INTRODUÇÃO: As exacerbações da doença pulmonar obstrutiva crônica (DPOC) afetam profundamente a força muscular periférica e a capacidade funcional destes pacientes, entretanto, intervenções para prevenir estas perdas funcionais e sistêmicas são pobremente compreendidas. OBJETIVOS: Avaliar os efeitos do treinamento resistido sobre a força muscular periférica, capacidade funcional e a qualidade de vida em pacientes com DPOC durante a hospitalização. Além disso, avaliar a resposta desta intervenção sobre a inflamação sistêmica e a atividade física de vida diária. MÉTODOS: Vinte e nove pacientes hospitalizados na enfermaria de um Hospital Universitário por exacerbação da DPOC foram randomizados em dois grupos: controle (GC) e treinamento (GT). Eles foram avaliados no 2° dia de internação, na alta hospitalar e após 30 dias. Nestas avaliações foram verificadas a força muscular periférica dos membros inferiores e superiores, a distância percorrida no teste de caminha de 6 minutos (TC6min), a inflamação sistêmica (TNF-, PCR, IL1, IL-12p70, IL-6, IL-8, IL10), a saúde relacionada a qualidade de vida e a atividade física de vida diária. RESULTADOS: Nossos resultados demonstram que durante a hospitalização todos os pacientes, independente do grupo, mantiveram-se a maior parte do tempo (87%) na posição deitada ou sentada. Os pacientes do GC demonstraram uma redução de força muscular dos membros inferiores (p<0,05), mas sem alteração na distância percorrida em 6 minutos (p>0,05) durante a hospitalização. Os pacientes do GT demonstraram uma melhora na força muscular dos membros inferiores e do TC6min (p<0,05) que permaneceram até 30 dias após a alta hospitalar. Diferentemente do GC, os pacientes do GT apresentaram uma melhora do domínio impacto no questionário de qualidade de vida após a alta hospitalar. Não houve diferença entre os grupos nos marcadores inflamatórios sistêmicos analisados durante a hospitalização e após 30 dias. Além disso, diversos pacientes de ambos os grupos permaneciam fisicamente inativos (70%) em casa. CONCLUSÃO: Nossos resultados sugerem que o treinamento resistido durante a hospitalização melhora a força muscular periférica e capacidade funcional. Entretanto, sem alterar a inflamação sistêmica e a atividade física de vida diária / BACKGROUND: Exacerbations of chronic obstructive pulmonary disease (COPD) profoundly affects the peripheral muscle strength and functional capacity of patients, however, interventions to prevent these losses and systemic function are poorly understood. OBJECTIVE: Evaluate the effects of resistance training on peripheral muscle strength, functional capacity and quality of life in COPD patients during hospitalization. In addition, evaluate the response of this intervention on systemic inflammation and physical activity of daily living. METHODS: Twenty-nine patients hospitalized in the ward of a university hospital for exacerbation of COPD were randomized into two groups: control (CG) and training (GT). They were evaluated at day 2 after admission, hospital discharge and after 30 days. These assessments were verified to peripheral muscle strength of upper and lower limbs, the distance walked in 6 minutes, systemic inflammation (TNF-, CRP, IL1, IL-12p70, IL-6, IL-8 and IL10), health-related quality of life and physical activity of daily living. RESULTS: Our results show that during hospitalization all patients, regardless of group, remained most of the time (87%) in the lying or sitting position. The CG patients showed a reduction in muscle strength of lower limbs (p <0.05) but no change in distance walked in 6 minutes (p> 0.05) during hospitalization. TG patients demonstrated improvement in muscle strength of lower limbs and distance walked in 6 minutes (p <0.05) that remained until 30 days after hospital discharge. Differently from GC, the GT patients showed an improvement in the domain impact on health-related quality of life after discharge. There was no difference between groups in systemic inflammatory markers analyzed during hospitalization and after 30 days. In addition, several patients in both groups remained physically inactive (70%) at home. CONCLUSION: Our results suggest that resistance training during hospitalization improves peripheral muscle strength and functional capacity. However, without altering systemic inflammation and physical activity of daily living

Bei IL-10 handelt es sich um ein antiinflammatorisches Zytokin, dessen immunmodulatorische Effekte bereits in zahlreichen Studien aufgezeigt werden konnten. Ziel dieser Studie war, die Unterschiede in der systemischen Inflammation und Organdysfunktion an Mäusen zu untersuchen, die nach Induktion eines hämorrhagischen Schocks, entweder inhalativ oder systemisch, mit IL-10 behandelt wurden. Männliche C57/BL6 Mäuse (6 Tiere pro Gruppe) wurden für 1.5 Stunden blutdruckkontrolliert in einen hämorrhagischen Schock versetzt. Nach anschließender Volumensubstitution wurde ihnen inhalativ oder intraarteriell rekombiniertes Maus - IL-10 verabreicht. Nach einer Gesamtversuchsdauer von 6 - bzw. 24 Stunden erfolgte die Tötung der Tiere. Die Ergebnisse der Studie zeigen, dass die lokale und systemische Verabreichung von IL-10 das Zytokinprofil der systemischen Inflammationsantwort unterschiedlich beeinflusst. Die Lunge kann durch inhalative Gabe von IL-10 geschützt werden, ohne die systemische Inflammationsantwort zu beeinflussen.

