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Keywords:

  • inflammatory bowel diseases;
  • adherent-invasive E. coli;
  • NOD2;
  • antibiotics;
  • dysbiosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Background:

Inflammatory bowel diseases (IBD) patients are abnormally colonized by adherent-invasive Escherichia coli (AIEC). NOD2 gene mutations impair intracellular bacterial clearance. We evaluated the impact of antibiotic treatment on AIEC colonization in wildtype (WT) and NOD2 knockout mice (NOD2KO) and the consequences on intestinal inflammation.

Methods:

After 3 days of antibiotic treatment, mice were infected for 2 days with 109 CFU AIEC and sacrificed 1, 5, and 60 days later. In parallel, mice were challenged with AIEC subsequent to a dextran sodium sulfate (DSS) treatment and sacrificed 9 days later. Ileum, colon, and mesenteric tissues were sampled for AIEC quantification and evaluation of inflammation.

Results:

Without antibiotic treatment, AIEC was not able to colonize WT and NOD2KO mice. Compared with nontreated animals, antibiotic treatment led to a significant increase in ileal and colonic colonization of AIEC in WT and/or NOD2KO mice. Persistent AIEC colonization was observed until day 5 only in NOD2KO mice, disappearing at day 60. Mesenteric translocation of AIEC was observed only in NOD2KO mice. No inflammation was observed in WT and NOD2KO mice treated with antibiotics and infected with AIEC. During DSS-induced colitis, colonization and persistence of AIEC was observed in the colon. Moreover, a dramatic increase in clinical, histological, and molecular parameters of colitis was observed in mice infected with AIEC but not with a commensal E. coli strain.

Conclusions:

Antibiotic treatment was necessary for AIEC colonization of the gut and mesenteric tissues and persistence of AIEC was dependent on NOD2. AIEC exacerbated a preexisting DSS-induced colitis in WT mice. (Inflamm Bowel Dis 2012)

Inflammatory bowel diseases (IBDs) are chronic conditions involving predominantly young people. Their pathophysiology is multifactorial including genetic, immunologic, and environmental factors, leading to a defect of the intestinal barrier and to an inappropriate response of the gut immune system to the intestinal flora.1 In patients with acute intestinal inflammation, bacteria with adherent and invasive-specific properties have been reported such as Shigella flexneri, Yersinia, and members of the E. coli family, such as EIEC (enteroinvasive E. coli) and AIEC (adherent-invasive E. coli).2, 3 In Crohn's disease (CD), AIEC have been described to preferentially colonize the ileum of 40% patients.4–8 AIEC are a family of genetically heterogeneous strains.9–11 One type strain, namely LF82, has been chosen among other AIEC strains, for its in vitro invasive ability, adhering and invading intestinal epithelial cells and replicating into macrophages triggering tumor necrosis factor (TNF) secretion.6, 12 CEACAM6 (carcinoembryonic antigen-related cell-adhesion molecule 6) and the endoplasmic reticulum-stress response chaperone Gp96, expressed on ileal epithelial cells and upregulated during IBD, were described to be receptors for AIEC.13, 14

More than 100 single nucleotide polymorphism genetic mutations have been described so far participating in IBD susceptibility, most of them controlling microbial recognition, autophagy, and innate immune pathways.15, 16 Among these susceptibility genes, NOD2 (nucleotide-binding oligomerization domain-2) represents the more investigated and replicated one.17, 18 NOD2 mutations are carried by 30%–40% of CD patients compared with 10%–15% in healthy controls, with a 4–14-fold increased risk to develop CD if respectively one or two NOD2 alleles are mutated.19, 20 NOD2 belongs to the nucleotide-binding domain leucin-rich repeat (LRR) family of cytoplasmic proteins and is expressed in macrophages, intestinal epithelial cells, dendritic cells, and T lymphocytes.21 NOD2 binding to the Rip2 kinase activates downstream signaling pathways, including nuclear factor kappaB (NF-κB) and mitogen-activated protein kinase (MAPK), resulting in the secretion of inflammatory cytokines and antimicrobial peptides contributing to subsequent elimination of bacteria. More than 90% of the NOD2 mutations associated with CD occur in the LRR domain, suggesting a defect of bacterial clearance in CD patients with NOD2 mutations.22 Monocytes isolated from CD patients carrying NOD2 mutations were demonstrated to have a disturbed cytokine secretion following infection with AIEC.23 These data provided a link between a genetic susceptibility and an environmental factor such as an invasive bacterium. However, several arguments against a primary role for NOD2 and AIEC in the early onset of IBD are now emerging.24 Indeed, on the one hand, NOD2 mutated mice, carrying either the knockout or the frameshift mutation, do not spontaneously develop colitis, and on the other hand, AIEC colonization may occur without signs of disease.25–27 However, it is well known that the mucosal biofilm acts as a protective barrier toward pathogens.28, 29 These properties have been experimentally shown by covering intestinal epithelial cell lines with Lactobacillus casei, resulting in noncolonization by LF82.30 Antibiotics consumption participates in the deregulation of the intestinal flora, first by disrupting not only the luminal but also the mucosa-associated flora, and after the end of the antibiotic treatment by a massive rebound effect increasing the risk of intestinal infections.31 Moreover, epidemiological studies have reported more frequent CD in adults who have received frequent antibiotic treatments during childhood.32

