Role of the RIG-like helicase pathway in the regulation of intestinal microbiota homeostasis and « sterile » inflammatory responses

La voie des RLR (RIG-I like Receptors) joue un rôle essentiel dans la détection des virus à ARN, déclenchant une réponse immunitaire antivirale grâce au recrutement de la protéine adaptatrice mitochondriale MAVS (Mitochondrial AntiViral Signaling protein). Nous avons mis en évidence que les souris déficientes pour la protéine MAVS (MAVS KO) présentaient un phénotype proallergénique dans un modèle d'inflammation stérile d'hypersensibilité retardée de contact (HSRC) qui reproduit la dermatite allergique de contact (DAC) chez l'homme. Nous avons caractérisé le système immunitaire des souris MAVS KO en condition d'équilibre et durant la réponse d'HSRC. Nous avons identifié un rôle du microbiote intestinal des souris MAVS KO dans l'exacerbation de réponse d'HSRC et mis en évidence une dysbiose du microbiote bactérien. Nous avons démontré que la dysbiose était responsable du phénotype inflammatoire observé, phénotype transmissible à des souris sauvages par des expériences de cohébergement et de transplantation fécale. Cette dysbiose induit une augmentation de la perméabilité intestinale chez les souris MAVS KO lors de la réponse d'HSRC, aboutissant à une translocation bactérienne dans les organes lymphoïdes et à la modulation des réponses immunitaires à l'origine de l'exacerbation de réponse d'hypersensibilité. La 2ème partie de ma thèse vise à étudier les conséquences de la déficience en MAVS sur le métabolisme glucidique. Nos expériences ont démontré que les souris MAVS KO développaient une surcharge pondérale et une insulino-résistance sous régime riche en lipides et sucrose, dépendants de la dysbiose intestinale. Au niveau cellulaire, une altération des interactions aux points de contact entre la mitochondrie et le réticulum endoplasmique a été observée. Nos résultats permettent d'envisager le développement de nouvelles approches thérapeutiques des pathologies allergiques et métaboliques humaines par la modulation du microbiote intestinalRIG-I like receptors (RLRs) play a major role in response to cytosolic viral RNAs by initiating an antiviral immune response through the recruitment of the mitochondrial adaptor protein MAVS (Mitochondrial AntiViral Signaling protein). We showed that MAVS-deficient mice developed an exacerbated response in a sterile inflammatory model of Contact Hypersensitivity (CHS), that reproduces the pathophysiology of allergic contact dermatitis (ACD) in human. We characterized the immune system of MAVS KO mice at steady state and during CHS response. We found that MAVS deficiency leads to changes in the gut bacterial composition suggesting an unexpected role of the RLR pathway in the regulation of intestinal homeostasis. We demonstrated that intestinal dysbiosis is responsible for the increased CHS response, and showed that the inflammatory phenotype of MAVS KO mice can be transferred to WT mice by cohousing and fecal transplantation. We demonstrated that the dysbiotic gut microbiota exerts its effect due to an increased intestinal permeability during DTH sensitization. The ensuing bacterial translocation within lymphoid organs enhances characteristic cytokines production that increases CHS response. The 2nd part of my thesis aimed to study the consequences of MAVS deficiency on glucose metabolism. Our experiments showed that MAVS KO mice exhibit disorders of glucose homeostasis during high fat diet (HFD) associated with the development of overweight and insulin resistance. We also observed alterations of MAM (Mitochondria-associated endoplasmic reticulum membranes), contact poins between mitochondria and endoplasmic reticulum. Recent preliminary data suggested that the metabolic disorders associated with MAVS deficiency are due to intestinal dysbiosis. Our results highlight a new role for the RLR pathway and allow to consider the development of new therapeutic approaches to human allergic and metabolic diseases by modulation of the intestinal microbiot

Prévalence des anticorps antinucléaires et taux d'IL-6, IL-10, IL-17 et BAFF chez des patients atteints de lymphomes malins non hodgkiniens (une étude rétrospective)

LYON1-BU Santé (693882101) / SudocSudocFranceF

Overexpression of the Transcription Factor Sp1 Activates the OAS-RNAse L-RIG-I Pathway

<div><p>Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells <i>in vitro</i> and <i>in vivo</i>. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.</p></div

A schematic overview of the signaling pathway triggered by increased Sp1 level.

<p>Upon Sp1 overexpression, <i>Oas2</i> and <i>Rnasel</i> genes are upregulated and small self-RNAs are produced. Sensing of small self-RNAs leads to the activation of the sensor RIG-I, the IRF3/7 transcription factors and downstream effector targets such as ISGs.</p

Functional analysis of Sp1 signature.

<p>The gene list enrichment analysis from the Gene Ontology of the SP1 specific signature was performed with g:Profiler. The moderate hierarchical filtering used here allows a compact representation of gene list enrichment results. Significantly enriched GO terms containing less than 5 genes were excluded.</p><p>Functional analysis of Sp1 signature.</p

Sp1 overexpression activates genes of the RIG-I pathway.