Background: Systemic inflammation can occur after transcatheter aortic valve replacement (TAVR) and correlates with adverse outcome. The impact of remote ischemic preconditioning (RIPC) on TAVR associated systemic inflammation is unknown and was focus of this study. Methods: We performed a prospective controlled trial at a single center and included 66 patients treated with remote ischemic preconditioning (RIPC) prior to TAVR, who were matched to a control group by propensity score. RIPC was applied to the upper extremity using a conventional tourniquet. Definition of systemic inflammation was based on leucocyte count, C-reactive protein (CRP), procalcitonin (PCT) and interleukin-6 (IL-6), assessed in the first 5 days following the TAVR procedure. Mortality was determined within 6 months after TAVR. RIPC group and matched control group showed comparable baseline characteristics. Results: Systemic inflammation occurred in 66% of all patients after TAVR. Overall, survival after 6 months was significantly reduced in patients with systemic inflammation. RIPC, in comparison to control, did not significantly alter the plasma levels of leucocyte count, CRP, PCT or IL-6 within the first 5 days after TAVR. Furthermore, inflammation associated survival after 6 months was not improved by RIPC. Of all peri-interventional variables assessed, only the amount of the applied contrast agent was connected to the occurrence of systemic inflammation. Conclusions: Systemic inflammation frequently occurs after TAVR and leads to increased mortality after 6 months. RIPC neither reduces the incidence of systemic inflammation nor improves inflammation associated patient survival within 6 months.

Abstract Sepsis is a common problem in the intensive care unit (ICU) still having a high mortality and causing high costs to health care system. Currently, there is no marker to distinguish sepsis from other causes of systemic inflammation. Leukocyte surface molecules have been proposed as markers of sepsis. The most promising markers have been neutrophil CD64 and CD11b on monocytes and neutrophils and HLA-DR on monocytes. In this thesis, leukocyte surface molecules were investigated using quantitative flow cytometry in critically ill patients with sepsis, non-septic ICU controls, and healthy volunteers. The surface molecules of interest were neutrophil CD11b and CD64, monocyte CD11b, CD14, CD40, CD64, CD80, HLA-DR, and lymphocyte CD69. First, a special emphasize was indicated in methodological aspects of the quantitative flow cytometry. Then, the surface molecule kinetics was investigated in different types of critically ill patients. Finally, the diagnostic performance of the molecules was determined and compared to that of traditionally used sepsis markers. Furthermore, an example of multiple marker analysis was introduced as a diagnostic tool. The optimal circumstances for leukocyte surface molecule analysis were +4&#176;C temperature throughout the collection and preparation of the samples using tubes containing acid citrate dextrose (ACD) as an anticoagulant, followed by flow cytometry within 6 hours from sampling. Monocyte CD11b and CD40, neutrophil CD11b and CD64, and CD69 on CD4+ T