The aim of the present study was first to determine the role of an antibiotic treatment on AIEC colonization in wildtype (WT) and NOD2KO (knockout) mice and its effect on the induction of an intestinal inflammation. The second aim was to assess the effect of a preexisting inflammation on the expression of AIEC pathogenicity.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Bacterial Strains

AIEC strains LF82 and 06362 were isolated from ileal lesions of patients with CD. Commensal E. coli K12 strain was used as a nonpathogenic reference. Bacterial strains were grown overnight in Luria-Bertani (LB) broth at 37°C. Cultured bacteria were harvested by centrifugation at 3000g for 10 minutes and pellets were resuspended in sterile NaCl 0.9% for intragastric administration.

Cell Lines and Cell Culture

The Intestine-407 (I-407) cell line (ATCC CCL6, Manassas, VA) was derived from human embryonic jejunum and ileum. The cells were maintained in an atmosphere containing 5% CO2 at 37°C in Basal Medium Eagle (Lifetechnologies, France) supplemented with 10% (v/v) heat-inactivated fetal calf serum (PAN biotech-GmbH, Germany), 1% L-glutamine (Lifetechnologies, France), 100,000 U/L penicillin, and 100 mg/L streptomycin (Lifetechnologies).

Mice

C57/Bl6 WT mice were purchased from Centre d'Elevage Janvier Laboratories (Le Genest-St. Isle, France). NOD2KO mice were bred in the Transgenose Institute, Centre National de la Recherche Scientifique of Orleans (France). All mice were housed in specific pathogen-free conditions at the Pasteur Institute of Lille (France). All experimental procedures were approved by and complied with the ethical and animal experiment regulations of the French Government.

Mice Infection Procedure

The 8–12-week-old WT and NOD2KO mice were orally given gentamicin (3 mg/kg/d) and vancomycin (40 mg/kg/d) once a day for 3 days.33 At the end of the third day of antibiotic treatment, mice were orally challenged with 109 CFU of E. coli AIEC (LF82 or 06362), once a day for 2 days (Supporting Fig. 1A). Controls consisted of nontreated (NT) mice, mice infected with AIEC without antibiotic treatment, and mice only treated with antibiotics (ATB), raised in the same environment. Animals were harvested at days 1, 5, and 60 after infection. Samples of colon and ileum were collected for mucosal-associated bacteria quantification, histological analysis, and TNF expression by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Mesenteric lymph nodes and mesenteric fat were collected for bacterial translocation assessment.

Protocol of DSS-induced Colitis

Mice were given 2% (wt/vol) of dextran sulfate sodium (DSS; molecular mass = 40 kDa, TdB, Uppsala, Sweden) in drinking water for 9 days, starting with the antibiotic treatment previously described, and were challenged with AIEC 06362 or K12 (Supporting Fig. 1B). Controls consisted of mice receiving DSS and antibiotic treatment (DSS+ATB) without AIEC challenge. Animals were harvested after 9 days of DSS treatment. Samples of colon, ileum, mesenteric lymph nodes, and mesenteric fat were collected and processed as above.

