A single faecal microbiota transplantation modulates the microbiome and improves clinical manifestations in a rat model of colitis

Inflammatory bowel disease; Faecal microbiota transplantation; Rat model of colitisEnfermedad inflamatoria intestinal; Trasplante de microbiota fecal; Modelo de colitis en ratasMalaltia inflamatòria intestinal; Trasplantament de microbiota fecal; Model de colitis en ratesBackground: Faecal microbiota transplantation (FMT) is a novel potential therapy for inflammatory bowel diseases, but it is poorly characterised. Methods: We evaluated the performance of the mouse and rat as a pre-clinical model for human microbiota engraftment. We then characterised the effect of a single human stool transfer (HST) on a humanised model of DSS-induced colitis. Colonic and faecal microbial communities were analysed using the 16S rRNA approach and clinical manifestations were assessed in a longitudinal setting. Findings: The microbial community of rats showed greater similarity to that of humans, while the microbiome of mice showed less similarity to that of humans. Moreover, rats captured more human microbial species than mice after a single HST. Using the rat model, we showed that HST compensated faecal dysbiosis by restoring alpha-diversity and by increasing the relative abundance of health-related microbial genera. To some extent, HST also modulated the microbial composition of colonic tissue. These faecal and colonic microbial communities alterations led to a relative restoration of colon length, and a significant decrease in both epithelium damage and disease severity. Remarkably, stopping inflammation by removing DSS before HST caused a faster and greater recovery of both microbiome and clinical manifestation features. Interpretation: Our results indicate that the rat outperforms the mouse as a model for human microbiota engraftment and show that the efficacy of HST can be enhanced when inflammation stimulation is withdrawn. Finally, our findings support a new therapeutic strategy based on the use FMT combined with anti inflammatory drugs.Study funded by the Instituto de Salud Carlos III/FEDER (PI17/00614), a government agency. The funder had no role in study design, data collection, data analysis, interpretation or writing of the report

Melanoma Cells Treated with GGTI and IFN-γ Allow Murine Vaccination and Enhance Cytotoxic Response against Human Melanoma Cells

International audienceBACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-gamma; and iv) anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells

CD4CD8αα Lymphocytes, A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease

It has become evident that bacteria in our gut affect health and disease, but less is known about how they do this. Recent studies in mice showed that gut Clostridium bacteria and their metabolites can activate regulatory T cells (Treg) that in turn mediate tolerance to signals that would ordinarily cause inflammation. In this study we identify a subset of human T lymphocytes, designated CD4CD8αα T cells that are present in the surface lining of the colon and in the blood. We demonstrate Treg activity and show these cells to be activated by microbiota; we identify F. prausnitzii, a core Clostridium strain of the human gut microbiota, as a major inducer of these Treg cells. Interestingly, there are fewer F. prausnitzii in individuals suffering from inflammatory bowel disease (IBD), and accordingly the CD4CD8αα T cells are decreased in the blood and gut of patients with IBD. We argue that CD4CD8αα colonic Treg probably help control or prevent IBD. These data open the road to new diagnostic and therapeutic strategies for the management of IBD and provide new tools to address the impact of the intestinal microbiota on the human immune system

Faecalibacterium prausnitzii Skews Human DC to Prime IL10-Producing T Cells Through TLR2/6/JNK Signaling and IL-10, IL-27, CD39, and IDO-1 Induction

The human colonic mucosa contains regulatory type 1-like (Tr1-like, i.e., IL-10-secreting and Foxp3-negative) T cells specific for the gut Clostridium Faecalibacterium prausnitzii (F. prausnitzii), which are both decreased in Crohn's disease patients. These data, together with the demonstration, in mice, that colonic regulatory T cells (Treg) induced by Clostridium bacteria are key players in colon homeostasis, support a similar role for F. prausnitzii-specific Treg in the human colon. Here we assessed the mechanisms whereby F. prausnitzii induces human colonic Treg. We demonstrated that F. prausnitzii, but not related Clostridia, skewed human dendritic cells to prime IL-10-secreting T cells. Accordingly, F. prausnitzii induced dendritic cells to express a unique array of potent Tr1/Treg polarizing molecules: IL-10, IL-27, CD39, IDO-1, and PDL-1 and, following TLR4 stimulation, inhibited their up-regulation of costimulation molecules as well as their production of pro-inflammatory cytokines IL-12 (p35 and p40) and TNFα. We further showed that these potent tolerogenic effects relied on F. prausnitzii-induced TLR2/6 triggering, JNK signaling and CD39 ectonucleotidase activity, which was induced by IDO-1 and IL-27. These data, together with the presence of F. prausnitzii-specific Tr1-like Treg in the human colon, point out to dendritic cells polarization by F. prausnitzii as the first described cellular mechanism whereby the microbiota composition may affect human colon homeostasis. Identification of F. prausnitzii-induced mediators involved in Tr1-like Treg induction by dendritic cells opens therapeutic avenues for the treatment of inflammatory bowel diseases

Rôle régulateur des protéines Rho dans la réponse immune anti-mélanome

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Microbiota-specific CD4CD8αα Tregs: role in intestinal immune homeostasis and implications for IBD