cells and natural killer (NK) cells separated sepsis from non-septic ICU controls and healthy volunteers, neutrophil CD64, having the best area under curve. Procalcitonin (PCT) was second best marker. Monocyte CD40 and NK CD69 may predict positive blood culture detection, whereas CD11b may predict early mortality. In multiple marker analysis, combination of positive neutrophil CD64, C-reactive protein (CRP) and PCT increased post-test probability for sepsis. In conclusion, pre-analytical and analytical factors have effects on results of leukocyte surface molecule analysis. Leukocyte surface molecules may improve sepsis diagnostics in ICU setting. Neutrophil CD64 was the most promising marker. Combination of CD64, CRP and PCT increased the detection of sepsis in ICU. / Tiivistelmä Sepsis on yleinen tehohoidon ongelma, johon liittyy korkea kuolleisuus ja suuret hoidolliset kustannukset. Toistaiseksi ei ole laboratoriomerkkiainetta, joka erottaisi sepsistä sairastavat muista kriittisesti sairaista, joilla on yleistynyt tulehdusvaste. Valkosolujen pintamolekyylien käyttöä sepsiksen laboratoriomerkkiaineena on tutkittu. Lupaavimmat näistä molekyyleistä ovat olleet neutrofiilien CD64, monosyyttien ja neutrofiilien CD11b ja monosyyttien HLA-DR. Tässä väitöskirjassa tutkittiin valkosolujen pintamolekyylejä kriittisesti sairailla sepsistä sairastavilla potilailla, niillä tehohoitopotilailla, joilla ei ollut sepsistä, ja terveillä vapaaehtoisilla virtaussytometriaa käyttäen. Mielenkiinnon kohteina olivat neutrofiilien CD11b ja CD64, monosyyttien CD11b, CD14, CD40, CD64, CD80 ja HLA-DR, sekä lymfosyyttien CD69. Ensimmäiseksi tutkittiin kvantitatiivista virtaussytometriaa menetelmänä. Sen jälkeen pintamolekyylien kinetiikkaa tutkittiin eri potilasryhmillä. Lopuksi määritettiin pintamolekyylien diagnostinen tehokkuus ja sitä verrattiin perinteisempiin sepsiksen diagnostiikassa käytettyihin laboratoriomerkkiaineisiin. Lisäksi selvitettiin usean merkkiaineen mallin diagnostista osuvuutta. Parhaat olosuhteet virtaussytometrialle olivat: +4 &#176;C:n lämpötila näytteenoton ja -käsittelyn aikana, näytteiden ottaminen putkiin, joissa on antikoagulanttina hapan sitraatti-dekstroosi (ACD) ja näytteiden analysointi kuuden tunnin kuluessa näytteenotosta. Monosyyttien CD11b ja CD40, neutrofiilien CD11b ja CD64 sekä CD4+ T-solujen ja NK-solujen CD69 erottivat sepsistä sairastavat tehohoitoverrokeista ja terveistä. Neutrofiilien CD64:llä oli paras erottelukyky. Prokalsitoniini (PCT) oli toiseksi paras merkkiaine. Monosyyttien CD40 ja NK-solujen CD69 voivat parantaa positiivisen veriviljelylöydöksen havaitsemista, kun taas CD11b voi ennustaa varhaista potilaan menehtymistä. Usean merkkiaineen mallissa neutrofiilien CD64 paransi C-reaktiivisen proteiinin (CRP) ja PCT:n tehoa sepsiksen diagnostiikassa. Loppupäätelmänä on, että valkosolujen pintamolekyylien analysointivaiheen eri muuttujilla on vaikutusta virtaussytometriatuloksiin. Valkosolujen pintamolekyylien käyttö voi parantaa sepsiksen diagnostiikkaa teho-osastolla. Neutrofiilien CD64 oli lupaavin merkkiaine. Neutrofiilien CD64:n, CRP:n ja PCT:n yhdistelmä paransi sepsiksen diagnostiikkaa teho-osastolla.

No description available.