<p>(A-B) BaF3 (A) and 3T3 (B) cells were transduced with retroviruses encoding wild-type Sp1, Sp1 Zn^2,3 mutant (Zn) that does not bind DNA, or empty vector (EV). Transduced cells (CD2 positive) were purified by magnetic selection 30 h (BaF3) or 72 h (3T3) later, and mRNA levels of indicated genes were measured by RT-qPCR (TLDA used in B) and normalized to housekeeping genes (HPRT in A and Rps-21 in B) mRNA levels. Data are representative of one out of three independent experiments in A. In B panel, data are representative of two independent experiments and statistical analysis was performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: **<i>P</i> <0.01. (C) BaF3-Sp1 inducible cell line was grown with (white bars) or without (black bars) dox for indicated times, and relative mRNA levels of indicated genes were quantified by RT-qPCR (TLDA). Data are representative of one out of four independent experiments.</p

Proteins of the antiviral RIG-I pathway are induced by increased Sp1 level.

<p>BaF3-Sp1 cells were grown in the presence or absence of dox. (A) Cell extracts were collected at indicated time and expression of the indicated proteins was analyzed by immunoblot. A549 cells treated 16 h with IFNa (1000U/ml) were used as positive control (IFN) for MDA5 and RIG-I induction. (B) BaF3-Sp1 cells were cultured for 20 h, fixed and stained for RIG-I (red) and analyzed by fluorescent microscopy. The percentage of cells presenting RIG-I punctuated staining is indicated, and corresponds to the mean of 8 independent acquisitions. Data are representative of one out of two independent experiments.</p

Immune cells are recruited by cells overexpressing Sp1.

<p>(A) BaF3-Sp1 cells were grown in the presence (+) or absence (-) of dox. CXCL4 production by BaF3-Sp1 was measured at the indicated times by ELISA. (B) BaF3-Sp1 cells were treated with or without dox for 24 h and incubated in the presence of neutrophils into transwell chambers during 3 h. Neutrophils recruitment (Ly6G⁺ CD11b⁺ cells) was assessed by flow cytometry. (C) BaF3 and BaF3-Sp1 cells were grown 18 h in absence or presence of dox and injected intra-peritoneally into mice. PBS injected mice were used as controls (Naive). Peritoneal washes were performed 6 h after injection. Cells were then harvested and cellular recruitment (CD11b⁺ Ly6G^- cells) was estimated by flow cytometry. These results are from one representative out of three independent experiments. Statistical analysis were performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: *<i>P</i> <0.05; **<i>P</i> <0.01; ***<i>P</i> = 0.0001; ****<i>P</i> <0.0001.</p

Sp1 activates the OAS-RNAse L pathway and the production of small self-RNAs.

<p>(A) BaF3-Sp1 inducible cell line was grown with (white bars) or without (black bars) dox for the indicated times, and relative mRNA levels of OAS2 and RNase L genes were quantified by RT-qPCR. (B, D, E) 3T3 (B) and MEFs (D, E) WT or deficient for the RNase L (RNase L KO) or MAVS (MAVS KO) were transduced with Sp1, Sp1 Zn^2,3 (Zn) or empty vector (EV). Transduced cells (CD2 positive) were purified 72 h post-transduction by magnetic selection and relative mRNA levels of indicated genes were quantified by RT-qPCR. (C) Small RNAs from BaF3-Sp1 cells following induction Sp1 or not were extracted at the indicated times, and transfected into 293HEK cells (5μg per condition). Luciferase reporter assay was performed to analyze the ISRE promoter activity. Data are mean values ± standard deviation (SD) from one experiment representative of two or three independent experiments. Statistical analysis were performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: *<i>P</i> <0.05; **<i>P</i> <0.01.</p

MAVS deficiency induces gut dysbiotic microbiota conferring a proallergic phenotype

International audienceProminent changes in the gut microbiota (referred to as "dysbiosis") play a key role in the development of allergic disorders, but the underlying mechanisms remain unknown. Study of the delayed-type hypersensitivity (DTH) response in mice contributed to our knowledge of the pathophysiology of human allergic contact dermatitis. Here we report a negative regulatory role of the RIG-I-like receptor adaptor mitochondrial antiviral signaling (MAVS) on DTH by modulating gut bacterial ecology. Cohousing and fecal transplantation experiments revealed that the dysbiotic microbiota of Mavs -/- mice conferred a proallergic phenotype that is communicable to wild-type mice. DTH sensitization coincided with increased intestinal permeability and bacterial translocation within lymphoid organs that enhanced DTH severity. Collectively, we unveiled an unexpected impact of RIG-I-like signaling on the gut microbiota with consequences on allergic skin disease outcome. Primarily, these data indicate that manipulating the gut microbiota may help in the development of therapeutic strategies for the treatment of human allergic skin pathologies