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Increased T helper (Th)1/Th17 immune responses are a hallmark of Crohn’s disease (CD) immunopathogenesis. CD90+ (myo-)fibroblasts (MFs) are abundant cells in the normal (N) intestinal mucosa contributing to mucosal tolerance via suppression of Th1 cell activity through cell surface membrane-bound PD-L1 (mPD-L1). CD-MFs have a decreased level of mPD-L1. Consequently, mPD-L1-mediated suppression of Th1 cells by CD-MFs is decreased, yet the mechanism responsible for the reduction in mPDL-1 is unknown. Increased expression of matrix metalloproteinases (MMPs) has been reported in CD. Herein we observed that when compared to N- and ulcerative colitis (UC)-MFs, CD-MFs increase in LPS-inducible levels of MMP-7 and -9 with a significant increase in both basal and inducible MMP-10. A similar pattern of MMP expression was observed in the CD-inflamed mucosa. Treatment of N-MFs with a combination of recombinant human MMP-7, -9 and -10 significantly decreased mPD-L1. In contrast, inhibition of MMP activity with MMP inhibitors or anti-MMP-10 neutralizing antibodies restores mPD-L1 on CD-MFs. CD-MFs demonstrated reduced capacity to suppress Th1 and Th17 responses from activated CD4+ T cells. By contrast, supplementation of the CD-MF:T-cell co-cultures with MMP inhibitors or anti-MMP neutralizing antibodies restored the CD-MF-mediated suppression. Our data suggest that (i) increased MMP-10 expression by CD-MFs and concomitant cleavage of PD-L1 from the surface of CD-MFs are likely to be one of the factors contributing to the decrease of mPD-L1-mediated suppression of Th1/Th17 cells in CD; and (ii) MMPs are likely to have a significant role in the intestinal mucosal immune responses.

Simple Summary Gut microbiota is emerging as new diagnostic and prognostic marker and/or therapeutic target to improve the management of cancer. This review aims to summarize microbial signatures that have been associated with digestive and other cancers. We report the clinical relevance of these microbial markers to predict the response to cancer therapy. Among these biomarkers, colibactin-producing E. coli are prevalent in the colonic mucosa of patients with colorectal cancer and they promote colorectal carcinogenesis in several pre-clinical models. Here we discuss the promising use of colibactin-producing E. coli as a new predictive factor and a therapeutic target in colon cancer management. Abstract The gut microbiota is crucial for physiological development and immunological homeostasis. Alterations of this microbial community called dysbiosis, have been associated with cancers such colorectal cancers (CRC). The pro-carcinogenic potential of this dysbiotic microbiota has been demonstrated in the colon. Recently the role of the microbiota in the efficacy of anti-tumor therapeutic strategies has been described in digestive cancers and in other cancers (e.g., melanoma and sarcoma). Different bacterial species seem to be implicated in these mechanisms: F. nucleatum, B. fragilis, and colibactin-associated E. coli (CoPEC). CoPEC bacteria are prevalent in the colonic mucosa of patients with CRC and they promote colorectal carcinogenesis in susceptible mouse models of CRC. In this review, we report preclinical and clinical data that suggest that CoPEC could be a new factor predictive of poor outcomes that could be used to improve cancer management. Moreover, we describe the possibility of using these bacteria as new therapeutic targets.

International audience

International audience; Colorectal cancer (CRC) is the second leading cause of cancer worldwide. CRC is still associated with a poor prognosis among patients with advanced disease. On the contrary, due to its slow progression from detectable precancerous lesions, the prognosis for patients with early stages of CRC is encouraging. While most robust methods are invasive and costly, actual patient-friendly screening methods for CRC suffer of lack of sensitivity and specificity. Therefore, the development of sensitive, non-invasive and cost-effective methods for CRC detection and prognosis are necessary for increasing the chances of a cure. Beyond its beneficial functions for the host, increasing evidence suggests that the intestinal microbiota is a key factor associated with carcinogenesis. Many clinical studies have reported a disruption in the gut microbiota balance and an alteration in the faecal metabolome of CRC patients, suggesting the potential use of a microbial-based test as a non-invasive diagnostic and/or prognostic tool for CRC screening. This review aims to discuss the microbial signatures associated with CRC known to date, including dysbiosis and faecal metabolome alterations, and the potential use of microbial variation markers for non-invasive early diagnosis and/or prognostic assessment of CRC and advanced adenomas. We will finally discuss the possible use of these markers as predicators for treatment response and their limitations.

International audience; 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 APC(Min/+) mice colon. T-cell density was evaluated by immunohistochemistry in human tumors known for their CoPEC status. APC(min/+) 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.

Introduction: Chronic inflammatory responses within the colonic environment are critical to colorectal cancer (CRC) tumor development. While the exact causes of CRC development are unknown, recent data suggest that dysbiosis in the colonic microbiome results in the overgrowth of bacteria from Enterebacteriaceae, Fusobacteriaceae and Bacteroidaceae taxonomic families, contributing to CRC development. Among bacteria of the Enterebacteriaceae family, adherent invasive Esherichia coli has been shown to promote tumor cell growth via production of colibactin. However, the contribution of E.coli to the tumor promoting inflammatory microenvironment (TME) during sporadic CRC development remains unknown. Cancer associated fibroblasts (CAFs) are a major component of TME and contribute to tumor-promoting inflammatory responses via NF-κB dependent production of IL-6. Herein we investigate the effect of E. coli on CAFs. We hypothesize that stimulation of CAFs by cancer derived E.coli is a key processes in supporting tumor-promoting inflammation in CRC. Methods: Bacterial DNA was extracted from tumor and adjacent normal tissue of 28 CRC patients. High throughput sequencing was done using metabarcoding of 16S rDNA for bacteria and analyzed using CLC Genomics Workbench 8.0.5 Microbial Genomics Module, SILVA v119 database for 16S. E.coli PS092717 strain, isolated from CRC tumor site, was used in co-culture experiments. Real time RT-PCR and multiplex cytokine array was used to analyze fibroblasts gene expression and secretion, respectively. Results: We observed a reduction in microbial diversity in tumor vs adjacent normal tissue, with an increased prevalence of Fusobacteria, and Enterobactereacea, but not Bacteroidacea. We then tested the effect of E.coli PS092717 on colonic primary normal fibroblasts and CAFs. Exposure of N-CMFs to E.coli for 24 h only moderately increased IL-6, while CAF responded with a greater increase in IL-6 production. Analysis of the signaling mechanism demonstrated that this process was NF-κB dependent, and was abrogated in the presence of NF-kB specific inhibitor, triptolide (20 ng/mL). Bromodomain containing protein 4 (Brd4) is an epigenetic regulator that can recruit canonical NF-κB transcription factor RelA. Therefore, we analyzed its involvement in the above processes. NF-κB mediated increase in IL-6 expression and secretion induced by E.coli PS092717 was strongly reduced in CAFs than N-CMFs in the presence of BRD4 specific inhibitor (1 µM ZL0590). A similar observation was made when a major components of the E.coli cell wall, lipopolysaccharide (LPS), was used. Conclusion Taken together our data suggests that, E.coli derived from cancer tissue and its cell wall component LPS can contribute to the tumor promoting inflammation via stimulation of IL-6 production by CAFs and this process is BRD4/NF-κB-dependent. Citation Format: Gabriela Uribe, Russel Rourke, Romain Villeger, George Golovko, Kamil Khanipov, Zhiqing Liu, Maria Pimenova, Yuriy Fofanov, Jia Zhou, Allen R. Brasier, Irina V. Pinchuk. Cancer derived Escherichia coli induces tumor-promoting inflammatory cytokine IL-6 in cancer associated fibroblasts (CAFs) in a NF-κB/BRD4 dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5065.

Prostaglandin E2 (PGE2 ) plays a critical role in intestinal mucosal tolerance and barrier integrity. Cyclooxygenase-2 (COX-2)-dependent PGE2 production involves mobilisation of arachidonic acid. Lactobacillus rhamnosus GG (LbGG) is one of the most widely used probiotics reported to colonise the colonic mucosa. LbGG contributes to the protection of the small intestine against radiation injury through the repositioning of mucosal COX-2 expressing cells. However, it is unknown if LbGG modulates PGE2 production in the colonic mucosa under homeostasis and the major cellular elements involved in these processes. Colonic epithelial and CD90+ mesenchymal stromal cells, also known as (myo) fibroblasts (CMFs), are abundant innate immune cells in normal colonic mucosa able to produce PGE2 . Herein, we tested the hypothesis that under colonic mucosal homeostasis, LbGG modulates the eicosanoid pathway resulting in increased PGE2 production in both epithelial and stromal cells. Among the five tested human colonic epithelial cell lines, only exposure of Caco-2 to LbGG for 24 hr led to the mobilisation of arachidonic acid with concomitant increase in the components within the leukotriene and COX-2-dependent PGE2 pathways. By contrast, CMFs isolated from the normal human colonic mucosa responded to LbGG with increased expression of COX-2 and PGE2 in the prostaglandin pathway, but not 5-LO in the leukotriene pathway. Oral gavage of C57BL/6 mice for 5 days with LbGG (5 × 108 Colony-Forming Unit (CFU)/dose) increased COX-2 expression in the colonic mucosa. The majority of cells upregulating COX-2 protein expression were located in the colonic lamina propria and colocalised with α-SMA+ cells corresponding to the CMF phenotype. This process was myeloid differentiation factor-88-dependent, because silencing of myeloid differentiation factor-88 expression in CMFs abrogated LbGG-induced upregulation of COX-2 in culture and in vivo. Taken together, our data suggest that LbGG increases release of COX-2-mediated PGE2 , contributing to the maintenance of mucosal homeostasis in the colon and CMFs are among the major contributors to this process.

International audience; Probiotics represent a potential strategy to influence the host's immune system thereby modulating immune response. Lipoteichoic Acid (LTA) is a major immune-stimulating component of Gram-positive cell envelopes. This amphiphilic polymer, anchored in the cytoplasmic membrane by means of its glycolipid component, typically consists of a poly (glycerol-phosphate) chain with d-alanine and/or glycosyl substitutions. LTA is known to stimulate macrophages in vitro, leading to secretion of inflammatory mediators such as Nitric Oxide (NO). This study investigates the structure-activity relationship of purified LTA from three probiotic Bacillus strains (Bacillus cereus CH, Bacillus subtilis CU1 and Bacillus clausii O/C). LTAs were extracted from bacterial cultures and purified. Chemical modification by means of hydrolysis at pH 8.5 was performed to remove d-alanine. The molecular structure of native and modified LTAs was determined by 1H NMR and GC-MS, and their inflammatory potential investigated by measuring NO production by RAW 264.7 macrophages. Structural analysis revealed several differences between the newly characterized LTAs, mainly relating to their d-alanylation rates and poly (glycerol-phosphate) chain length. We observed induction of NO production by LTAs from B. subtilis and B. clausii, whereas weaker NO production was observed with B. cereus. LTA dealanylation abrogated NO production independently of the glycolipid component, suggesting that immunomodulatory potential depends on d-alanine substitutions. d-alanine may control the spatial configuration of LTAs and their recognition by cell receptors. Knowledge of molecular mechanisms behind the immunomodulatory abilities of probiotics is essential to optimize their use.

