ATG16L1 in myeloid cells limits colorectal tumor growth in ApcMin/+ mice infected with colibactin-producing Escherichia coli via decreasing inflammasome activation.

Escherichia coli strains producing the genotoxin colibactin, designated as CoPEC (colibactin-producing E. coli), have emerged as an important player in the etiology of colorectal cancer (CRC). Here, we investigated the role of macroautophagy/autophagy in myeloid cells, an important component of the tumor microenvironment, in the tumorigenesis of a susceptible mouse model infected with CoPEC. For that, a preclinical mouse model of CRC, the ApcMin/+ mice, with Atg16l1 deficiency specifically in myeloid cells (ApcMin/+/Atg16l1[∆MC]) and the corresponding control mice (ApcMin/+), were infected with a clinical CoPEC strain 11G5 or its isogenic mutant 11G5∆clbQ that does not produce colibactin. We showed that myeloid cell-specific Atg16l1 deficiency led to an increase in the volume of colonic tumors in ApcMin/+ mice under infection with 11G5, but not with 11G5∆clbQ. This was accompanied by increased colonocyte proliferation, enhanced inflammasome activation and IL1B/IL-1ß secretion, increased neutrophil number and decreased total T cell and cytotoxic CD8+ T cell numbers in the colonic mucosa and tumors. In bone marrow-derived macrophages (BMDMs), compared to uninfected and 11G5∆clbQ-infected conditions, 11G5 infection increased inflammasome activation and IL1B secretion, and this was further enhanced by autophagy deficiency. These data indicate that ATG16L1 in myeloid cells was necessary to inhibit colonic tumor growth in CoPEC-infected ApcMin/+ mice via inhibiting colibactin-induced inflammasome activation and modulating immune cell response in the tumor microenvironment. Abbreviation: AOM, azoxymethane; APC, APC regulator of WNT signaling pathway; ATG, autophagy related; Atg16l1[∆MC] mice, mice deficient for Atg16l1 specifically in myeloid cells; CASP1, caspase 1; BMDM, bone marrow-derived macrophage; CFU, colony-forming unit; CoPEC, colibactin-producing Escherichia coli; CRC, colorectal cancer; CXCL1/KC, C-X-C motif chemokine ligand 1; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MC, myeloid cell; MOI, multiplicity of infection; PBS, phosphate-buffered saline; pks, polyketide synthase; qRT-PCR, quantitative real-time reverse-transcription polymerase chain reaction; siRNA, small interfering RNA; TME, tumor microenvironment; TNF/TNF-α, tumor necrosis factor.

The colibactin-producing Escherichia coli alters the tumor microenvironment to immunosuppressive lipid overload facilitating colorectal cancer progression and chemoresistance.

Intratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land's cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

Colibactin-producing Escherichia coli enhance resistance to chemotherapeutic drugs by promoting epithelial to mesenchymal transition and cancer stem cell emergence.

Human colorectal cancers (CRCs) are readily colonized by colibactin-producing E. coli (CoPEC). CoPEC induces DNA double-strand breaks, DNA mutations, genomic instability, and cellular senescence. Infected cells produce a senescence-associated secretory phenotype (SASP), which is involved in the increase in tumorigenesis observed in CRC mouse models infected with CoPEC. This study investigated whether CoPEC, and the SASP derived from CoPEC-infected cells, impacted chemotherapeutic resistance. Human intestinal epithelial cells were infected with the CoPEC clinical 11G5 strain or with its isogenic mutant, which is unable to produce colibactin. Chemotherapeutic resistance was assessed in vitro and in a xenograft mouse model. Expressions of cancer stem cell (CSC) markers in infected cells were investigated. Data were validated using a CRC mouse model and human clinical samples. Both 11G5-infected cells, and uninfected cells incubated with the SASP produced by 11G5-infected cells exhibited an increased resistance to chemotherapeutic drugs in vitro and in vivo. This finding correlated with the induction of the epithelial to mesenchymal transition (EMT), which led to the emergence of cells exhibiting CSC features. They grew on ultra-low attachment plates, formed colonies in soft agar, and overexpressed several CSC markers (e.g. CD133, OCT-3/4, and NANOG). In agreement with these results, murine and human CRC biopsies colonized with CoPEC exhibited higher expression levels of OCT-3/4 and NANOG than biopsies devoid of CoPEC. Conclusion: CoPEC might aggravate CRCs by inducing the emergence of cancer stem cells that are highly resistant to chemotherapy.