Microbiologic Analysis

Samples of colon, ileum, mesenteric lymph nodes, and mesenteric fat were introduced immediately into preweighed vials containing 1.5 mL of cysteinated Ringer's solution. After physical disruption of the ileum and colon specimens, 10-fold dilutions were performed in the same diluent (decimal dilutions from 10−2 to 10−5). Each dilution was spread onto plates of nonselective blood agar (modified Columbia agar) incubated at 37°C for 1 week under anaerobic conditions, McConkey plates (BioMerieux, Marcy l'Etoile, France) incubated at 37°C for 48 hours under aerobic conditions, and MRS plates (Man, Rogosa, Sharpe) incubated at 37°C for 48 hours under CO2-enriched conditions.34 Total counts were performed, and different types of colonies were subcultured and identified following established morphological and biochemical criteria. Suspicious E. coli colonies were identified by the API system (BioMérieux sa, Marcy l'Etoile, France). AIEC colonies were differentiated from noninvasive E. coli by invasion assays. Quantitative results are expressed in log colony forming unit (CFU)/g. The threshold of detection is 104 CFU/g.

After disruption of mesenteric lymph nodes and mesenteric fat in the Ringer's solution, 1 mL was grown in BH enrichment broth (Brain-Heart) and 0.1 mL was spread onto plate of nonselective blood agar and incubated at 37°C for 1 week under anaerobic conditions. If a trouble was observed in the BH broth, 0.1 mL was spread onto plate of nonselective blood agar and incubated at 37°C for 1 week under anaerobic conditions. Subcultured bacteria were identified as above. The threshold of detection was 102 CFU/g. All samples were analyzed in a blind manner.

Invasion Assays

Monolayers of I-407 were seeded in 24-well tissue culture plates at a density of 4.105 cells/well and incubated for 20 hours. The cell monolayers were washed twice with phosphate-buffered saline (PBS). Bacteria were grown overnight in LB broth at 37°C. Each monolayer was infected with 1 ml of the cell culture medium (BME) without antibiotics at a multiplicity of infection of 10 bacteria per epithelial cell. After a 3-hour incubation period at 37°C with 10% CO2, infected monolayers were washed with PBS. For measurement of invasion, fresh cell culture medium (BME) containing 100 μg/mL of gentamicin (Invitrogen, France) was added to kill extracellular bacteria. After incubation for an additional hour, monolayers were washed twice with PBS and 1 mL of 1% Triton X-100 (Sigma Chemical, France) was added to each well for 5 minutes to lyse the eukaryotic cells. Samples were removed, diluted, and plated onto LB agar plates to determine the number of CFU recovered from the lysed monolayers. Invasion levels were expressed as the percentage of the original inoculum invading cells and thus resistant to gentamicin treatment. Bacterial strain was considered as invasive when invasion was >0.1%.6

mRNA Quantification

Total RNA were isolated from colonic and ileal tissues using the “Nucleospin RNA II” kit from Macherey-Nagel (Hoerdt, France) following the manufacturer's instructions. Reverse transcription of mRNA was carried out in a final volume of 20 μL from 1 μg total RNA using the “High Capacity DNA Reverse Transcription Kit” (Applied Biosystems, France) according to the manufacturer's instructions. PCR was performed using an ABI 7000 prism sequence detection system (Applied Biosystems) with SYBR green (Applied Biosystems). The sense and antisense oligonucleotides (Eurogentec) used were, respectively: β-actin, 5′-GAATGGGTCAGAAGG ACTCCTATGT-3′ and 5′-CCATGTCGTCCCAGTTGGTAA-3′; TNF, 5′-TGGGAGTAGACAAGGTACAACCC-3′ and 5′-CATCTTCTCAAAATTCGAGTGACA A-3′. All results were normalized to the unaffected housekeeping β-actin gene.

Clinical Assessment of Colitis and Histological Evaluation of Colonic Damage

Disease activity index (DAI) and histological scores were determined as previously described.35 Rectal bleeding was assessed with Hemocult II test (SKD SARL). The DAI score ranged from 0 (healthy) to 12 (greatest activity of colitis). After mouse sacrifice, colon and ileum were excised and rolls of the proximal colon and ileum were fixed in buffered 4% paraformaldehyde, paraffin-embedded, cut into 4-μm slices, and stained with MGG (May-Grünwald Giemsa). The histological severity of colitis was graded in a double-blinded fashion. The tissue samples were assessed for the extent and depth of inflammation and the degree of lamina propria and submucosal mononuclear cellular infiltration.

Statistical Tests

Statistical analysis was performed using the Wilcoxon–Mann–Whitney exact test with the software Graphpad Prism 5 (San Diego, CA). A P-value < 0.05 was considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Colonization of WT and NOD2KO Mice by AIEC Strain 06362

In order to analyze the ability of AIEC to colonize mice, we orally challenged WT and NOD2KO mice with the AIEC strain 06362 and assessed the number of AIEC adherent to the colon and ileum at days 1, 5, and 60 after infection. We noticed that AIEC was not able to colonize mice, as no AIEC bacteria were detected either in the colon or in the ileum of WT and NOD2KO mice at each time of necropsy (Fig. 1A,B). These results indicated that AIEC did not spontaneously colonize mice.

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Figure 1. AIEC 06362 colonization and persistence in the ileum and colon. Quantification of ileal (A) and colonic (B) mucosal-associated AIEC in WT and NOD2KO mice at day 1 (D1), 5 (D5), and 60 (D60) with or without (nontreated = NT) oral infection with 109 AIEC 06362. Results are expressed as CFU/g of tissue. Number of mice per group: n = 4–7 in the NT WT group, n = 5–6 in the other groups. Results are expressed as log CFU/g of tissue with a detection threshold of 4 log CFU/g. Error bars represent SEM.

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Colonization of WT and NOD2KO Mice by AIEC Strain 06362 After Antibiotic Treatment

We inoculated WT and NOD2KO mice with AIEC 06362, after the antibiotic treatment by two wide spectrum nonabsorbed antibiotics, gentamicin and vancomycin, and quantified AIEC in the ileum and colon. Antibiotic treatment significantly enhanced the colonization of the ileum by AIEC at day 1 postinfection in WT and NOD2KO mice (Fig. 2A). A persistence of AIEC 06362 was only observed in NOD2KO mice at day 5. In the colon, antibiotic treatment significantly enhanced AIEC colonization in NOD2KO mice but not in WT mice at day 1, but AIEC did not persist at day 5 (Fig. 2B). In the ileum and colon, AIEC were not detected at day 60.

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Figure 2. AIEC 06362 colonization and persistence in the ileum and colon after antibiotic treatment. Quantification of ileal (A) or colonic (B) mucosal-associated AIEC in WT and NOD2KO mice at day 1 (D1), 5 (D5), and 60 (D60) after antibiotic (ATB) treatment and oral infection with 109 AIEC 06362. Results are expressed as log CFU/g of tissue with a detection threshold of 4 log CFU/g. n = 5–6 mice per group. Error bars represent SEM. *P < 0.05; **P < 0.01.

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Mesenteric Translocation of AIEC 06362 in WT and NOD2KO Mice

We assessed bacterial translocation to the mesenteric lymph nodes and mesenteric fat. As shown in Figure 3, AIEC was not observed, at a significant level, in WT mice with or without antibiotic treatment. However, in NOD2KO mice the antibiotic treatment induced a colonization of the mesenteric lymph nodes and mesenteric fat with AIEC at day 1, but the bacteria did not persist significantly at days 5 and 60. Taken together, our results of bacterial translocation revealed an antibiotic-induced increase of AIEC 06362 colonization in mesenteric tissues dependent on NOD2 parallel to gut colonization as previously observed.

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Figure 3. AIEC 06362 bacterial colonization and persistence in the mesenteric lymph nodes and mesenteric fat after antibiotic treatment. Quantification of AIEC in mesenteric lymph nodes (MLN) (A) or mesenteric fat (B) of WT and NOD2KO mice at day 1 (D1), 5 (D5), and 60 (D60) after antibiotic (ATB) treatment and oral infection with 109 AIEC 06362. Results are expressed as log CFU/g of tissue with a detection threshold of 2 log CFU/g. Number of mice per group: n = 4–7 in the NT (nontreated) WT group and 5–6 in the other groups. Error bars represent SEM. *P < 0.05; **P < 0.01.

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Colonization of NOD2KO Mice by Opportunistic Bacteria After Antibiotic Treatment

To investigate the effect of antibiotic treatment and AIEC infection on the bacterial flora of mice, we analyzed the cultivable flora in the gut and mesenteric tissues during the course of our experiments. Two days after the end of the antibiotic treatment, corresponding to D1 after the end of infection, we observed an emergence of enterobacteria other than AIEC (noninvasive E. coli, Enterobacter cloacae, Serratia odorifera, Klebsiella oxytoca) in the ileum and colon of WT and NOD2KO mice (Fig. 4A,B). NOD2KO mice were more affected than WT mice by the antibiotic treatment. Indeed, instead of WT mice, in which enterobacteria had dramatically decreased at D5, in NOD2KO mice enterobacteria stayed at a high level. Translocation of enterobacteria to mesenteric tissues was only observed in NOD2KO mice, suggesting a susceptibility of NOD2KO mice to colonization and persistence of potentially harmful bacteria (Fig. 5).

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Figure 4. Enterobacteria other than AIEC colonization in the ileum and colon after treatment. Quantification of ileal (A) or colonic (B) mucosal-associated Enterobacteria other than AIEC (noninvasive E. coli, Enterobacter cloacae, Serratia odorifera, Klebsiella oxytoca) in WT and NOD2KO mice at day 1 (D1), 5 (D5), and 60 (D60) after antibiotic (ATB) treatment and oral infection with 109 AIEC 06362. Results are expressed as log CFU/g of tissue with a detection threshold of 4 log CFU/g. Number of mice per group: n = 5 in NT (nontreated) WT and NOD2KO, WT and NOD2KO AIEC 06362-infected groups, and n = 6 in the other groups. Error bars represent SEM. *P < 0.05; **P < 0.01.

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Figure 5. Enterobacteria other than AIEC colonization in the mesenteric tissues after treatment. Quantification of Enterobacteria other than AIEC (noninvasive E. coli, Enterobacter cloacae, Serratia odorifera, Klebsiella oxytoca) in the mesenteric tissues (mesenteric lymph nodes and mesenteric fat) of WT and NOD2KO mice at day 1 (D1), 5 (D5), and 60 (D60), after antibiotic (ATB) treatment and oral infection with 109 AIEC 06362. Results are expressed as log CFU/g of tissue with a detection threshold of 2 log CFU/g. Number of mice per group: n = 5 in NT (nontreated) WT and NOD2KO, WT and NOD2KO AIEC 06362-infected groups, and n = 6 in the other groups. Error bars represent SEM. *P < 0.05; **P < 0.01.

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Inflammation in WT and NOD2KO Mice Colonized by AIEC 06362

We assessed the occurrence of inflammation from day 1 to day 60 after AIEC administration. Body weight, a clinical parameter of inflammation, increased similarly in all animals, reflecting their healthy condition (Fig. 6A). TNF-α mRNA levels were similar in the ileum or colon of WT and NOD2KO animals at day 1 and day 5 after AIEC administration compared with uninfected control animals (Fig. 6B). Histological assessment of inflammation at day 1, 5, and 60 postinfection found no modification of ileon and colon architecture, epithelial lesions, or inflammatory infiltrate (Fig. 6C). Together, our results do not suggest an inflammation induced by the colonization of the gut and mesenteric tissues by AIEC 06362.

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Figure 6. Evaluation of clinical (body weight), molecular (TNF expression) and histological parameters of intestinal inflammation. (A) Body weight evolution is expressed as a percentage of variation in WT and NOD2KO mice. (B) TNF mRNA expression by real-time PCR in the ileum and colon of WT and NOD2KO mice at day1 (D1) and 5 (D5). mRNA levels were normalized to β-actin. (C) Photographs showing representative colonic and ileal sections of WT and NOD2KO mice treated by antibiotics (ATB) and infected with AIEC 06362 at day 1. Number of mice per group: n = 4 in the NT (nontreated) WT group, n = 5 in the NT NOD2KO group and n = 6 in the other groups. Error bars represent SEM. **P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Colonization of WT and NOD2KO Mice by AIEC LF82 After Antibiotic Treatment

The same experiments of colonization were performed with the AIEC-type strain LF82 and a similar profile as for AIEC 06362 was found. No colonization was observed without antibiotic treatment (data not shown), and a significant colonization of the ileum and colon of WT and NOD2KO was observed after antibiotic treatment at day 1, which persisted until day 5 only in the ileum of NOD2KO mice (Supporting Fig. 2A,B). Moreover, antibiotic treatment induced a mesenteric translocation of AIEC at day 1 but no persistence at day 5, only in NOD2KO mice (Supporting Fig. 3A,B). Consistent with AIEC 06362, no sign of intestinal inflammation was observed (data not shown).