International audience; Recently, preclinical and clinical studies targeting several types of cancer strongly supported the key role of the gut microbiota in the modulation of host response to anti-tumoral therapies such as chemotherapy, immunotherapy, radiotherapy and even surgery. Intestinal microbiome has been shown to participate in the resistance to a wide range of anticancer treatments by direct interaction with the treatment or by indirectly stimulating host response through immunomodulation. Interestingly, these effects were described on colorectal cancer but also in other types of malignancies. In addition to their role in therapy efficacy, gut microbiota could also impact side effects induced by anticancer treatments. In the first part of this review, we summarized the role of the gut microbiome on the efficacy and side effects of various anticancer treatments and underlying mechanisms. In the second part, we described the new microbiota-targeting strategies, such as probiotics and prebiotics, antibiotics, fecal microbiota transplantation and physical activity, which could be effective adjuvant therapies developed in order to improve anticancer therapeutic efficiency.

Introduction. Retinol (RO) and its active metabolite, all-trans retinoic acid (atRA), have been widely studied as cancer chemotherapeutic agents. atRA is a critical immunoregulatory molecule reported to decrease cancer cell proliferation and inhibit tumor-promoting inflammation. However, recent studies report a loss of efficacy of atRA treatment to counteract colorectal cancer (CRC) progress, and the mechanisms underlying this failure are not fully understood. Alcohol dehydrogenase 1B (ADH1B), a major isoform of alcohol dehydrogenase expressed in normal colon, is responsible for the conversion of RO to atRA. We recently reported that stromal (myo)fibroblasts are major ADH1B expressers in normal colonic mucosa and that this expression is lost during the neoplastic transformation process of CRC. Thus, we hypothesized that aberrant retinoid signaling in cancer-associated fibroblasts (CAFs) is among key processes supporting tumor-promoting inflammation in CRC. Methods. Real-time PCR, confocal microscopy and cytokine luminex arrays were used to evaluate alterations in retinoid pathway gene expression and activity to define its relevance to the tumor-associated inflammation in human normal and CRC colonic tissues, and in normal primary (myo)fibroblasts (N-CMFs) and CAFs. Results. We observed a strong downregulation of ADH1B expression in CAFs in situ and in culture along with an increased expression of the tumor growth promoting inflammatory cytokine IL-6. Addition of the ADH1B substrate, RO, or its metabolite, atRA, to N-CMF cultures decreased the LPS-inducible IL-6 expression by N-CMFs. No downregulation of LPS-inducible IL-6 expression by RO was observed in CAFs, while atRA treatment partially inhibits IL-6 expression. Silencing of adh1b gene in N-CMFs led to the increased production of the LPS-inducible IL-6. This suggests that lack of ADH1B expression in CAFs prevents conversion of RO into atRA, resulting in the disruption of the negative regulation of IL-6. A decrease in downstream enzyme ALDH1A expression, which converts ADH1B’s byproduct retinaldehyde, to atRA, was also observed in CAFs. Finally, only a partial inhibition of IL-6 production by exogenous atRA was observed in CAFs. When compared to N-CMFs, CAFs expressed higher levels of CYP26B1, a cytochrome that is responsible for atRA degradation. Treatment of CAFs with atRA resulted in an eight fold higher upregulation of CYP26B1 expression than in N-CMFs. Conclusions. Taken together, our data suggests that CYP26B1 overexpression in CAFs along with ADH1B downregulation support aberrant retinoic acid metabolism in CRC. Adh1blow/Cyp26b1high activity of CAFs leads to a decrease of atRA availability in the tumor microenvironment, supporting IL-6-driven tumor growth promoting inflammation and resistance to exogenous atRA as a chemotherapeutic agent in CRC. Citation Format: Romain Villeger, Gabriella Uribe, Judy A. Trieu, Paul Johnson, Suimmin Qiu, Don W. Powell, Ellen J. Beswick, Iryna V. Pinchuk. Adh1blow/Cyp26b1high activity of CD90+ (myo)fibroblasts supports tumor-promoting inflammation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4924. doi:10.1158/1538-7445.AM2017-4924