Role of adherent and invasive Escherichia coli in Crohn's disease: lessons from the postoperative recurrence model.

OBJECTIVE: We used the postoperative recurrence model to better understand the role of adherent and invasive Escherichia coli (AIEC) bacteria in Crohn's disease (CD), taking advantage of a well-characterised postoperative cohort. DESIGN: From a prospective, multicentre cohort of operated patients with CD, AIEC identification was performed within the surgical specimen (M0) (N=181 patients) and the neoterminal ileum (n=119 patients/181) during colonoscopy performed 6 months after surgery (M6). Endoscopic postoperative recurrence was graded using Rutgeerts' index. The mucosa-associated microbiota was analysed by 16S sequencing at M0 and M6. Relative risks or ORs were adjusted on potential confounders. RESULTS: AIEC prevalence was twofold higher within the neoterminal ileum at M6 (30.3%) than within the surgical specimen (14.9%) (p<0.001). AIEC within the neoterminal ileum at M6 was associated with higher rate of early ileal lesions (i1) (41.6% vs 17.1%; aRR 3.49 (95% CI 1.01 to 12.04), p=0.048) or ileal lesions (i2b+i3) (38.2% vs 17.1%; aRR 3.45 (95% CI 1.06 to 11.30), p=0.040) compared with no lesion (i0). AIEC within the surgical specimen was predictive of higher risk of i2b-endoscopic postoperative recurrence (POR) (aOR 2.54 (95% CI 1.01 to 6.44), p=0.049) and severe endoscopic POR (aOR 3.36 (95% CI 1.25 to 9.06), p=0.017). While only 5.0% (6/119) of the patients were AIEC-positive at both M0 and M6, 43.7% (52/119), patients with history of positive test for AIEC (M0 or M6) had higher risk of ileal endoscopic POR (aOR 2.32 (95% CI 1.01 to 5.39), p=0.048)), i2b-endoscopic postoperative recurrence (aOR 2.41 (95% CI 1.01 to 5.74); p=0.048) and severe endoscopic postoperative (aOR=3.84 (95% CI 1.32 to 11.18), p=0.013). AIEC colonisation was associated with a specific microbiota signature including increased abundance of Ruminococcus gnavus. CONCLUSION: Based on the postoperative recurrence model, our data support the idea that AIEC are involved in the early steps of ileal CD. TRIAL REGISTRATION NUMBER: NCT03458195.

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

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.

Anti-TNF Agents Restrict Adherent-invasive Escherichia coli Replication Within Macrophages Through Modulation of Chitinase 3-like 1 in Patients with Crohn's Disease.

BACKGROUND AND AIMS: The mechanism of action of anti-tumour necrosis factor [anti-TNF] agents could implicate macrophage modulation in Crohn's disease [CD]. As CD macrophages are defective in controlling CD-associated adherent-invasive Escherichia coli [AIEC], anti-TNF agents could limit AIEC replication within macrophages. We assessed the effect of anti-TNF agents on AIEC survival within monocyte-derived macrophages [MDMs] from CD patients and attempted to identify the proteins involved. METHODS: Peripheral blood MDMs were obtained from 44 CD patients [22 with and 22 without anti-TNF agents]. MDMs were infected with reference strain AIEC-LF82. Proteomic analysis was performed before and 6 h after AIEC-LF82 infection. RESULTS: AIEC-LF82 survival was lower in MDMs from CD patients receiving anti-TNF agents compared to those who did not [-73%, p = 0.006]. After AIEC-LF82 infection, the levels of CD82 [p = 0.007], ILF3 [Interleukin enhancer-binding factor 3; p = 0.001], FLOT-1 [Flotillin-1; p = 0.007] and CHI3L1 [Chitinase 3-like 1; p = 0.035] proteins were different within CD-MDMs depending on anti-TNF exposure. FLOT-1 [ϱ = -0.44; p = 0.038] and CHI3L1 [ϱ = 0.57, p = 0.006] levels were inversely and positively correlated with AIEC survival within MDMs from CD patients with or without anti-TNF, respectively. We observed a dose-dependent decrease of AIEC-LF82 survival after adjunction of anti-TNF within MDMs, inducing an increase of FLOT-1 and decrease of CHI3L1 mRNA levels. Neutralization of intra-macrophagic CHI3L1 protein using anti-CHI3L1 antibodies reduced AIEC survival within macrophages 6 h after infection [p < 0.05]. CONCLUSION: Anti-TNF agents are able to restrict replication of pathobionts, such as AIEC, within macrophages by modulating FLOT-1 and CHI3L1 expression in CD patients.

When idiopathic male infertility is rooted in maternal malnutrition during the perinatal period in mice†.

Infertility represents a growing burden worldwide, with one in seven couples presenting difficulties conceiving. Among these, 10-15% of the men have idiopathic infertility that does not correlate with any defect in the classical sperm parameters measured. In the present study, we used a mouse model to investigate the effects of maternal undernutrition on fertility in male progeny. Our results indicate that mothers fed on a low-protein diet during gestation and lactation produce male offspring with normal sperm morphology, concentration, and motility but exhibiting an overall decrease of fertility when they reach adulthood. Particularly, in contrast to control, sperm from these offspring show a remarkable lower capacity to fertilize oocytes when copulation occurs early in the estrus cycle relative to ovulation, due to an altered sperm capacitation. Our data demonstrate for the first time that maternal nutritional stress can have long-term consequences on the reproductive health of male progeny by affecting sperm physiology, especially capacitation, with no observable impact on spermatogenesis and classical quantitative and qualitative sperm parameters. Moreover, our experimental model could be of major interest to study, explain, and ultimately treat certain categories of infertilities.

Beneficial Effects of Natural Mineral Waters on Intestinal Inflammation and the Mucosa-Associated Microbiota.

Natural mineral water (NMWs) intake has been traditionally used in the treatment of various gastrointestinal diseases. We investigated the effect of two French NMWs, one a calcium and magnesium sulphate, sodium chloride, carbonic, and ferruginous water (NMW1), the other a mainly bicarbonate water (NMW2) on the prevention of intestinal inflammation. Intestinal epithelial cells stimulated with heat inactivated Escherichia coli or H2O2 were treated with NMWs to evaluate the anti-inflammatory effects. Moderate colitis was induced by 1% dextran sulfate sodium (DSS) in Balbc/J mice drinking NMW1, NWW2, or control water. General signs and histological features of colitis, fecal lipocalin-2 and pro-inflammatory KC cytokine levels, global mucosa-associated microbiota, were analyzed. We demonstrated that both NMW1 and NMW2 exhibited anti-inflammatory effects using intestinal cells. In induced-colitis mice, NMW1 was effective in dampening intestinal inflammation, with significant reductions in disease activity scores, fecal lipocalin-2 levels, pro-inflammatory KC cytokine release, and intestinal epithelial lesion sizes. Moreover, NMW1 was sufficient to prevent alterations in the mucosa-associated microbiota. These observations, through mechanisms involving modulation of the mucosa-associated microbiota, emphasize the need of investigation of the potential clinical efficiency of such NMWs to contribute, in human beings, to a state of low inflammation in inflammatory bowel disease.

Colibactin-Producing Escherichia coli Induce the Formation of Invasive Carcinomas in a Chronic Inflammation-Associated Mouse Model.

BACKGROUND: Escherichia coli producing the genotoxin colibactin (CoPEC or colibactin-producing E. coli) abnormally colonize the colonic mucosa of colorectal cancer (CRC) patients. We previously showed that deficiency of autophagy in intestinal epithelial cells (IECs) enhances CoPEC-induced colorectal carcinogenesis in ApcMin/+ mice. Here, we tested if CoPEC trigger tumorigenesis in a mouse model lacking genetic susceptibility or the use of carcinogen. METHODS: Mice with autophagy deficiency in IECs (Atg16l1∆IEC) or wild-type mice (Atg16l1flox/flox) were infected with the CoPEC 11G5 strain or the mutant 11G5∆clbQ incapable of producing colibactin and subjected to 12 cycles of DSS treatment to induce chronic colitis. Mouse colons were used for histological assessment, immunohistochemical and immunoblot analyses for DNA damage marker. Results: 11G5 or 11G5∆clbQ infection increased clinical and histological inflammation scores, and these were further enhanced by IEC-specific autophagy deficiency. 11G5 infection, but not 11G5∆clbQ infection, triggered the formation of invasive carcinomas, and this was further increased by autophagy deficiency. The increase in invasive carcinomas was correlated with enhanced DNA damage and independent of inflammation. Conclusions: CoPEC induce colorectal carcinogenesis in a CRC mouse model lacking genetic susceptibility and carcinogen. This work highlights the role of (i) CoPEC as a driver of CRC development, and (ii) autophagy in inhibiting the carcinogenic properties of CoPEC.

