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

Lymphocytes T régulateurs induits par Faecalibacterium prausnitzii : modalités de leur induction et signification fonctionnelle

Comprendre les mécanismes impliqués dans l’homéostasie des muqueuses est un enjeu majeur pour prévenir ou traiter les maladies inflammatoires chroniques de l’intestin (MICI). Mon équipe d’accueil a montré la présence dans la muqueuse colique et le sang de lymphocytes T régulateurs (Treg) CD4CD8α (DP8a) sécréteurs d’IL-10 induits par la bactérie commensale Faecalibacterium prausnitzii (F prau). De plus, ces Treg sont diminués dans la muqueuse colique et le sang des patients atteints de MICI. Ces données, et la diminution de F prau observée dans ces pathologies, suggèrent l’existence d’un lien entre la diminution de F prau et celle des Treg spécifiques et un rôle de ces cellules dans la prévention de l’inflammation. Mon projet de thèse a consisté à rechercher comment F prau induit des Treg et à mieux caractériser et quantifier les PBL DP8α spécifiques de F prau, afin de questionner leur signification fonctionnelle. Nous montrons que l’exposition des cellules dendritiques (DC) à F prau pendant leur différenciation induit la sécrétion d’IL-10 et d’IDO-1, une résistance à la maturation, la non sécrétion de TNFα et IL-12 en réponse au LPS et favorisent le priming de lymphocytes sécréteurs d’IL-10 par ces cellules. Ces effets de F prau ne sont pas ou peu partagés par des bactéries proches de F prau testées en parallèle, suggérant que l’effet dominant de F prau dans l’induction des Treg DP8α dépend de sa capacité unique à tolériser les DC via l’induction de molécules immunomodulatrices dont plusieurs ont été identifiées par PCR quantitative et puces affymétrix comparant des DC exposées ou non à F prau et à une bactérie proche. Par ailleurs, nous montrons que plusieurs récepteurs de chimiokines et l’intégrine a4b7 marquent les PBL DP8α spécifiques de F prau et que ces lymphocytes représentent une fraction variables des PBL DP8α, eux-mêmes en proportion très variable, et augmentant avec l’âge, entre les individus. Malgré cela, la fréquence des DP8α totaux semble diminuée dans la spondylarthrite, les MICI et les cancers coliques. La quantification plus précise des Treg DP8α originaires du colon dans le sang des patients grâce aux marqueurs identifiés, permettra de rechercher la valeur prognostiqe de cette fréquence et sa corrélation avec le niveau de F prau dans le microbiote fécal.Understanding mechanisms underlying mucosalhomeostasis represent a critical step towards prevention andtreatment of inflammatory bowel diseases (IBD). My hostlaboratory has shown the presence, in colonic mucosa, of IL-10-producing regulatory T cells (Treg) CD4CD8α (DP8α) induced bythe commensal bacteria F prau. Moreover, these Treg are lessfrequent in the colonic mucosa as well as in the blood of IBDpatients. These data, together with the fact that F prau is reducedin these diseases, suggest a link between the decrease of F prauand of specific Treg as well as a role for these cells in containinginflammation. My thesis project consisted in understanding how Fprau induces Treg and in further characterizing and quantifying Fprau specific PBL to define their function. Here we show that Fprau exposure during dendritic cell (DC) differentiation induces IL-10 and IDO-1 production, and a poor maturation including a lack ofTNFα and IL-12 secretion in response to LPS. We also show thatF prau-conditioned DC prime CD4 T cells towards an IL-10-producing phenotype. These F prau-mediated effects are not allshared by other close bacteria, suggesting that the critical role forF prau in inducing DP8α Treg depends on its unique ability totolerize DC. This tolerization could happen via induction ofimmunoregulatory molecules identified by qPCR and affymetrixgene array. Also, we show that several chemokine receptors aswell as α4β7 identify F prau-specific PBL-derived DP8α cells.Indeed, these lymphocytes represent a variable fraction of DP8αcells, the inter-individual frequency of which considerably fluctuateand increases with age. Nonetheless, frequency of total DP8αcells seems diminished in spondyloarthritis, IBD and colorectalcancers. Accurate quantification of DP8α Treg originating from thecolon in patients’ blood, using identified markers, should allow tostudy their prognosis value as well as their correlation with F praulevels within fecal microbiota

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

Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions

International audienceIn mice, microbiota-induced Tregs both maintain intestinal homeostasis and provide resistance to immuno-pathologies in the adult. Identifying their human functional counterpart therefore represents an important goal. We discovered, in the human colonic lamina propria and blood, a FoxP3-negative IL-10-secreting Treg subset, which co-expresses CD4 and CD8α (hence named DP8α) and displays a TCR-reactivity against Faecalibacterium prausnitzii , indicating a role for this symbiotic bacterium in their induction. Moreover, supporting their role in intestinal homeostasis, we previously reported both their drastic decrease in IBD patients and their protective role in vivo against intestinal inflammation, in mice. Here, we aimed at identifying the genomic, phenotypic and functional signatures of these microbiota-induced Tregs, towards delineating their physiological role(s) and clinical potential. Human F. prausnitzii -reactive DP8α Treg clones were derived from both the colonic lamina propria and blood. RNA-sequencing, flow cytometry and functional assays were performed to characterize their response upon activation and compare them to donor- and tissue-matched FoxP3 + Treg clones. DP8α Tregs exhibited a unique mixed Tr1-like/cytotoxic CD4 + T cell-profile and shared the RORγt and MAF master genes with mouse gut microbiota-induced FoxP3 + Tregs. We revealed their potent cytotoxic, chemotactic and IgA-promoting abilities, which were confirmed using in vitro assays. Therefore, besides their induction by a Clostridium bacterium, DP8α Tregs also partake master genes with mouse microbiota-induced Tregs. The present identification of their complete signature and novel functional properties, should be key in delineating the in vivo roles and therapeutic applications of these unique human microbiota-induced Tregs through their study in pathological contexts, particularly in inflammatory bowel diseases

DataSheet_1_Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions.pdf

In mice, microbiota-induced Tregs both maintain intestinal homeostasis and provide resistance to immuno-pathologies in the adult. Identifying their human functional counterpart therefore represents an important goal. We discovered, in the human colonic lamina propria and blood, a FoxP3-negative IL-10-secreting Treg subset, which co-expresses CD4 and CD8α (hence named DP8α) and displays a TCR-reactivity against Faecalibacterium prausnitzii, indicating a role for this symbiotic bacterium in their induction. Moreover, supporting their role in intestinal homeostasis, we previously reported both their drastic decrease in IBD patients and their protective role in vivo against intestinal inflammation, in mice. Here, we aimed at identifying the genomic, phenotypic and functional signatures of these microbiota-induced Tregs, towards delineating their physiological role(s) and clinical potential. Human F. prausnitzii-reactive DP8α Treg clones were derived from both the colonic lamina propria and blood. RNA-sequencing, flow cytometry and functional assays were performed to characterize their response upon activation and compare them to donor- and tissue-matched FoxP3+ Treg clones. DP8α Tregs exhibited a unique mixed Tr1-like/cytotoxic CD4+ T cell-profile and shared the RORγt and MAF master genes with mouse gut microbiota-induced FoxP3+ Tregs. We revealed their potent cytotoxic, chemotactic and IgA-promoting abilities, which were confirmed using in vitro assays. Therefore, besides their induction by a Clostridium bacterium, DP8α Tregs also partake master genes with mouse microbiota-induced Tregs. The present identification of their complete signature and novel functional properties, should be key in delineating the in vivo roles and therapeutic applications of these unique human microbiota-induced Tregs through their study in pathological contexts, particularly in inflammatory bowel diseases.</p

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

International audienceThe 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

Proliferative memory SAMHD1 low CD4 + T cells harbour high levels of HIV-1 with compartmentalized viral populations

International audienceWe previously reported the presence of memory CD4+ T cells that express low levels of SAMHD1 (SAMHD1low) in peripheral blood and lymph nodes from both HIV-1 infected and uninfected individuals. These cells are enriched in Th17 and Tfh subsets, two populations known to be preferentially targeted by HIV-1. Here we investigated whether SAMHD1low CD4+ T-cells harbour replication-competent virus and compartimentalized HIV-1 genomes. We sorted memory CD4+CD45RO+SAMHD1low, CD4+CD45RO+SAMHD1+ and naive CD4+CD45RO-SAMHD1+ cells from HIV-1-infected patients on anti-retroviral therapy (c-ART) and performed HIV-1 DNA quantification, ultra-deep-sequencing of partial env (C2/V3) sequences and phenotypic characterization of the cells. We show that SAMHD1low cells include novel Th17 CCR6+ subsets that lack CXCR3 and CCR4 (CCR6+DN). There is a decrease of the % of Th17 in SAMHD1low compartment in infected compared to uninfected individuals (41% vs 55%, p<0.05), whereas the % of CCR6+DN increases (7.95% vs 3.8%, p<0.05). Moreover, in HIV-1 infected patients, memory SAMHD1low cells harbour high levels of HIV-1 DNA compared to memory SAMHD1+ cells (4.5 vs 3.8 log/106cells, respectively, p<0.001), while naïve SAMHD1+ showed significantly lower levels (3.1 log/106cells, p<0.0001). Importantly, we show that SAMHD1low cells contain p24-producing cells. Moreover, phylogenetic analyses revealed well-segregated HIV-1 DNA populations with compartmentalization between SAMHD1low and SAMHD1+ memory cells, and limited viral exchange. As expected, the % of Ki67+ cells was significantly higher in SAMHD1low compared to SAMHD1+ cells. There was positive association between levels of HIV-1 DNA and Ki67+ in memory SAMHD1low cells, but not in memory and naïve SAMHD1+ CD4+ T-cells. Altogether, these data suggest that proliferative memory SAMHD1low cells contribute to viral persistence

Expression of CCR6 and CXCR6 by Gut-Derived CD4+/CD8α+ T-Regulatory Cells, Which Are Decreased in Blood Samples From Patients With Inflammatory Bowel Diseases

International audienceBACKGROUND & AIMS:Faecalibacterium prausnitzii, a member of the Clostridium IV group of the Firmicutes phylum that is abundant in the intestinal microbiota, has anti-inflammatory effects. The relative level of F prausnitzii is decreased in fecal samples from patients with inflammatory bowel diseases (IBDs) compared with healthy individuals. Reduced F prausnitzii was correlated with relapse of Crohn's disease after surgery. We identified, in human colonic mucosa and blood, a population of T regulatory type 1-like T regulatory (TREG) cells that express CD4 and CD8α (DP8α T cells) and are specific for F prausnitzii. We aimed to determine whether they are altered in patients with IBD.METHODS:We isolated DP8α T cells from human colon lamina propria and blood samples and used flow cytometry to detect markers of cells that are of colon origin. We quantified DP8α cells that express colon-specific markers in blood samples from 106 patients with IBD, 12 patients with infectious colitis, and 35 healthy donors (controls). We identified cells that respond to F prausnitzii. Cells were stimulated with anti-CD3, and their production of interleukin 10 was measured by enzyme-linked immunosorbent assay. We compared the frequency and reactivity of cells from patients vs controls using the 2-sided Student t test or 1-way analysis of variance.RESULTS:Circulating DP8α T cells that proliferate in response to F prausnitzii express the C-C motif chemokine receptor 6 (CCR6) and C-X-C motif chemokine receptor 6 (CXCR6). These cells also have features of TREG cells, including production of IL-10 and inhibition of T-cell proliferation via CD39 activity. The proportion of circulating CCR6+/CXCR6+ DP8α T cells was significantly reduced (P < .0001) within the total population of CD3+ T cells from patients with IBD compared with patients with infectious colitis or controls. A threshold of <7.875 CCR6+/CXCR6+ DP8α T cells/10,000 CD3+ cells discriminated patients with IBD from those with infectious colitis with 100% specificity and 72.2% sensitivity.CONCLUSIONS:We identified a population of gut-derived TREG cells that are reduced in blood samples from patients with IBD compared with patients with infectious colitis or controls. These cells should be studied further to determine the mechanisms of this reduction and how it might contribute to the pathogenesis of IBD and their prognostic or diagnostic value