in response to AIEC infection. Here, we aimed at investigating whether gut microbiota modification contributes to AIEC-induced intestinal inflammation in these mouse models. For this, eif2ak4+/+ and eif2ak4−/− mice or CEABAC10 Tg mice invalidated for Eif2ak4 gene (Tg/eif2ak4−/−) or not (Tg/eif2ak4+/+) were infected with the AIEC reference strain LF82 or the non-pathogenic E. coli K12 MG1655 strain. In all mouse groups, LF82 colonized the gut better and longer than MG1655. No difference in fecal microbiota composition was observed in eif2ak4+/+ and eif2ak4−/− mice before infection and at day 1 and 4 post-infection. LF82-infected eif2ak4−/− mice exhibited altered fecal microbiota composition at day 14 and 21 post-infection and increased fecal lipocalin-2 level at day 21 post-infection compared to other groups, indicating that intestinal inflammation developed after microbiota modification. Similar results were obtained for LF82-infected Tg/eif2ak4−/− mice. These results suggest that in genetically predisposed hosts, AIEC colonization might induce chronic intestinal inflammation by altering the gut microbiota composition.