Colibactin-positive Escherichia coli induce a procarcinogenic immune environment leading to immunotherapy resistance in colorectal cancer.

Colibactin-producing E. coli (CoPEC) are frequently detected in colorectal cancer (CRC) and exhibit procarcinogenic properties. Because increasing evidence show the role of immune environment and especially of antitumor T-cells in CRC development, we investigated the impact of CoPEC on these cells in human CRC and in the APCMin/+ mice colon. T-cell density was evaluated by immunohistochemistry in human tumors known for their CoPEC status. APCmin/+ mice were chronically infected with a CoPEC strain (11G5). Immune cells (neutrophils and T-cell populations) were then quantified by immunofluorescent staining of the colon. The quantification of lymphoid populations was also performed in the mesenteric lymph nodes (MLNs). Here, we show that the colonization of CRC patients by CoPEC is associated with a decrease of tumor-infiltrating T lymphocytes (CD3+ T-cells). Similarly, we demonstrated, in mice, that CoPEC chronic infection decreases CD3+ and CD8+ T-cells and increases colonic inflammation. In addition, we noticed a significant decrease in antitumor T-cells in the MLNs of CoPEC-infected mice compared to that of controls. Moreover, we show that CoPEC infection decreases the antimouse PD-1 immunotherapy efficacy in MC38 tumor model. Our findings suggest that CoPEC could promote a procarcinogenic immune environment through impairment of antitumor T-cell response, leading to tumoral resistance to immunotherapy. CoPEC could thus be a new biomarker predicting the anti-PD-1 response in CRC.

Autophagy of Intestinal Epithelial Cells Inhibits Colorectal Carcinogenesis Induced by Colibactin-Producing Escherichia coli in ApcMin/+ Mice.

BACKGROUND & AIMS: Colibactin-producing Escherichia coli (CoPEC) colonize the colonic mucosa of a higher proportion of patients with vs without colorectal cancer (CRC) and promote colorectal carcinogenesis in susceptible mouse models of CRC. Autophagy degrades cytoplasmic contents, including intracellular pathogens, via lysosomes and regulates intestinal homeostasis. We investigated whether inhibiting autophagy affects colorectal carcinogenesis in susceptible mice infected with CoPEC. METHODS: Human intestinal epithelial cells (IECs) (HCT-116) were infected with a strain of CoPEC (11G5 strain) isolated from a patient or a mutant strain that does not produce colibactin (11G5ΔclbQ). Levels of ATG5, ATG16L1, and SQSTM1 (also called p62) were knocked down in HCT-116 cells using small interfering RNAs. ApcMin/+ mice and ApcMin/+ mice with IEC-specific disruption of Atg16l1 (ApcMin/+/Atg16l1ΔIEC) were infected with 11G5 or 11G5ΔclbQ. Colonic tissues were collected from mice and analyzed for tumor size and number and by immunohistochemical staining, immunoblot, and quantitative reverse transcription polymerase chain reaction for markers of autophagy, DNA damage, cell proliferation, and inflammation. We analyzed levels of messenger RNAs (mRNAs) encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse-transcription polymerase chain reaction. RESULTS: Patient colonic mucosa with CoPEC colonization had higher levels of mRNAs encoding proteins involved in autophagy than colonic mucosa without these bacteria. Infection of cultured IECs with 11G5 induced autophagy and DNA damage repair, whereas infection with 11G5ΔclbQ did not. Knockdown of ATG5 in HCT-116 cells increased numbers of intracellular 11G5, secretion of interleukin (IL) 6 and IL8, and markers of DNA double-strand breaks but reduced markers of DNA repair, indicating that autophagy is required for bacteria-induced DNA damage repair. Knockdown of ATG5 in HCT-116 cells increased 11G5-induced senescence, promoting proliferation of uninfected cells. Under uninfected condition, ApcMin/+/Atg16l1ΔIEC mice developed fewer and smaller colon tumors than ApcMin/+ mice. However, after infection with 11G5, ApcMin/+/Atg16l1ΔIEC mice developed more and larger tumors, with a significant increase in mean histologic score, than infected ApcMin/+ mice. Increased levels of Il6, Tnf, and Cxcl1 mRNAs, decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in the colonic mucosa of 11G5-infected ApcMin/+/Atg16l1ΔIEC mice vs 11G5-infected ApcMin/+ mice. CONCLUSION: Infection of IECs and susceptible mice with CoPEC promotes autophagy, which is required to prevent colorectal tumorigenesis. Loss of ATG16L1 from IECs increases markers of inflammation, DNA damage, and cell proliferation and increases colorectal tumorigenesis in 11G5-infected ApcMin/+ mice. These findings indicate the importance of autophagy in response to CoPEC infection, and strategies to induce autophagy might be developed for patients with CRC and CoPEC colonization.

Prognostic value of a combination of innovative factors (gut microbiota, sarcopenia, obesity, metabolic syndrome) to predict surgical/oncologic outcomes following surgery for sporadic colorectal cancer: a prospective cohort study protocol (METABIOTE).

INTRODUCTION: Colorectal cancer (CRC) is still associated with poor prognosis, especially in patients with advanced disease. Development of new prognostic tools replacing or supplementing those routinely used is definitely needed, with the aim to optimise and personalise treatment strategies. Gut microbiota composition and body composition profile (obesity, sarcopenia and metabolic syndrome) have recently been reported separately as new relevant prognostic factors for postoperative surgical and oncologic outcomes following CRC surgery. However interactions that exist between these factors have been poorly studied. The purpose of this translational prospective cohort study (METABIOTE) is to investigate potential interactions between gut microbiota, body composition profile and postoperative outcomes and recurrence in patients undergoing surgery for non-metastatic sporadic CRC. METHODS AND ANALYSIS: This single-centre project aims to prospectively enrol 300 consecutive patients undergoing surgery for non-metastatic sporadic CRC at the University Hospital of Clermont-Ferrand, France for the identification of specific microbial signatures (from tumour, colonic mucosa and stools samples) associated with particular metabolic profiles that could impact postoperative morbidity and oncologic outcomes, using microbiological, molecular and imaging approaches. The primary outcome is the 5-year overall survival (OS). Other outcomes are 5-year CRC-related OS, 5-year disease-free survival, 30-day postoperative morbidity, 90-day postoperative mortality and length of hospital stay. ETHICS AND DISSEMINATION: This study protocol was reviewed and approved by an independent French regional review board (n°2018-A00352-53, 'Comité de Protection des Personnes Ile de France VII' on 4 July 2018, declared to the competent French authority ('Agence Nationale de Sécurité du Médicament et des produits de santé', France), and registered on the Clinical Trials web-based platform (NCT03843905). Oral and written informed consent will be obtained from each included patient. Study results will be reported to the scientific community at conferences and in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: NCT03843905..

Tissue-Specific Oxidative Stress Modulation by Exercise: A Comparison between MICT and HIIT in an Obese Rat Model.

BACKGROUND AND AIM: Exercise is an effective strategy to reduce obesity-induced oxidative stress. The purpose of this study was to compare the effects of two training modalities (moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT)) on the pro/antioxidant status of different tissues in obese Zucker rats. METHODS: Eight-week-old male Zucker rats (fa/fa, n = 36) were subdivided in three groups: MICT, HIIT, and control (no exercise) groups. Trained animals ran on a treadmill (0° slope), 5 days/week for 10 weeks (MICT: 51 min at 12 m·min-1; HIIT: 6 sets of 3 min at 10 m·min-1 followed by 4 min at 18 m·min-1). Epididymal (visceral) and subcutaneous adipose tissue, gastrocnemius muscle, and plasma samples were collected to measure oxidative stress markers (advanced oxidation protein products (AOPP), oxidized low-density lipoprotein (oxLDL)), antioxidant system markers (ferric-reducing ability of plasma (FRAP), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities), and prooxidant enzymes (NADPH oxidase and xanthine oxidase (XO) activities, myeloperoxidase content). RESULTS: Compared with the control, MICT increased GPx and catalase activities and the FRAP level in epididymal adipose tissue. HIIT increased the AOPP level in subcutaneous adipose tissue. In the muscle, HIIT increased both SOD and GPx activities and reduced the AOPP level, whereas MICT increased only SOD activity. Finally, plasma myeloperoxidase content was similarly decreased by both training modalities, whereas oxLDL was reduced only in the MICT group. CONCLUSION: Both HIIT and MICT improved the pro/antioxidant status. However, HIIT was more efficient than MICT in the skeletal muscle, whereas MICT was more efficient in epididymal adipose tissue. This suggests that oxidative stress responses to HIIT and MICT are tissue-specific. This could result in ROS generation via different pathways in these tissues. From a practical point of view, the two training modalities should be combined to obtain a global response in people with obesity.

High intensity interval training promotes total and visceral fat mass loss in obese Zucker rats without modulating gut microbiota.

AIMS: Increased visceral adipose tissue and dysbiosis in the overweight and obese promote chronic inflammation. The aim of this study was to compare the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on the gut-adipose tissue cross-talk in obese Zucker rats. METHODS: Obese male Zucker rats (n = 36) were divided in three groups: MICT (12m.min-1 for 51min), HIIT (6 sets at 18 m.min-1 for 4min followed by 3min at 10m.min-1) and controls (CONT; no exercise). The animals ran on a treadmill 5 days/week for 10 weeks. Body composition, glycaemic control, lipid profile, inflammation, lipolysis signalling in subcutaneous and visceral adipose tissue, intestinal permeability (tight junctions and plasma lipopolysaccharide binding protein; LBP), and gut microbiota composition were assessed in the three groups. RESULTS: After 10 weeks of exercise, total and epididymal fat mass decreased only in the HIIT group. The α/ß adrenergic receptor RNA ratio in subcutaneous adipose tissue increased only in the HIIT group. The expression level of phosphorylated hormone-sensitive lipase was not modified by training. Both HIIT and MICT decreased inflammation (plasma myeloperoxidase and keratinocyte-derived chemokine secretion in adipose tissue) and improved glucose metabolism. Zonula occludens-1 and occludin were upregulated in the HIIT group. Plasma LBP was similarly reduced in both training groups. HIIT and MICT did not affect gut microbiota composition. CONCLUSION: In obese Zucker rats, HIIT and MICT improved inflammation and glucose metabolism. In contrast, only HIIT decreased total and visceral fat mass. These adaptations were not associated with modifications in gut microbiota composition.

Preventive Effect of Spontaneous Physical Activity on the Gut-Adipose Tissue in a Mouse Model That Mimics Crohn's Disease Susceptibility.

Crohn's disease is characterized by abnormal ileal colonization by adherent-invasive E. coli (AIEC) and expansion of mesenteric adipose tissue. This study assessed the preventive effect of spontaneous physical activity (PA) on the gut-adipose tissue in a mouse model that mimics Crohn's disease susceptibility. Thirty-five CEABAC10 male mice performed spontaneous PA (wheel group; n = 24) or not (controls; n = 11) for 12 weeks. At week 12, mice were orally challenged with the AIEC LF82 strain for 6 days. Body composition, glycaemic control, intestinal permeability, gut microbiota composition, and fecal short-chain fatty acids were assessed in both groups. Animals were fed a high fat/high sugar diet throughout the study. After exposure to AIEC, mesenteric adipose tissue weight was lower in the wheel group. Tight junction proteins expression increased with spontaneous PA, whereas systemic lipopolysaccharides were negatively correlated with the covered distance. Bifidobacterium and Lactobacillus decreased in controls, whereas Oscillospira and Ruminococcus increased in the wheel group. Fecal propionate and butyrate were also higher in the wheel group. In conclusion, spontaneous physical activity promotes healthy gut microbiota composition changes and increases short-chain fatty acids in CEABAC10 mice fed a Western diet and exposed to AIEC to mimic Crohn's disease.

Interactions between microsatellite instability and human gut colonization by Escherichia coli in colorectal cancer.

Recent studies suggest that colonization of colonic mucosa by pathogenic Escherichia coli could be involved in the development of colorectal cancer (CRC), especially through the production of genotoxins such as colibactin and/or by interfering with the DNA mismatch repair (MMR) pathway that leads to microsatellite instability (MSI). The present study, performed on 88 CRC patients, revealed a significant increase in E. coli colonization in the MSI CRC phenotype. In the same way, E. coli persistence and internalization were increased in vitro in MMR-deficient cells. Moreover, we demonstrated that colibactin-producing E. coli induce inhibition of the mutL homologue 1 (MLH1) MMR proteins, which could lead to genomic instability. However, colibactin-producing E. coli were more frequently identified in microsatellite stable (MSS) CRC. The present study suggests differences in the involvement of colibactin-producing E. coli in colorectal carcinogenesis according to the CRC phenotype. Further host-pathogen interactions studies should take into account CRC phenotypes.

Association of colorectal cancer with pathogenic Escherichia coli: Focus on mechanisms using optical imaging.

AIM: To investigate the molecular or cellular mechanisms related to the infection of epithelial colonic mucosa by pks-positive Escherichia coli (E. coli) using optical imaging. METHODS: We choose to evaluate the tumor metabolic activity using a fluorodeoxyglucose analogue as 2-deoxyglucosone fluorescent probes and to correlate it with tumoral volume (mm(3)). Inflammation measuring myeloperoxidase (MPO) activity and reactive oxygen species production was monitored by a bioluminescent (BLI) inflammation probe and related to histological examination and MPO levels by enzyme-linked immunosorbent assay (ELISA) on tumor specimens. The detection and quantitation of these two signals were validated on a xenograft model of human colon adenocarcinoma epithelial cells (HCT116) in nude mice infected with a pks-positive E. coli. The inflammatory BLI signal was validated intra-digestively in the colitis-CEABAC10 DSS models, which mimicked Crohn's disease. RESULTS: Using a 2-deoxyglucosone fluorescent probe, we observed a high and specific HCT116 tumor uptake in correlation with tumoral volume (P = 0.0036). Using the inflammation probe targeting MPO, we detected a rapid systemic elimination and a significant increase of the BLI signal in the pks-positive E. coli-infected HCT116 xenograft group (P < 0.005). ELISA confirmed that MPO levels were significantly higher (1556 ± 313.6 vs 234.6 ± 121.6 ng/mL P = 0.001) in xenografts infected with the pathogenic E. coli strain. Moreover, histological examination of tumor samples confirmed massive infiltration of pks-positive E. coli-infected HCT116 tumors by inflammatory cells compared to the uninfected group. These data showed that infection with the pathogenic E. coli strain enhanced inflammation and ROS production in tumors before tumor growth. Moreover, we demonstrated that the intra-digestive monitoring of inflammation is feasible in a reference colitis murine model (CEABAC10/DSS). CONCLUSION: Using BLI and fluorescence optical imaging, we provided tools to better understand host-pathogen interactions at the early stage of disease, such as inflammatory bowel disease and colorectal cancer.

Assessment of the Relation between the Expression of Oxaliplatin Transporters in Colorectal Cancer and Response to FOLFOX-4 Adjuvant Chemotherapy: A Case Control Study.

BACKGROUND: Adjuvant chemotherapy for colorectal cancer is mainly based on the combination of 5-fluorouracil, folinic acid and oxaliplatin (FOLFOX-4). The pharmacological target of oxaliplatin remains intracellular and therefore dependent on its entry into cells. The intracellular distribution of oxaliplatin is mediated by organic cation transporters 1, 2 and 3 (OCT1, 2 and 3), copper transporter 1 (CTR1) and ATPase Cu2+ transporting beta polypeptide (ATP7B) and may modulate the efficacy of oxaliplatin-based chemotherapy. The aim of this study was to perform a retrospective study to assess the relation between the expression of oxaliplatin transporters in colorectal cancer before chemotherapy and the response to FOLFOX-4 adjuvant chemotherapy in responder and non-responder patients. METHODS: This retrospective study was conducted at a single center (University Hospital of Clermont-Ferrand, France). The target population was patients with resectable colorectal cancer operated between 2006 and 2013. Inclusion criteria were defined for the responder patients as no cancer recurrence 3 years after the end of chemotherapy, and for the non-responder patients as cancer recurrence within 1 year. Other inclusion criteria were stages IIb-IV cancers, first-line adjuvant FOLFOX-4 chemotherapy, and the availability of resected primary tumor samples. Exclusion criteria were preoperative chemotherapy and/or radiotherapy, a targeted therapy, other anticancer drugs, cancer recurrence between the first and the third year after the end of chemotherapy and follow-up < 3 years. Immunostaining of oxaliplatin transporters (OCT1, 2, 3, CTR1 and ATP7B) and Ki-67 was assessed in tumor samples. RESULTS: Retrospectively, 31 patients have been selected according to inclusion and exclusion criteria (15 responders and 16 non-responders). Before FOLFOX-4 regimen, OCT3 expression was significantly lower in responder patients compared to non-responders (p<0.001). According to multivariate analysis, OCT3 remains an independent criterion for adjuvant FOLFOX chemotherapy response (p = 0.039). No significant relation is reported between chemotherapy response and the expression of OCT1 (p = 0.49), OCT2 (p = 0.09), CTR1 (p = 0.45), ATP7B (p = 0.94) and Ki-67 (p = 0.34) in tumors. CONCLUSIONS: High expression of OCT3 could be an independent factor related to resistance to FOLFOX-4 chemotherapy.

Western diet induces a shift in microbiota composition enhancing susceptibility to Adherent-Invasive E. coli infection and intestinal inflammation.

Recent advances have shown that the abnormal inflammatory response observed in CD involves an interplay among intestinal microbiota, host genetics and environmental factors. The escalating consumption of fat and sugar in Western countries parallels an increased incidence of CD during the latter 20(th) century. The impact of a HF/HS diet in mice was evaluated for the gut micro-inflammation, intestinal microbiota composition, function and selection of an E. coli population. The HF/HS diet created a specific inflammatory environment in the gut, correlated with intestinal mucosa dysbiosis characterized by an overgrowth of pro-inflammatory Proteobacteria such as E. coli, a decrease in protective bacteria, and a significantly decreased of SCFA concentrations. The expression of GPR43, a SCFA receptor was reduced in mice treated with a HF/HS diet and reduced in CD patients compared with controls. Interestingly, mice treated with an agonist of GPR43 were protected against DSS-induced colitis. Finally, the transplantation of feces from HF/HS treated mice to GF mice increased susceptibility to AIEC infection. Together, our results demonstrate that a Western diet could aggravate the inflammatory process and that the activation of the GPR43 receptor pathway could be used as a new strategy to treat CD patients.

Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation.

AIM: To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients. METHODS: Phylogroups and the presence of cyclomodulin-encoding genes of mucosa-associated E. coli from colon cancer and diverticulosis specimens were determined by PCR. Adhesion and invasion experiments were performed with I-407 intestinal epithelial cells using gentamicin protection assay. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression in T84 intestinal epithelial cells was measured by enzyme-linked immunosorbent assay and by Western Blot. Gut colonization, inflammation and pro-carcinogenic potential were assessed in a chronic infection model using CEABAC10 transgenic mice. Cell proliferation was analyzed by real-time mRNA quantification of PCNA and immunohistochemistry staining of Ki67. RESULTS: Analysis of mucosa-associated E. coli from colon cancer and diverticulosis specimens showed that whatever the origin of the E. coli strains, 86% of cyclomodulin-positive E. coli belonged to B2 phylogroup and most harbored polyketide synthase (pks) island, which encodes colibactin, and/or cytotoxic necrotizing factor (cnf) genes. In vitro assays using I-407 intestinal epithelial cells revealed that mucosa-associated B2 E. coli strains were poorly adherent and invasive. However, mucosa-associated B2 E. coli similarly to Crohn's disease-associated E. coli are able to induce CEACAM6 expression in T84 intestinal epithelial cells. In addition, in vivo experiments using a chronic infection model of CEACAM6 expressing mice showed that B2 E. coli strain 11G5 isolated from colon cancer is able to highly persist in the gut, and to induce colon inflammation, epithelial damages and cell proliferation. CONCLUSION: In conclusion, these data bring new insights into the ability of E. coli isolated from patients with colon cancer to establish persistent colonization, exacerbate inflammation and trigger carcinogenesis.