Réalisé en cotutelle avec le Dr James G Martin de l'Université McGill (Meakins-Christie laboratories) / L’asthme chez l’homme et le souffle chez le cheval sont des maladies inflammatoires chroniques des voies respiratoires partageant plusieurs caractéristiques physiopathologiques dont la bronchoconstriction réversible, l’inflammation des voies respiratoires inférieures, l’hyperréactivité bronchique et le remodelage tissulaire. Les phénotypes cliniques d’asthme se caractérisent en partie selon le type d’inflammation affectant les voies respiratoires et la présence ou non d’allergie. Le souffle chez le cheval s’avère être un modèle adapté pour l’étude des mécanismes impliqués dans l’asthme neutrophilique, lesquels demeurent particulièrement mal compris, en contraste avec ceux associés avec l’asthme éosinophilique. La réponse immunologique sous-jacente au souffle implique entre autres l’expression de cytokines de type Th2, suggestives d’une réponse allergique (immunité acquise). La poussière environnementale qui provoque les symptômes du souffle contient également des agents non-spécifiques dérivés de bactéries, champignons et moisissures, susceptibles d’activer des mécanismes immunitaires innés chez les chevaux atteints du souffle. Nous avons étudié le rôle des neutrophiles dans l’inflammation associée à la réponse innée et acquise chez le cheval atteint du souffle. Dans un premier temps, l’effet de produits dérivés de bactéries sur l’activation des neutrophiles sanguins provenant de chevaux normaux et atteints de souffle a été étudié dans le but d’évaluer la contribution de la réponse innée dans la physiopathologie du souffle. Nous avons évalué l’effet de l’IL-4, une cytokine de type Th2, sur les neutrophiles des deux groupes de chevaux afin d’évaluer de quelle manière le neutrophile peut participer à la réponse acquise associée à la réponse allergique. Finalement, nous avons étudié l’expression des isoformes de l'arginase par les neutrophiles équins car cette enzyme métabolise la L-arginine et est potentiellement impliquée dans le bronchospasme et le remodelage tissulaire associés à l’asthme. Nos résultats suggèrent que les neutrophiles et les mononucléaires sanguins isolés des chevaux atteints du souffle possèdent une réponse inflammatoire exagérée en réponse aux lipopolyssacharides et peptides formylés et surexpriment les cytokines pro-inflammatoires IL-1β, TNF et IL-8. Cette réponse innée aberrante est associée à une inflammation systémique caractérisée par des concentrations sériques élevées de TNF chez les chevaux atteints du souffle en période de rémission clinique. De plus, nos résultats montrent que l’IL-4 active le neutrophile équin et favorise son chimiotactisme de manière autocrine. L’IL-4 induit un phénotype d’activation typique dans le neutrophile équin, caractérisé par l’expression accrue des cytokines pro-inflammatoires (IL-8 et TNF) ainsi que de récepteurs potentiellement impliqués dans la réponse allergique (IL-4Rα et CD23). Enfin, nous montrons que que l’arginase 1 n’est pas un marqueur de l’activation des neutrophiles équins par l’IL-4, mais que ces cellules expriment constitutivement l’isoforme 2 fonctionnelle de l’arginase. La surrégulation des deux isoformes au niveau des poumons périphériques semble être associée à la pathologie du souffle, ce qui est en accord avec les modèles d’asthme chez la souris, le rat et le cobaye. L'ensemble de ces travaux suggère que les neutrophiles sont des cellules effectrices importantes de la réponse innée et acquise dans la pathophysiologie du souffle, un modèle naturel d’asthme neutrophilique. / Human asthma and equine heaves are chronic pulmonary diseases sharing several pathophysiological properties including lower airway inflammation, reversible bronchoconstriction, bronchial hyperresponsiveness, and tissue remodeling. Clinical phenotypes of asthma are characterized in part by the inflammatory cell populations infiltrating the airways, and the presence or absence of allergy. Heaves is a suitable animal model for the study of the poorly defined pathophysiological processes leading to airway neutrophilia. The immune response in heaves involves Th2 cytokine expression, which is, among other features, associated to allergic inflammation (acquired immunity). Environmental dust exposure leading to clinical exacerbation of heaves contains non-specific agents derived from bacteria, molds or fungi which could also activate innate immune responses in heaves affected horses. We studied the role of neutrophils in innate and acquired immune responses in heaves affected-horse. First, innate immune responses of neutrophils isolated from normal and heaves-affected horses to bacterial-derived products were studied. We also assessed the effect of IL-4, a Th2 cytokine, on equine neutrophils isolated from both groups of horses. Finally, we evaluated the arginase isoforms expressed by equine neutrophils as this enzyme that takes part to the L-arginine metabolism and is thought to contribute to bronchospasm and tissue remodeling associated with asthma. Our results suggest that both neutrophils and mononuclear cells from heaves-affected horses, when compared to healthy horses, have an excessive inflammatory response to lipopolyssacharides and formylated peptides characterized by increased IL-1β, IL-8 and TNF expression. This altered innate response was associated with systemic inflammation in asymptomatic susceptible horses as high serum TNF concentrations were detected. Furthermore, we found that equine neutrophils are activated by IL-4 and release neutrophil chemotactic factors in response to this cytokine. IL-4 also induces a distinctive activation phenotype in neutrophils that is characterized by increased expression of the pro-inflammatory cytokines (IL-8 and TNF) and receptors (IL-4Rα and CD23) potentially involved in the allergic response. Finally, we showed that arginase 1 is not a marker of IL-4-activated equine neutrophils although they constitutively express a functionally active isoform 2 of the enzyme. The up-regulation of arginase isoforms in the peripheral lungs of horses with heaves suggests a role for arginase in this model, as it is described in the mouse, rat and guinea pig models. Taken together, this work suggest that neutrophils could play an important role in both innate and acquired immune responses associated with heaves pathophysiology, a natural model of neutrophilic asthma.

Depuis 2013, le traitement « universel » est recommandé en France. Le moment de l’initiation thérapeutique est une question qui reste cependant d’actualité pour les patients se présentant en primo-infection. Cette thèse s’attache à étudier la prise en charge thérapeutique du VIH au stade de la primo-infection (PIV) sous différents angles :1) le suivi par les cliniciens des recommandations d’initiation des traitements antirétroviraux depuis 1996 en fonction de l’évolution de ces recommandations; 2) l’impact d’un traitement ARV transitoire en PIV sur la réponse immuno-virologique lors de la reprise du traitement et 3) l’identification de nouveaux biomarqueurs comme facteurs pronostiques de progression de l’infection VIH. La majorité des travaux présentés dans cette thèse repose sur les données de la cohorte ANRS PRIMO qui comporte environ 1 500 patients infectés par le VIH inclus en PIV entre juin 1996 et décembre 2013, dans 94 hôpitaux français. Tous les patients étaient naïfs de traitement antirétroviral à l'inclusion.La première partie de la thèse analyse la mise en œuvre des recommandations d’initiation du traitement ARV entre 1996 et 2010 par les médecins en France, dans deux situations distinctes : au stade chronique et lors de la primo-infection par le VIH-1. Nous avons montré que les recommandations d’initiation du traitement ARV étaient largement suivies. Néanmoins, il existe un effet d’inertie dans leurs applications lors des changements de recommandation. Il reste à améliorer le délai de mise sous traitement lorsque le taux de CD4 atteint le seuil recommandé. Au stade chronique, le traitement était plus fréquemment initié chez les patients présentant un critère d’initiation dès le diagnostic d’infection par le VIH (96%), que chez les patients qui atteignaient un critère d’initiation au cours du suivi (78%, p<0.001). Nous avons identifié comme facteurs de risque de ne pas être traité en phase chronique malgré une indication de traitement : une charge virale < 5log (versus >5), un plus faible niveau d’éducation et des conditions de vie précaires.L’impact de l’interruption d’un traitement antirétroviral initié en PIV sur la restauration des CD4 après reprise du traitement a été exploré en modélisant l’évolution des CD4 avec des modèles linéaires à effets mixtes avec intercept et pente aléatoires. Les patients qui avaient initié un traitement ARV pendant la phase chronique avaient une meilleure réponse immunologique que les patients reprenant un 2ème traitement après un traitement transitoire en PIV : à 36 mois, les gains en √CD4 cellules/mm3 et en pourcentage de CD4 étaient significativement plus élevés. Cependant, il s’agissait de différences modestes en termes cliniques, qui ne conduisent pas à recommander d’arrêter la recherche clinique sur les arrêts de traitement cherchant à induire des contrôleurs post traitement. Après un état des lieux des mécanismes complexes d’activation/inflammation du système immunitaire pendant la primo-infection nous avons cherché à identifier de nouveaux biomarqueurs prédictifs de l’évolution de l’infection. Le taux de sCD14 (marqueur d’activation monocyte/macrophage et marqueur indirect de translocation microbienne) au moment de la PIV a été identifié comme marqueur potentiel de prédiction du déclin des CD4 et du risque de mortalité d’origine cardio-vasculaire. En conclusion, bien que des progrès considérables aient été réalisés dans la prise en charge du VIH, d'autres études sont nécessaires pour optimiser et adapter le traitement au profil du patient dès les premiers stades de l’infection VIH. / In France, since 2013, HIV treatment has been recommended for all HIV-infected patients independently of their CD4 count. However, when to start anti-retroviral (ARV) treatment is still an issue. This thesis aims to explore the therapeutic management of HIV at the stage of PHI in different aspects: 1) we explored how physicians in France have applied the evolving guidelines for ART initiation since 1996 2) the impact of a transient ARV treatment at PHI on immuno-virological response during 2nd treatment and 3) identification of new biomarkers prognostic of HIV progression.Most of the work presented in this thesis is based on data from the ongoing ANRS PRIMO cohort that enrolled more than 1 500 HIV infected patients enrolled at PHI since June 1996 in 94 French hospitals. All patients were antiretroviral therapy naive at baseline.The first part of the thesis analyzes the implementation of the recommendations of ARV treatment initiation between 1996 and 2010 by physicians in France, in two distinct situations: in the chronic HIV-1 infection and during primary HIV-1 infection. We have shown that the recommendations of ARV treatment initiation were widely followed. Nevertheless, there was inertia in guidelines application when changes in the recommendations took place. The time to treatment when CD4 cell counts reach the threshold to treat can be improved. 96% of the patients initiated ART when they had a CD4 cell count below the threshold to treat at entry, while treatment was less timely initiated when the CD4 threshold was reached during active follow-up (78%, p <0.001).We identified as risk factors for not being timely treated in chronic phase despite an indication for treatment: a viral load <5log (versus> 5), a lower education level and poor living conditions.The impact of ARV interruption after a first treatment initiated at PHI on the CD4 count restoration after resumption was explored by modeling the evolution of CD4 cells with linear mixed effects models with random intercept and slope. Patients who initiated ARV treatment during the chronic phase had a better immune response than patients who initiated a second course treatment after a transient ART at PHI: at 36 months, the gains in √CD4 cells / mm3 and CD4 percentage were significantly higher. However, this difference was clinically modest and further research on treatment interruptions seeking to induce post-treatment controllers is still an issue but only in research settings and under close medical surveillance. After an overview of the complex mechanisms of activation / inflammation of the immune system during primary infection we sought to identify new predictive biomarkers of disease progression. The level of sCD14 (marker of monocyte/macrophage activation and an indirect marker of microbial translocation) at the time of PHI was identified as predictive marker of CD4 decline and of risk of cardio-vascular mortality. In conclusion, although considerable progress has been made in the management of HIV, further studies are needed to optimize and adapt the treatment to the patient profile in the early stages of HIV infection.

L’asthme chez l’homme et le souffle chez le cheval sont des maladies inflammatoires chroniques des voies respiratoires partageant plusieurs caractéristiques physiopathologiques dont la bronchoconstriction réversible, l’inflammation des voies respiratoires inférieures, l’hyperréactivité bronchique et le remodelage tissulaire. Les phénotypes cliniques d’asthme se caractérisent en partie selon le type d’inflammation affectant les voies respiratoires et la présence ou non d’allergie. Le souffle chez le cheval s’avère être un modèle adapté pour l’étude des mécanismes impliqués dans l’asthme neutrophilique, lesquels demeurent particulièrement mal compris, en contraste avec ceux associés avec l’asthme éosinophilique. La réponse immunologique sous-jacente au souffle implique entre autres l’expression de cytokines de type Th2, suggestives d’une réponse allergique (immunité acquise). La poussière environnementale qui provoque les symptômes du souffle contient également des agents non-spécifiques dérivés de bactéries, champignons et moisissures, susceptibles d’activer des mécanismes immunitaires innés chez les chevaux atteints du souffle. Nous avons étudié le rôle des neutrophiles dans l’inflammation associée à la réponse innée et acquise chez le cheval atteint du souffle. Dans un premier temps, l’effet de produits dérivés de bactéries sur l’activation des neutrophiles sanguins provenant de chevaux normaux et atteints de souffle a été étudié dans le but d’évaluer la contribution de la réponse innée dans la physiopathologie du souffle. Nous avons évalué l’effet de l’IL-4, une cytokine de type Th2, sur les neutrophiles des deux groupes de chevaux afin d’évaluer de quelle manière le neutrophile peut participer à la réponse acquise associée à la réponse allergique. Finalement, nous avons étudié l’expression des isoformes de l'arginase par les neutrophiles équins car cette enzyme métabolise la L-arginine et est potentiellement impliquée dans le bronchospasme et le remodelage tissulaire associés à l’asthme. Nos résultats suggèrent que les neutrophiles et les mononucléaires sanguins isolés des chevaux atteints du souffle possèdent une réponse inflammatoire exagérée en réponse aux lipopolyssacharides et peptides formylés et surexpriment les cytokines pro-inflammatoires IL-1β, TNF et IL-8. Cette réponse innée aberrante est associée à une inflammation systémique caractérisée par des concentrations sériques élevées de TNF chez les chevaux atteints du souffle en période de rémission clinique. De plus, nos résultats montrent que l’IL-4 active le neutrophile équin et favorise son chimiotactisme de manière autocrine. L’IL-4 induit un phénotype d’activation typique dans le neutrophile équin, caractérisé par l’expression accrue des cytokines pro-inflammatoires (IL-8 et TNF) ainsi que de récepteurs potentiellement impliqués dans la réponse allergique (IL-4Rα et CD23). Enfin, nous montrons que que l’arginase 1 n’est pas un marqueur de l’activation des neutrophiles équins par l’IL-4, mais que ces cellules expriment constitutivement l’isoforme 2 fonctionnelle de l’arginase. La surrégulation des deux isoformes au niveau des poumons périphériques semble être associée à la pathologie du souffle, ce qui est en accord avec les modèles d’asthme chez la souris, le rat et le cobaye. L'ensemble de ces travaux suggère que les neutrophiles sont des cellules effectrices importantes de la réponse innée et acquise dans la pathophysiologie du souffle, un modèle naturel d’asthme neutrophilique. / Human asthma and equine heaves are chronic pulmonary diseases sharing several pathophysiological properties including lower airway inflammation, reversible bronchoconstriction, bronchial hyperresponsiveness, and tissue remodeling. Clinical phenotypes of asthma are characterized in part by the inflammatory cell populations infiltrating the airways, and the presence or absence of allergy. Heaves is a suitable animal model for the study of the poorly defined pathophysiological processes leading to airway neutrophilia. The immune response in heaves involves Th2 cytokine expression, which is, among other features, associated to allergic inflammation (acquired immunity). Environmental dust exposure leading to clinical exacerbation of heaves contains non-specific agents derived from bacteria, molds or fungi which could also activate innate immune responses in heaves affected horses. We studied the role of neutrophils in innate and acquired immune responses in heaves affected-horse. First, innate immune responses of neutrophils isolated from normal and heaves-affected horses to bacterial-derived products were studied. We also assessed the effect of IL-4, a Th2 cytokine, on equine neutrophils isolated from both groups of horses. Finally, we evaluated the arginase isoforms expressed by equine neutrophils as this enzyme that takes part to the L-arginine metabolism and is thought to contribute to bronchospasm and tissue remodeling associated with asthma. Our results suggest that both neutrophils and mononuclear cells from heaves-affected horses, when compared to healthy horses, have an excessive inflammatory response to lipopolyssacharides and formylated peptides characterized by increased IL-1β, IL-8 and TNF expression. This altered innate response was associated with systemic inflammation in asymptomatic susceptible horses as high serum TNF concentrations were detected. Furthermore, we found that equine neutrophils are activated by IL-4 and release neutrophil chemotactic factors in response to this cytokine. IL-4 also induces a distinctive activation phenotype in neutrophils that is characterized by increased expression of the pro-inflammatory cytokines (IL-8 and TNF) and receptors (IL-4Rα and CD23) potentially involved in the allergic response. Finally, we showed that arginase 1 is not a marker of IL-4-activated equine neutrophils although they constitutively express a functionally active isoform 2 of the enzyme. The up-regulation of arginase isoforms in the peripheral lungs of horses with heaves suggests a role for arginase in this model, as it is described in the mouse, rat and guinea pig models. Taken together, this work suggest that neutrophils could play an important role in both innate and acquired immune responses associated with heaves pathophysiology, a natural model of neutrophilic asthma. / Réalisé en cotutelle avec le Dr James G Martin de l'Université McGill (Meakins-Christie laboratories)

This thesis investigates how air pollution, both natural and anthropogenic, affects changes in the proximal small intestine and ileum microbiota profile, as well as intestinal barrier integrity, histological changes, and inflammation. APO-E KO mice on a high fat diet were randomly selected to be exposed by whole body inhalation to either wood smoke (WS) or mixed vehicular exhaust (MVE), with filtered air (FA) acting as the control. Intestinal integrity and histology were assessed by observing expression of well- known structural components tight junction proteins (TJPs), matrix metallopeptidase-9 (MMP-9), and gel-forming mucin (MUC2), as well known inflammatory related factors: TNF-α, IL-1β, and toll-like receptor (TLR)-4. Bacterial profiling was done using DNA analysis of microbiota within the ileum, utilizing 16S metagenomics sequencing (Illumina miSeq) technique. Overall results of this experiment suggest that air pollution, both anthropogenic and natural, cause a breach in the intestinal barrier with an increase in inflammatory factors and a decrease in beneficial bacteria. This evidence suggests the possibility of air pollution being a potential causative agent of intestinal disease as well as a possible contributing mechanism for induction of systemic inflammation.

Various molecular pathways in the brain shape our understanding of good and bad, as well as our motivation to seek and avoid such stimuli. This work evolves around how systemic inflammation causes aversion; and why general unpleasant states such as sickness, stress, pain and nausea are encoded by our brain as undesirable; and contrary to these questions, how drugs of abuse can subjugate the motivational neurocircuitry of the brain. A common feature of these various disease states is involvement of the motivational neurocircuitry - from mesolimbic to striatonigral pathways. Having an intact motivational system is what helps us evade negative outcomes and approach natural positive reinforcers, which is essential for our survival. During disease-states the motivational neurocircuitry may be overthrown by the molecular mechanisms that originally were meant to aid us. In study I, to investigate how inflammation is perceived as aversive, we used a behavioral test based on Pavlovian place conditioning with the aversive inflammatory stimulus E. coli lipopolysaccharide (LPS). Using a combination of cell-type specific gene deletions, pharmacology, and chemogenetics, we uncovered that systemic inflammation triggered aversion by MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Moreover, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, inflammation-induced aversion was not an indirect consequence of fever or anorexia but constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic circuitry is a key mechanism underlying inflammation-induced aversion. In study II, we investigate the role of peripheral IFN-γ in LPS induced conditioned place aversion by employing a strategy based on global and cell-type specific gene deletions, combined with measures of gene-expression. LPS induced IFN-ɣ expression in the blood, and deletion of IFN-ɣ or its receptor prevented conditioned place aversion (CPA) to LPS. LPS increased the expression of chemokine Cxcl10 in the striatum of normal mice. This induction was absent in mice lacking IFN-ɣ receptors or Myd88 in blood brain barrier endothelial cells. Furthermore, inflammation-induced aversion was blocked in mice lacking Cxcl10 or its receptor Cxcr3. Finally, mice with a selective deletion of the IFN-ɣ receptor in brain endothelial cells did not develop inflammation-induced aversion. Collectively, these findings demonstrate that circulating IFN-ɣ binding to receptors on brain endothelial cells which induces Cxcl10, is a central link in the signaling chain eliciting inflammation-induced aversion. In study III, we explored the role of melanocortin 4 receptors (MC4Rs) in aversive processing using genetically modified mice in CPA to various stimuli. In normal mice, robust aversions were induced by systemic inflammation, nausea, pain and kappa opioid receptor-induced dysphoria. In sharp contrast, mice lacking MC4Rs displayed preference towards most of the aversive stimuli, but were indifferent to pain. The unusual flip from aversion to reward in mice lacking MC4Rs was dopamine-dependent and associated with a change from decreased to increased activity of the dopamine system. The responses to aversive stimuli were normalized when MC4Rs were re-expressed on dopamine D1 receptor-expressing cells or in the striatum of mice otherwise lacking MC4Rs. Furthermore, activation of arcuate nucleus proopiomelanocortin neurons projecting to the ventral striatum increased the activity of striatal neurons in a MC4R-dependent manner and elicited aversion. Our findings demonstrate that melanocortin signaling through striatal MC4Rs is critical for assigning negative motivational valence to harmful stimuli. The neurotransmitter acetylcholine has been implied in reward learning and drug addiction. However, the role of cholinergic receptor subtypes in such processes remains elusive. In study IV we investigated the function of muscarinic M4Rs on dopamine D1R expressing neurons and acetylcholinergic neurons, using transgenic mice in various reward-enforced behaviors and in a “waiting”-impulsivity test. Mice lacking M4-receptors from D1-receptor expressing neurons exhibited an escalated reward seeking phenotype towards cocaine and natural reward, in Pavlovian conditioning and an operant self-administration task, respectively. In addition, the M4-D1RCre mice showed impaired waiting impulsivity in the 5-choice-serial-reaction-time-task. On the contrary, mice without M4Rs in acetylcholinergic neurons were unable to learn positive reinforcement to natural reward and cocaine, in an operant runway paradigm and in Pavlovian conditioning.  Immediate early gene expression mirrored the behavioral findings arising from M4R-D1R knockout, as cocaine induced cFos and FosB was significantly increased in the forebrain of M4-D1RCre mice, whereas it remained normal in the M4R-ChatCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality.