Recherche de thérapies innovantes dans un modèle murin de myopathies inflammatoires

Inflammatory myopathies are acquired diseases characterized by motor deficit involving an autoimmune myopathy.They are responsible for impairing disability and may be associated with life-threatening complications.Treatment, when they exist, based on the use of corticosteroids in high doses and for long periods.However, 60 % of patients relapse and 20-30 % are readily corticosteroid.Other immunosuppressive treatments are then required (methotrexate , azathioprime , cyclosporine).The side effects of these treatments are inevitable and sometimes severe, which is why the development of new approaches treatment is essential. To test these new approaches, the laboratory developed a mouse model of Experimental Autoimmune Myositis with same Clinical and histological characteristics to polymyositis.In this model we tested the effect of a sub-population of T lymphocyte, nammed regulatory T cells, plays a crucial role in peripheral tolerance self-antigens. In order to control autoimmune manifestations that can replicate the mechanisms of action of Tregs , we sought to amplify in vivo, pharmacologically.At first, we observed the beneficial effect of rapamycin on our model, notably with increase of Tregs.In a second step, we have shown the beneficial role IVIg in the same model in curative.Finally, we investigated the effect of arsenic trioxide ( Trisenox ) in this animal model.Thus, all these data provide insight into the pathophysiology of myositis and to develop a clinical trial with rapamycin .Les myopathies inflammatoires sont des maladies acquises caractérisées par un déficit moteur impliquant une atteinte musculaire auto-immune. Elles sont responsables d'un handicap invalidant et peuvent s'accompagner de complications engageant le pronostic vital. Les traitements, quand ils existent, reposent sur l'utilisation de corticoïdes à fortes doses et au long cours. Cependant, 60% des patients rechutent et 20 à 30 % sont d'emblée corticorésistants. D'autres traitements immunosuppresseurs sont alors nécessaires. Les effets secondaires de ces traitements sont inévitables et parfois sévères. Afin de tester des nouvelles approches, le laboratoire a développé un modèle de Myosite Auto-immune Expérimentale dont les caractéristiques sont similaires à celles de la polymyosite. Dans ce modèle nous avons testé l'effet d'une sous population lymphocytaire T, appelée les lymphocytes T régulateurs, joue un rôle déterminant dans la tolérance périphérique aux antigènes du soi. Afin de contrôler les manifestations auto-immunes, nous avons cherché à les amplifier les Tregs in vivo, pharmacologiquement. D'abord, nous avons observé l'effet bénéfique de la rapamycine sur la sévérité de notre modèle en permettant en particulier d'augmenter le pourcentage de Tregs. Dans un second temps, nous avons montré le rôle bénéfique des immunoglobulines en intraveineux dans ce même modèle en traitement curatif. Enfin, nous nous sommes intéressés à l'effet de l'arsenic trioxyde (Trisenox) dans ce modèle animal.Ainsi, l'ensemble ces données permettent de mieux comprendre la physiopathologie des myosites et de mettre au point un essai clinique avec la rapamycine

Leishmania guyanensis suppressed inducible nitric oxide synthase provoked by its viral endosymbiont

Inducible nitric oxide synthase (iNOS) is essential to the production of nitric oxide (NO), an efficient effector molecule against intracellular human pathogens such a

Exacerbated leishmaniasis caused by a viral endosymbiont can be prevented by immunization with Its viral capsid

Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities

Research for innovative therapies in a murine model of inflammatory myopathies

Les myopathies inflammatoires sont des maladies acquises caractérisées par un déficit moteur impliquant une atteinte musculaire auto-immune. Elles sont responsables d'un handicap invalidant et peuvent s'accompagner de complications engageant le pronostic vital. Les traitements, quand ils existent, reposent sur l'utilisation de corticoïdes à fortes doses et au long cours. Cependant, 60% des patients rechutent et 20 à 30 % sont d'emblée corticorésistants. D'autres traitements immunosuppresseurs sont alors nécessaires. Les effets secondaires de ces traitements sont inévitables et parfois sévères. Afin de tester des nouvelles approches, le laboratoire a développé un modèle de Myosite Auto-immune Expérimentale dont les caractéristiques sont similaires à celles de la polymyosite. Dans ce modèle nous avons testé l'effet d'une sous population lymphocytaire T, appelée les lymphocytes T régulateurs, joue un rôle déterminant dans la tolérance périphérique aux antigènes du soi. Afin de contrôler les manifestations auto-immunes, nous avons cherché à les amplifier les Tregs in vivo, pharmacologiquement. D'abord, nous avons observé l'effet bénéfique de la rapamycine sur la sévérité de notre modèle en permettant en particulier d'augmenter le pourcentage de Tregs. Dans un second temps, nous avons montré le rôle bénéfique des immunoglobulines en intraveineux dans ce même modèle en traitement curatif. Enfin, nous nous sommes intéressés à l'effet de l'arsenic trioxyde (Trisenox) dans ce modèle animal.Ainsi, l'ensemble ces données permettent de mieux comprendre la physiopathologie des myosites et de mettre au point un essai clinique avec la rapamycine.Inflammatory myopathies are acquired diseases characterized by motor deficit involving an autoimmune myopathy.They are responsible for impairing disability and may be associated with life-threatening complications.Treatment, when they exist, based on the use of corticosteroids in high doses and for long periods.However, 60 % of patients relapse and 20-30 % are readily corticosteroid.Other immunosuppressive treatments are then required (methotrexate , azathioprime , cyclosporine).The side effects of these treatments are inevitable and sometimes severe, which is why the development of new approaches treatment is essential. To test these new approaches, the laboratory developed a mouse model of Experimental Autoimmune Myositis with same Clinical and histological characteristics to polymyositis.In this model we tested the effect of a sub-population of T lymphocyte, nammed regulatory T cells, plays a crucial role in peripheral tolerance self-antigens. In order to control autoimmune manifestations that can replicate the mechanisms of action of Tregs , we sought to amplify in vivo, pharmacologically.At first, we observed the beneficial effect of rapamycin on our model, notably with increase of Tregs.In a second step, we have shown the beneficial role IVIg in the same model in curative.Finally, we investigated the effect of arsenic trioxide ( Trisenox ) in this animal model.Thus, all these data provide insight into the pathophysiology of myositis and to develop a clinical trial with rapamycin

Beneficial Role of Rapamycin in Experimental Autoimmune Myositis

<div><p>Introduction</p><p>We developed an experimental autoimmune myositis (EAM) mouse model of polymyositis where we outlined the role of regulatory T (Treg) cells. Rapamycin, this immunosuppressant drug used to prevent rejection in organ transplantation, is known to spare Treg. Our aim was to test the efficacy of rapamycin <i>in vivo</i> in this EAM model and to investigate the effects of the drug on different immune cell sub-populations.</p><p>Methods</p><p>EAM is induced by 3 injections of myosin emulsified in CFA. Mice received rapamycin during 25 days starting one day before myosin immunization (preventive treatment), or during 10 days following the last myosin immunization (curative treatment).</p><p>Results</p><p>Under preventive or curative treatment, an increase of muscle strength was observed with a parallel decrease of muscle inflammation, both being well correlated (R² = −0.645, p<0.0001). Rapamycin induced a general decrease in muscle of CD4 and CD8 T cells in lymphoid tissues, but spared B cells. Among T cells, the frequency of Treg was increased in rapamycin treated mice in draining lymph nodes (16.9±2.2% vs. 9.3±1.4%, p<0.001), which were mostly activated regulatory T cells (CD62L^lowCD44^high: 58.1±5.78% vs. 33.1±7%, treated vs. untreated, p<0.001). In rapamycin treated mice, inhibition of proliferation (Ki-67⁺) is more important in effector T cells compared to Tregs cells (p<0.05). Furthermore, during preventive treatment, rapamycin increased the levels of KLF2 transcript in CD44^low CD62L^high naive T cell and in CD62L^low CD44^high activated T cell.</p><p>Conclusions</p><p>Rapamycin showed efficacy both as curative and preventive treatment in our murine model of experimental myositis, in which it induced an increase of muscle strength with a parallel decrease in muscle inflammation. Rapamycin administration was also associated with a decrease in the frequency of effector T cells, an increase in Tregs, and, when administered as preventive treatment, an upregulation of KFL2 in naive and activated T cells.</p></div

Beneficial effect of rapamycin (3 mg/kg/day) in curative treatment of EAM.

<p>Rapamycin (or water for control animals) was given orally for 10 days at a dose of 3 mg/kg. A: strength of mice evaluated by inverted screen test (time to fall in seconds). B: Gastrocnemius muscle inflammatory infiltrates evaluated by histological grading after haematoxylin-eosin staining. C: Percent of Treg cells (CD3⁺CD4⁺CD25⁺FoxP3⁺) in draining lymph nodes from controls and rapamycin-treated (3 mg/kg/day) mice.</p

Rapamycin treatment induced a T cells lymphopenia sparing B cells.

<p>A: absolute lymphocyte number in draining lymph nodes, spleen, and non-draining lymph nodes is decreased in rapamycin-treated mice compare to controls in a dose-dependent fashion. B: quantification of the different sub-populations of lymphocytes: B lymphocytes (B, CD19⁺B220⁺), T (CD3⁺), T CD4⁺ (CD3⁺CD4⁺) and T CD8⁺ (CD3⁺CD8⁺) cells. * p<0.05, *** p<0.001. White histogram bars: control mice, black histogram bars: rapamycin treated mice (3 mg/kg/day). C: percent of T or B lymphocytes in draining lymph nodes. D: Representative dot plot of flow cytometry analysis of draining lymph nodes for percentage of pre-activated CD4⁺ T cell (CD3⁺CD4⁺FoxP3⁻CD69⁺) and percentage of pre-activated CD4⁺ T cell (CD3⁺CD4⁺FoxP3⁻CD69⁺) in controls and rapamycin (3 mg/kg/day) treated mice.</p

Effect of rapamycin on Treg cells.

<p>Representative dot plot of flow cytometry analysis of draining lymph nodes for percent of Treg (CD4⁺CD25⁺FoxP3⁺) in CD4⁺ (A) and in CD4⁺CD44^high (B) in controls and rapamycin (3 mg/kg/day) treated mice. Percent of Treg (CD4⁺CD25⁺FoxP3⁺) in CD4⁺ (A, right) and in CD4⁺CD44^high (B, right) in controls and rapamycin (3 mg/kg/day) treated mice. C: Histogram representative of the difference in activation of Treg (CD62L^low cell) and naive Treg (CD62L^high) in controls and rapamycin (3 mg/kg/day) treated mice. D: Suppressive test. Horizontal lines indicate means. Suppressive activity of sorted CD4⁺CD25⁺ T cells (Treg) from controls (white bars) and rapamycin (3 mg/kg/day, black bars) mice on the proliferation of autologous CD4⁺CD25⁻ T cells (responders) stimulated with irradiated (15 Gy) splenocytes. Proliferation of responder cells (Teff) was measured by 3H-Tymidine incorporation (counts per minute, cpm). Results are indicated as percentage inhibition (±SD) compared to a max (ratio 1∶0). Different Teff∶Treg ratios were tested. E: Percent of Ki-67positive cells (i.e. proliferative cells) in activated effector (aT) and activated regulatory (aTreg) T cells in controls and rapamycin (3 mg/kg/day) treated mice.</p