Colonization of WT Mice by AIEC 06362 After DSS Treatment

We analyzed AIEC 06362 colonization following DSS treatment in WT mice. At the end of the DSS treatment, corresponding to day 5 after infection, we observed a significant gut colonization of AIEC (Fig. 7A). By comparison with the antibiotic treatment alone, in which AIEC was not detected at day 5 postinfection (Fig. 2B), we showed that DSS induced a persistence of AIEC in the colon. Regarding mesenteric translocation, DSS treatment did not induce a significant colonization of the mesenteric fat by AIEC (Fig. 7B). However, we noticed an increase in translocation of opportunistic bacteria (AIEC, noninvasive E. coli, Cronobacter sakazakii, Enterobacter cloacae, Lactobacillus sp, Enterococcus sp, Clostridium clostridioforme, Prevotella, Ruminococcus) in WT mice following DSS treatment (Fig. 7C).

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Figure 7. Colonization of the colon and mesenteric tissues after DSS and antibiotic treatment. Quantification of colonic-associated AIEC (A), mesenteric tissues-associated AIEC (B), or mesenteric tissues total bacteria (AIEC, E. coli, Cronobacter sakazakii, Enterobacter cloacae, Lactobacillus sp, Enterococcus sp, Clostridium clostridioforme, Prevotella, Ruminococcus) (C) in WT mice receiving 2% DSS and antibiotic treatment (ATB) at day 5 after oral infection with 109 AIEC 06362. Results are expressed as log CFU/g of tissue with a detection threshold of 4 log CFU/g for colonic tissue and 2 log CFU/g for mesenteric tissues. Number of mice per group: n = 9 for the DSS-, antibiotic-treated and AIEC 06362-infected group, and n = 7 for the other groups. Error bars represent SEM. *P < 0.05; **P < 0.01; ***P <0.001.

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Inflammation in WT Mice Colonized by AIEC 06362 After DSS Treatment

We assessed the occurrence of inflammation during DSS treatment on clinical parameters (body weight and DAI), TNF expression, and histological analysis. Body weight variations suggested a persistent inflammation in AIEC 06362-treated mice. AIEC 06362 infection induced a highly significant weight loss in comparison to noninfected and commensal E. coli strain K12-infected mice (Fig. 8A). DAI, TNF expression, and histological score, assessed at day 5, were significantly higher in AIEC 06362-infected mice compared with the other groups (Fig. 8B–D).

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Figure 8. Evaluation of clinical and molecular parameters of colitis. (A) Body weight evolution is expressed as a percentage of variation. (B) Disease activity index (DAI) reflecting clinical parameters (weight loss, stool consistency, and bleeding) at day 5 postinfection. (C) TNF mRNA expression by real-time PCR in the colon at day 5. mRNA levels were normalized to bactin. (D) Histological score based on extent and depth of the epithelium inflammation and degree of lamina propria and submucosal mononuclear cellular infiltration. The histological score corresponds to the sum of each item. Number of mice per group: n = 9 for the DSS-, antibiotic-treated (ATB) and AIEC 06362-infected group, and n = 7 for the other groups. (NT = nontreated) ***P < 0.001.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

In this study we postulated colonization of NOD2KO mice by AIEC after antibiotic treatment that aimed to disrupt the normal intestinal microflora. To avoid strain specificity, we tested two different AIEC strains (06362 and LF82) and observed the same behavior. We used an antibiotic treatment including nonabsorbable gentamicin and vancomycin, which has been demonstrated to induce a profound rupture of colonization resistance.36, 37 Indeed, immediately after microflora disruption and AIEC inoculation, high levels of these bacteria were present on the ileal and colonic mucosa in both WT and NOD2KO mice. In contrast to WT mice, colonization persisted for a longer time in NOD2KO mice and translocation was enhanced, showing a lack of elimination of bacteria penetrating the tissues linked to NOD2 deficiency. Increased translocation was not specific to AIEC but occurred also for other enterobacteria such as, noninvasive E. coli, Enterobacter, Serratia, or Klebsiella. Bacterial translocation was achieved in mesenteric lymph nodes and mesenteric fat; it has been previously described that mesenteric fat showed a higher colonization level than mesenteric lymph nodes.38 Our results confirmed these observations by revealing a 10-fold higher level of bacteria in mesenteric fat.