International audienceIn studies in murine models, active suppression by IL-10-secreting Foxp3 regulatory T cells (Tregs) has emerged as an essential mechanism in colon homeostasis. However, the role of the equivalent subset in humans remains unclear, leading to suggestions that other subsets and/or mechanisms may substitute for Foxp3 Tregs in the maintenance of colon homeostasis. We recently described a new subset of CD4CD8αα T cells reactive to the gut bacterium Faecalibacterium prausnitzii and endowed with regulatory/suppressive functions. This subset is abundant in the healthy colonic mucosa, but less common in that of patients with inflammatory bowel disease (IBD). We discuss here the physiological significance and potential role of these Tregs in preventing inflammation of the gut mucosa and the potential applications of these discoveries for IBD management. DIVERSITY OF PERIPHERALLY DERIVED Tregs (pTregs) CD4 regulatory T cells (Tregs) inhibit inflammatory responses (1). They can be subdivided into natural Tregs, which differentiate in the thymus (tTreg) and peripherally derived Tregs (pTregs), which differentiate in secondary lymphoid organs or tissues (2). These populations differ in terms of their non-redundant roles: tTregs play an essential role in maintaining tolerance toward self-structures, whereas pTregs are involved in the responses to externally delivered antigens or commensal microbes. Furthermore, the tTreg population appears to be stable, whereas that of pTregs may be more labile (3). This functional dichotomy results from differences in differentiation due to exposure to different TCR ligands (self and non-self antigens, respectively) and specific factors (cytokines, route of exposure, and antigen-presenting cells) in contrasting settings (4). The two Treg subsets can also be distinguished on the basis of the presence or absence of constitutive expression of the Foxp3 transcription factor. Constitutive Foxp3 expression and more particularly, the demethylation of a specific region of the Foxp3 locus are characteristic features of tTregs (5). Three main subsets of CD4 pTregs have been described in mice: Foxp3 + CD25 + lymphocytes (3, 6), which are particularly abundant in the colon lamina propria (LP) (7) and two Foxp3 − subsets: the type 1 regulatory T (Tr1) cells and the T helper 3 (Th3) cells. The Tr1 subset secretes IL-10 and TGF-β in the absence of IL-4 and IL-17 (8–10) and is abundant in the small intestine (7). The Th3 subset may also secrete IL-10, but it differs from Tr1 in its expression of membrane-bound TGF-β (11, 12). The Tr1 Tregs are induced in vitro by IL-10 (8–10) and in vivo by TGF-β and IL-27 (9, 13) in the context of diverse immune responses (14) and upon chronic stimulation with antigens in the presence of IL-10 (10). The suppressive action of Tr1 Tregs is essentially IL-10-dependent, but it is also at least partly governed by TGF-β (8, 9). Moreover, the suppressive function of these cells may b

Statins Stimulate In Vitro Membrane FasL Expression and Lymphocyte Apoptosis through RhoA/ROCK Pathway in Murine Melanoma Cells12

The capacity of FasL molecules expressed on melanoma cells to induce lymphocyte apoptosis contributes to either antitumor immune response or escape depending on their expression level. Little is known, however, about the mechanisms regulating FasL protein expression. Using the murine B16F10 melanoma model weakly positive for FasL, we demonstrated that in vitro treatment with statins, inhibitors of 3-hydroxy-3-methylgutaryl CoA reductase, enhances membrane FasL expression. C3 exotoxin and the geranylgeranyl transferase I inhibitor GGTI-298, but not the farnesyl transferase inhibitor FTI-277, mimic this effect. The capacity of GGTI-298 and C3 exotoxin to inhibit RhoA activity prompted us to investigate the implication of RhoA in FasL expression. Inhibition of RhoA expression by small interfering RNA (siRNA) increased membrane FasL expression, whereas overexpression of constitutively active RhoA following transfection of RhoAV14 plasmid decreased it. Moreover, the inhibition of a RhoA downstream effector p160ROCK also induced this FasL overexpression. We conclude that the RhoA/ROCK pathway negatively regulates membrane FasL expression in these melanoma cells. Furthermore, we have shown that B16F10 cells, through the RhoA/ROCK pathway, promote in vitro apoptosis of Fas-sensitive A20 lymphoma cells. Our results suggest that RhoA/ROCK inhibition could be an interesting target to control FasL expression and lymphocyte apoptosis induced by melanoma cells

Tumor-reactive CD4+CD8αβ+ CD103+ αβT cells: A prevalent tumor-reactive T-cell subset in metastatic colorectal cancers

International audienceHigh level of T-cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of thisinfiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype andfunctional features of tumor-reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated fromCRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivityof CRC TIL and tumor-reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions(up to 18%) of double positive CD41CD8ab1 (DP) ab T cells. Interestingly, a high proportion (16–20%) of this TIL subsetdisplayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in mostCRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DPTIL were polyclonal, restricted by HLA class-I, proliferated poorly and secreted higher amounts of IL-4 and IL-13 than singlepositive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increasedfrequencies of tumor-reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of thiscancer. Based on their high secretion of IL-4 and IL-13 and on previously described roles of these cytokines in cancers, wepostulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors

Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors