Stimulation of innate immune cells Induced by probiotics: participation of toll- like receptors

Objective: The present work aimed to study the functionality of macrophages from different locations (peritoneum, spleen and Peyer´s patches) when they were stimulated with probiotics microorganisms: Lactobacillus casei CRL 431 and Lactobacillus paracasei CNCM I-1518 or a Probiotic Fermented Milk (PFM) through Toll-Like Receptors (TLRs), prior challenged with agonists or antagonists of TLRs. Methods: BALB/c mice received in the drinking water, the probiotic bacteria (L. casei CRL 431 and L. paracasei CNCM I-1518) or the PFM. We focused our investigation mainly on the phagocytic activity of macrophages from peritoneum, spleen and Peyer?s patches and cytokine production were evaluated prior challenged with TLR2 and TLR4 agonists or antagonists. The microbicidal activity of macrophages and against an infection with Salmonella typhimurium was also studied. To assess the role of TLR in probiotic stimulation, we evaluated the phagocytic activity, cytokine production and Immunoglobin G (IgG) anti-OVA in C57BL/6 knockout mice to MyD88, TLR2 and TLR4. Results: In BALB/c mice, the best effect in the phagocytosis assay was obtained with the probiotic bacteria L. casei CRL 431, this effect was reinforced with TLR2 agonist. The production of different cytokines (IL-10 and IL-6) was improved with the probiotic treatments and this production was ameliorated with TLRs agonists. The antimicrobial activity was increased with L. casei CRL 431 and L. paracasei CNCM I-1518, TLR2 and TLR4 antagonists had a negative effect on microbicidal activity. The administration of probiotic bacteria or PFM improved the host response against S. typhimurium controlling the infection during the first hours post-infection. In C57BL/6 knockout mice, phagocytic activity was significantly diminished in comparison to wild type mice and the probiotic bacteria or PFM administration was not able to improve this activity. The IL-10 production was increased at a concentration of 108 cells/ml of L. casei CRL 431 in TLR2-/- and TLR4-/-, but not in MyD88-/- mice. The administration of probiotic bacteria or PFM did not play a stimulating effect in the systemic immune response against to OVA antigen in knockout mice. Conclusions: Probiotics modulate the different signaling pathways of innate immune cells through the TLRs. The macrophages activation depends on location of them and that different probiotic strains of Lactobacilli can evoke different intensity of responses. The data suggest that probiotic not only promote a major expression of TLRs but also use these receptors via the innate immune cells as macrophages to stimulate and modulate the immune response.Fil: Maldonado Galdeano, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; ArgentinaFil: Lemme Dumit, José María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; ArgentinaFil: Thieblemont, Nathalie. Inserm; Francia. Center of Excellence; FranciaFil: Carmuega, Esteban. Centro de Estudios Nutricionales Infantiles; ArgentinaFil: Weill, Ricardo. DANONE; ArgentinaFil: Perdigon, Gabriela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; Argentin

Systemic Toll-Like Receptor Stimulation Suppresses Experimental Allergic Asthma and Autoimmune Diabetes in NOD Mice

BackgroundInfections may be associated with exacerbation of allergic and autoimmune diseases. Paradoxically, epidemiological and experimental data have shown that some microorganisms can also prevent these pathologies. This observation is at the origin of the hygiene hypothesis according to which the decline of infections in western countries is at the origin of the increased incidence of both Th1-mediated autoimmune diseases and Th2-mediated allergic diseases over the last decades. We have tested whether Toll-like receptor (TLR) stimulation can recapitulate the protective effect of infectious agents on allergy and autoimmunity. Methods and Findings Here, we performed a systematic study of the disease-modifying effects of a set of natural or synthetic TLR agonists using two experimental models, ovalbumin (OVA)-induced asthma and spontaneous autoimmune diabetes, presenting the same genetic background of the non obese diabetic mouse (NOD) that is highly susceptible to both pathologies. In the same models, we also investigated the effect of probiotics. Additionally, we examined the effect of the genetic invalidation of MyD88 on the development of allergic asthma and spontaneous diabetes. We demonstrate that multiple TLR agonists prevent from both allergy and autoimmunity when administered parenterally. Probiotics which stimulate TLRs also protect from these two diseases. The physiological relevance of these findings is further suggested by the major acceleration of OVA-induced asthma in MyD88 invalidated mice. Our results strongly indicate that the TLR-mediated effects involve immunoregulatory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β and different subsets of regulatory T cells, notably CD4+CD25+FoxP3+ T cells for TLR4 agonists and NKT cells for TLR3 agonists. Conclusions/Significance These observations demonstrate that systemic administration of TLR ligands can suppress both allergic and autoimmune responses. They provide a plausible explanation for the hygiene hypothesis. They also open new therapeutic perspectives for the prevention of these pathologies

Etude des voies de signalisation TLR/MyD88 en situation normale et immunopathologique

Les Récepteurs Toll-Like (TLR) permettent d'initier et d'orienter les réponses immunes dirigées contre les pathogènes. Ils participent également à la régulation des réponses induites, comme en témoigne leur implication dans de nombreuses maladies du système immunitaire.Notre recherche a eu pour objectif d'étudier le rôle des voies de signalisation TLR en situation normale et immunopathologique. Nous avons démontré que : 1) les agonistes des TLR protègent efficacement de l'apparition du diabète spontané chez la souris NOD via l'activation et/ou le recrutement de cellules régulatrices et la production de cytokines immunorégulatrices; 2) Les cellules iNKT sont activées par l'agoniste TLR7 ; 3) La signalisation via TLR7 protège de l'asthme allergique. 4) la voie signalisation via TLR2/MyD88 a un effet majeur sur le développement de l'athérosclérose, ainsi que sur la production de chimiokines et cytokines ; 5) la voie de signalisation MyD88 est impliquée dans le développement et la fonction des lymphocytes iNKT. En conclusion, notre travail met en évidence les propriétés immunomodulatrices des voies de signalisation TLR

Granulomatosis with polyangiitis (Wegener granulomatosis): A proteinase-3 driven disease?

International audienc

Le rôle des basophiles et effets régulateurs induit par les probiotiques dans le modèle expérimental d'asthme allergique

Dans cette thèse, nous avons étudié le rôle ambivalent des microorganismes dans la pathologie de l'asthme allergique. Nous avons ainsi montré le rôle protecteur des probiotiques, bactéries non pathogènes dont la signalisation utilise la molécule MyD88, dans un modèle d'inflammation allergique induit par l'ovalbumine. Cette protection se caractérise par une diminution de l'hyperreactivité bronchique, de l'infiltration des eosinophiles et de la production de cytokines de type Th2 (IL-4, IL-5, IL-13) dans les poumons. Quant aux mécanismes responsables de cette protection, nous avons montré qu'elle était associée à une réduction des cytokines pro-inflammatoires (IL-17 et TNF-a) et à une augmentation des cellules T régulatrices CD4*CD25*FoxP3*. Cette protection est transférable par les cellules T CD4* et est dépendante de l'IL-10. Dans une étude indépendante, nous avons démontré l'implication des basophiles dans l'asthme allergique à la fois après transfert adoptif et après déplétion. Nous avons également testé la réponse des basophiles murins à différents agomstes TLR et identifié une molécule qui était capable d'activer cette cellule. Ainsi, nous avons démontré que l'ARN synthétique double brin, le poly(A:U), qui est décrit comme un ligand TLR3/TLR7, induit une production d'IL-4, d'IL-6, d'IL-13 et d'histamine par les basophiles in vitro. Cette stimulation dépend du senseur viral RIG-I et de l'adaptateur moléculaire CARDIF. Nous avons enfin démontré la pertinence de ces observations puisque Pactivation des basoplulcs in vivo par le poly(A:U) exacerbe tous les paramètres de l'asthme expérimental allergique. Nous suggérons que nos résultats sur le poly(A:U), considéré comme mimant l'effet d'un virus, pourraient constituer une approche du mécanisme responsable de l'aggravation de la pathologie bien connue chez les patients asthmatiques durant une infection virale.We have shown here that non-pathogenic microorganisms such as probiotics can protect mice from experimental allergic asthma. We have observed that oral administration of the preparation containing lactobacillus, bifidobacterium and spretococcus prevents from allergic asthma induced by ovalbumin as shown by the decreased broncho-hyperactivity, the eosmophilia in the bronchoalveolar liquid (BAL) and the production of Th2 type cytokines (IL-4, IL-5, IL-13) and chemokines (eotaxin) in the lungs. Probiotic administration also decreased the level of pro-inflammatory cytokines IL-6, IL-17 and TNFa in sera and increased the frequency of CD4+CD25+FoxP3+T cells in the spleen. In addition, we have shown that the asthma protection was MyD88- and IL-10-dependent. We have also analyzed the potential role and activation of basophils in experimental allergic asthma. We have demonstrated by adoptive transfer and by depletion of these cells the role of basophils in this model. We have further characterized the double-stranded RNA poly(A:U) as a potent agonist of purified murine basophils since it induced a strong IL-4, IL-6, IL-13 and histamine production in vitro. Poly(A:U). which is described as TLR3/TLR7 ligand, activated basophils through the RIG-I/CARDIF pathway. The relevance of this stimulation has been illustrated in the model of allergic asthma, since poly(A:U)-activated basophils exacerbated asthma responses by increasing TH2 cytokine and chemokine production in lungs as well as eosinophilia in the BAL. We suggest that this mechanism may account for the aggravating effect of respiratory viral infections well known in asthma patients.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Modulation de la réponse immunitaire par des agonistes de la voie de signalisation TLR/IL - 1R dans le modèle d'asthme

PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Modulation de la réponse immunitaire par TLR7 (rôle dans le diabète de type 1 et l'asthme allergique)

Conformément à l'hypothèse de l'hygiène, les infections peuvent protéger des désordres immunologiques. Nous montrons que la stimulation des TLR2, 3, 4 et 7 qui détectent les infections, peut protéger les souris Non obese-diabetic (NOD), développant un diabète de type 1 spontané. Cet effet serait porté par des populations de cellules régulatrices et des cytokines immunorégulatrices. Nous avons observé que la protection induite par des agonistes de TLR2 et 3 est dépendante des cellules iNKT avec une implication de l'IL-4 pour TLR3. Par ailleurs, nous avons décrit dans un modèle d'asthme allergique que la protection induite par un agoniste de TLR7 était portée par les lymphocytes iNKT et l'IFN-y. Nous montrons in vitro que ce produit cible directement les iNKT et que la réponse ne nécessite pas la présence de cellules présentatrices de l'antigène. En conclusion, nos travaux soulignent que le ciblage des TLR module les réponses immunitaires, notamment celles dues à une perte de tolérance.According to hygiene hypothesis, infections can protect from immune disorders. We demonstrate that stimulation of TLR2, 3, 4 and 7 that are implicated in sensing infections, can protect Non obese-diabetic (NOD) mice, which develop a spontaneous type 1 diabetes. This effect would depend on the activation of regulatory cells and immunoregulatory cytokines. We have further demonstrated that TLR2- and TLR3-induced protection were dependent on iNKT cells with an implication of IL-4 for TLR3. In an allergic asthma model, we have shown that TLR7-induced protection was dependent on iNKT cells and IFN-y. We show in vitro that this product directly target iNKT cells without requirement of antigen presenting cells. Taken together, our work emphasize the modulatory role of TLR in immune responses, and especially those due to a rupture of tolerance.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Granulomatosis with polyangiitis (Wegener granulomatosis): A proteinase-3 driven disease?

International audienceGranulomatosis with polyangiitis (GPA, Wegener granulomatosis) is a systemic autoimmune vasculitis that affects small arteries, arterioles, and capillaries, most notably in the kidneys and lungs. In this disease, proteinase-3 (PR3), produced by neutrophils, is targeted by antineutrophil cytoplasmic antibodies (ANCA). Recent work by our group has shown how PR3 impairs the resolution of inflammation and deregulates the immune system. Normally, the clearance of activated neutrophils triggers an anti-inflammatory, pro-resolution process. In patients with GPA, however, macrophages phagocytose apoptotic neutrophils then release massive amounts of pro-inflammatory mediators, notably interleukin-1, thereby generating a pro-inflammatory microenvironment conducive to autoimmunity. This deregulation of immune processes is accompanied with activation of plasmacytoid dendritic cells and with polarization of T-helper-2 (Th2), Th9, and Th17 cells. These recent data highlight the dual role of PR3, both auto-antigenic and auto-inflammatory, thus potentially opening up new therapeutic avenues