Our data showed that a modification of the intestinal protective biofilm was necessary to allow AIEC to invade the gut. We suggest that a permeable biofilm might be a risk factor leading somehow to develop IBD. Clinical studies showed a higher antibiotic intake in the 2–5 years prior to diagnosis in CD patients compared with controls,39 and an increased risk of pediatric CD if antibiotics are used before the age of 5 years, especially in the first year of life.32, 40 These retrospective data were confirmed by a recent prospective study showing that antibiotics in childhood increased the risk of pediatric CD.41 Another mechanism destroying normal intestinal flora is acute infection by enteropathogens like Salmonella or Campylobacter, which also appear as risk factors for developing IBD.42, 43

When animals were followed up to 2 months, AIEC colonization no longer persisted in either mouse model. Furthermore, at no time were clinical, histological, or biological signs of inflammation observed, despite the fact that AIEC gained access to the epithelial layer.

To better understand the respective role of AIEC and inflammation in the pathophysiology of CD, we built in WT mice (excluding additional genetic susceptibility) a model of DSS-induced inflammation with subsequent AIEC colonization. We observed a nearly 10-fold higher level and a longer-lasting colonization by AIEC in the colonic tissue of DSS-treated mice. This heavier colonization by AIEC was associated with dramatic increase in all measured parameters of inflammation. Our observations are indeed in accordance with the data published by Carvalho et al,44 where a daily LF82-challenge aggravated a 2% DSS-induced colitis in WT mice. Our results evidence a persistent colonization of the colonic mucosa by AIEC, 5 days after infection in our model of DSS-induced colitis.

Since we demonstrated in this work that AIEC persistence did not occur in the absence of inflammation in WT mice, we believe its persistence is a consequence of inflammation, through a leaky mucosal barrier allowing nonspecific translocation of several bacteria other than AIEC. Our results reinforce the hypothesis that AIEC need a particular context to participate to the inflammatory process, such as CEACAM overexpression, DSS, or antibiotic treatment.14, 24

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

We thank all members of CDTA Orléans for technical advice. We thank the staff of the laboratory of bacteriology and especially Isabelle Houcke, Severine Mahieux, and Charles Paul-Constant for skillful technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Additional supporting information may be found in the online version of this article.

FilenameFormatSizeDescription
IBD_22908_sm_SuppFig1.TIF56KSupporting Figure 1: Study design. (A) WT and NOD2KO mice were orally given antibiotics (ATB) once a day for three days. At the end of the 3th day of antibiotic treatment, mice were orally challenged with 109 CFU of E. coli AIEC (strain LF82 or strain 06362), once a day for two days. Animals were harvested at day 1,5 and 60 after infection. (B) WT mice were given 2% (wt/vol) of dextran sulfate sodium (DSS), for 9 days, in drinking water starting with the antibiotic treatment previously described, and were challenged with AIEC 06362 or E. coli K12 Animals were harvested after 9 days of DSS treatment.
IBD_22908_sm_SuppFig2.TIF82KSupporting Figure 2: AIEC LF82 colonization and persistence in the ileum and colon after antibiotic treatment. Quantification of ileal (A) or colonic (B) mucosal-associated AIEC in WT and NOD2KO mice receiving antibiotic treatment (ATB) at day 1 (D1), 5 (D5) and 60 (D60) after oral infection with 109 AIEC LF82. Results are expressed as log CFU per gram of tissue with a detection threshold of 4 log CFU/g. Number of mice per group: n= 4 to 6 in the WT and NOD2KO AIEC LF82-infected and n=6 in the other groups. Error bars represent SEM. *, p < 0.05; **, p < 0.01.
IBD_22908_sm_SuppFig3.TIF80KSupporting Figure 3: AIEC LF82 translocation to the mesenteric lymph nodes and mesenteric fat after antibiotic treatment. Quantification of AIEC LF82 bacteria in the mesenteric lymph nodes (MLN) (A) or mesenteric fat (B) of WT and NOD2KO mice receiving antibiotic treatment at day 1 (D1), 5 (D5) and 60 (D60) after oral infection with 109 AIEC LF82. Results are expressed as log CFU per gram of tissue with a detection threshold of 2 log CFU/g. Number of mice per group :n= 4 to 6 in the WT and NOD2KO AIEC LF82-infected and n=6 in the other groups. Error bars represent SEM. *, p < 0.05; **, p < 0.01.

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