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Este estudo teve por finalidade estudar os efeitos da fototerapia com laser em baixa intensidade (Low Level Laser Therapy LLLT) sobre a inflamação e reparação após lesão criogênica realizada no sitema nervoso central (SNC) de ratos. Foram realizados 3 experimentos. Em todos os experimentos foi utilizado um modelo de deformação cortical direta induzida por lesão criogênica. A LLLT foi realizada com laser de diodo em baixa intensidade emitindo no vermelho visível (InGaAlP, 660 nm) ou no infravermelho (AlGaAr, 780 nm). Os parâmetros de irradiação foram: potência de 40 mW, área do feixe de 0,04 cm2, densidades de energia de 3 J/cm2 (3 s) ou 5 J/cm2 (5 s) determinando energias por ponto de 0,12 J e 0,20 J, respectivamente. Foram realizadas 2 irradiações com intervalo de 3 h, no modo contato e em 2 pontos por lesão. No experimento 1, 50 ratos da linhagem Wistar foram utilizados para determinar os parâmetros de LLLT capazes de influenciar na dinâmica da produção de citocinas proinflamatórias (TNF-a, IL-1b, IL-6) e antiinflamatória (IL-10). As citocinas foram mensuradas pelo teste ELISA no cérebro e no sangue dos animais, 6 e 24h após a lesão. Os grupos experimentais foram: controle (não irradiado) e 4 grupos irradiados com 3 J/cm2 ou 5 J/cm2 para cada comprimento de onda (n=10 por grupo). Para os experimentos 2 e 3 foram utilizados 40 ratos (20 não irradiados controles e 20 irradiados). A LLLT foi realizada somente com o parâmetro de irradiação do laser no infravermelho e a densidade de energia de 3 J/cm2. Nestes experimentos o processo de reparação das lesões criogênicas no SNC foi acompanhado em 6 h, 1, 7 e 14 dias após a última irradiação (n=5 por grupo por tempo experimental). No experimento 2 foi realizada a análise morfométrica da região lesionada do SNC. No experimento 3 foi analisada a distribuição das células inflamatórias (linfócitos T, leucócitos e macrófagos). Os dados de cada experimento foram comparados estatísticamente por análise de variância (ANOVA) ou Kruskal-Wallis seguido dos testes de Tukey ou de Dunn, respectivamente (F=5%). O trauma criogênico foi capaz de criar lesões focais no córtex representadas por necrose, edema, hemorragia e infiltrado inflamatório. Os achados mais marcantes foram: no experimento 1 com a irradiação do laser no infravermelho e 3 J/ cm2, o TNF- a e a IL-6 se mantiveram nos mesmos níveis em 6 e 24 h, enquanto no controle houve aumento significativo. Experimento 2: as lesões não tratadas apresentaram maior perda tecidual em 6 h que as irradiadas. Experimento 3: as lesões irradiadas apresentaram menor quantidade de leucócitos e linfócitos T nas primeiras 24 h do que nas lesões controle. A quantidade de macrófagos foi similar nos dois grupos. Conclusões: Levando em consideração as condições experimentais deste estudo concluiu-se que LLLT exerce efeitos nos processos de inflamação e reparação diminuindo a concentração de citocinas próinflamatórias (TNF-F e IL-6) no sangue e mantendo a de IL-1I no cérebro. Adicionalmente, diminui a perda tecidual inicial pós- lesão criogênica e a infiltração inicial de leucócitos e linfócitos T. / This study aimed to study the effects of phototherapy with low-intensity laser (Low Level Laser Therapy - LLLT) on inflammation and repair after cryogenic injury held in central nervous system (CNS) of rats. There were 3 experiments. In all experiments a model of deformation induced by direct cortical cryogenic injury was used. The LLLT was carried out with a low intensity diode laser emitting in the visible red (InGaAlP, 660 nm) or infrared (AlGaAs, 780 nm). The irradiation parameters were: power of 40 mW, beam area of 0.04 cm2, energy densities of 3 J/cm2 (3 s) or 5 J/cm2 (5 s) providing energy per point of 0.12 J and 0.20 J, respectively. Two irradiations were performed at 3 h-intervals, in contact mode and in 2 points for lesion. In experiment 1, 50 Wistar rats were used to determine the parameters of LLLT able to influence the dynamics of production proinflammatory cytokines (TNF-, IL-1 and IL-6) and antiinflammatory cytokine (IL- 10). Cytokines were measured by ELISA in the brain and blood of animals, 6 and 24 hours after injury. The experimental groups were: control (non-irradiated) and 4 irradiated groups [3 J/cm2 and 5 J/cm2 for each wavelength (n = 10 per group)]. For experiments 2 and 3 40 rats (20 non-irradiated - controls and 20 irradiated).were used. The LLLT was performed only with the parameter of laser irradiation on the infrared and the energy density of 3 J/cm2. In these experiments, the process of repair of cryogenic injury in the CNS was followed in 6 h, 1, 7 and 14 days after the last irradiation (n = 5 per group and time trial). In experiment 2 a morphometric analysis of the injured area of the CNS was done. In experiment 3 the distribution of inflammatory cells (lymphocytes, leukocytes and macrophages) was analyzed. Data from each experiment were compared statistically by analysis of variance (ANOVA) or Kruskal-Wallis followed by tests of Tukey or Dunn, respectively ( = 5%). Cryogenic trauma was able to create focal lesions in the cortex represented by necrosis, edema, hemorrhage and inflammatory infiltrate. The most striking findings were: in experiment 1 with the laser irradiation in the infrared and 3 J/cm 2, TNF- and IL-6 remained at the same levels at 6 and 24 h, while in the control there was a significant increase. Experiment 2: untreated lesions showed greater tissue loss than irradiated lesions in 6 h. Experiment 3: lesions irradiated had fewer leukocytes and lymphocytes in the first 24 h than control lesions. The amount of macrophages was similar in both groups. Conclusions: Considering the experimental conditions of this study it was concluded that LLLT has effects in the processes of inflammation and repair by decreasing the concentration of proinflammatory cytokines (TNF- and IL-6) in blood and holding the IL-1 in the brain. Additionally, decreases the initial tissue loss after cryogenic injury and the initial infiltration of leukocytes and lymphocytes T.

A lesão granulomatosa é caracterizada como um agregado compacto de fagócitos maduros formado em resposta à um estímulo persistente. Os mediadores pró-inflamatórios da família da IL-1, ao promoverem a ativação da imunidade inata e o remodelamento tecidual descontrolados, geram a fisiopatologia da paracoccidioidomicose, doença pulmonar granulomatosa causada pelo fungo P. brasiliensis. A principal via inflamatória envolvida na secreção de IL-1?, IL-18 e IL-1? é a ativação dos inflammasomas, complexos protéicos conhecidos pela sua capacidade de ativar proteoliticamente a enzima caspase-1. Neste estudo abordamos os mecanismos subjacentes às vias canônica e não canônica do inflamassoma, avaliando a importância funcional das caspases 1, 11 e 8 na resistência do hospedeiro durante a infecção por P. brasiliensis. Demonstramos que a resposta imunológica protetora mediada pelo IFN-?, após a liberação de IL-18 pela via da caspase-1, é o principal mecanismo responsável pelo controle fúngico quando o dano celular desencadeado pelo fungo é reconhecido pelo inflamassoma de NLRP3. No entanto, apesar do papel importante da caspase-1 na maturação da IL-1?, a falta de caspase-1 apenas reduz parcialmente os níveis de IL-1? durante a infecção por P. brasiliensis. Ao contrário do esperado, a deficiência de caspase-11 não prejudicou a produção de IL-1?, mas em vez disso, preveniu, na célula, a morte por piroptose e a secreção IL-1?, citocina importante para restringir o crescimento fúngico através da síntese de IL-17. Por fim, observamos que a ativação de caspase-8 pela sinalização de dectina-1 / Syk, além de mediar a maturação da IL-1?, que acontece de maneira independente de caspase-1 e 11, é necessária também para o funcionamento eficiente da via canônica de caspase-1, demonstrando uma rede interligada entre as vias canônica de caspse-1 e não canônica de caspase-8 para coordenar o processamento da IL-1?. Em conjunto, nossos resultados mostram contribuições distintas das vias canônica e não canônica do inflamassoma na produção de citocinas da família da IL-1, enfatizam a versatilidade desta plataforma em recrutar várias proteínas efetoras para adequar a resposta imunológica antifúngica e evidenciam a complexidade envolvida nas interações patógeno-hospedeiro. / Granuloma is a lesion characterized by a compact aggregate of mature phagocytes arising in response to a persistent stimulus. By driving uncontrolled innate immunity and tissue remodelling, IL-1 family pro-inflammatory mediators govern the pathophysiology of paracoccidioidomycosis, a granulomatous lung disorders caused by Paracocccidioides brasiliensis. A major inflammatory pathway involved in IL-1?, IL-18 e IL-1? secretion is the activation of inflammasomes, large multimolecular complexes best known for their ability to control activation of the proteolytic enzyme caspase-1. In this study we addressed the mechanisms that underlie canonical and non-canonical inflammasome pathways, assessing the functional importance of caspase-1, caspase-11 and caspase-8 in the regulation of inflammasome-mediated host resistance during P. brasiliensis infection. We found that IFN-?-mediated protective immune response following by caspase-1- dependent IL-18 release after is the key mechanism responsible for the fungal control after P. brasiliensis-induced cell damage recognition by NLRP3 canonical inflammasome pathway. Nonetheless, despite the important role of caspase-1 in the IL-1? maturation, the lack of caspase-1 only partially reduced IL-1? levels during P. brasiliensis infection. Unlike caspase-1, caspase-11 deficiency did not impair IL-1? production, cytokine strictly secreted by canonical caspase-1 inflammasome pathway. Instead, P. brasiliensis-triggered caspase-11 activation in an ill-defined manner leads to a rapid pore-mediated cell lysis and is required for IL-1? production during P. brasiliensis infection. IL-1?, in turn, is important for promoting the restriction of fungal growth trough IL-17-based inflammation. Finally, the caspase-8 induction by dectin-1/Syk signaling besides playing a role in mediating the caspase-1/11-independent IL-1? maturation is also required to efficient canonical caspase-1 inflammasome pathway, demonstrating a connected network between non-canonical caspase-8 and canonical caspase-1 inflammasome pathways to coordinate IL-1?. Taken together, our results revealed distincts contributions of both canonical and non-canonical inflammasome pathways in IL-1 family cytokine production and emphasizes the versatility of this platform to recruit several effector proteins to tailor the antifungal immune response, sheding new light on the complexity of this hostpathogen interaction.

Este estudo teve por finalidade estudar os efeitos da fototerapia com laser em baixa intensidade (Low Level Laser Therapy LLLT) sobre a inflamação e reparação após lesão criogênica realizada no sitema nervoso central (SNC) de ratos. Foram realizados 3 experimentos. Em todos os experimentos foi utilizado um modelo de deformação cortical direta induzida por lesão criogênica. A LLLT foi realizada com laser de diodo em baixa intensidade emitindo no vermelho visível (InGaAlP, 660 nm) ou no infravermelho (AlGaAr, 780 nm). Os parâmetros de irradiação foram: potência de 40 mW, área do feixe de 0,04 cm2, densidades de energia de 3 J/cm2 (3 s) ou 5 J/cm2 (5 s) determinando energias por ponto de 0,12 J e 0,20 J, respectivamente. Foram realizadas 2 irradiações com intervalo de 3 h, no modo contato e em 2 pontos por lesão. No experimento 1, 50 ratos da linhagem Wistar foram utilizados para determinar os parâmetros de LLLT capazes de influenciar na dinâmica da produção de citocinas proinflamatórias (TNF-a, IL-1b, IL-6) e antiinflamatória (IL-10). As citocinas foram mensuradas pelo teste ELISA no cérebro e no sangue dos animais, 6 e 24h após a lesão. Os grupos experimentais foram: controle (não irradiado) e 4 grupos irradiados com 3 J/cm2 ou 5 J/cm2 para cada comprimento de onda (n=10 por grupo). Para os experimentos 2 e 3 foram utilizados 40 ratos (20 não irradiados controles e 20 irradiados). A LLLT foi realizada somente com o parâmetro de irradiação do laser no infravermelho e a densidade de energia de 3 J/cm2. Nestes experimentos o processo de reparação das lesões criogênicas no SNC foi acompanhado em 6 h, 1, 7 e 14 dias após a última irradiação (n=5 por grupo por tempo experimental). No experimento 2 foi realizada a análise morfométrica da região lesionada do SNC. No experimento 3 foi analisada a distribuição das células inflamatórias (linfócitos T, leucócitos e macrófagos). Os dados de cada experimento foram comparados estatísticamente por análise de variância (ANOVA) ou Kruskal-Wallis seguido dos testes de Tukey ou de Dunn, respectivamente (F=5%). O trauma criogênico foi capaz de criar lesões focais no córtex representadas por necrose, edema, hemorragia e infiltrado inflamatório. Os achados mais marcantes foram: no experimento 1 com a irradiação do laser no infravermelho e 3 J/ cm2, o TNF- a e a IL-6 se mantiveram nos mesmos níveis em 6 e 24 h, enquanto no controle houve aumento significativo. Experimento 2: as lesões não tratadas apresentaram maior perda tecidual em 6 h que as irradiadas. Experimento 3: as lesões irradiadas apresentaram menor quantidade de leucócitos e linfócitos T nas primeiras 24 h do que nas lesões controle. A quantidade de macrófagos foi similar nos dois grupos. Conclusões: Levando em consideração as condições experimentais deste estudo concluiu-se que LLLT exerce efeitos nos processos de inflamação e reparação diminuindo a concentração de citocinas próinflamatórias (TNF-F e IL-6) no sangue e mantendo a de IL-1I no cérebro. Adicionalmente, diminui a perda tecidual inicial pós- lesão criogênica e a infiltração inicial de leucócitos e linfócitos T. / This study aimed to study the effects of phototherapy with low-intensity laser (Low Level Laser Therapy - LLLT) on inflammation and repair after cryogenic injury held in central nervous system (CNS) of rats. There were 3 experiments. In all experiments a model of deformation induced by direct cortical cryogenic injury was used. The LLLT was carried out with a low intensity diode laser emitting in the visible red (InGaAlP, 660 nm) or infrared (AlGaAs, 780 nm). The irradiation parameters were: power of 40 mW, beam area of 0.04 cm2, energy densities of 3 J/cm2 (3 s) or 5 J/cm2 (5 s) providing energy per point of 0.12 J and 0.20 J, respectively. Two irradiations were performed at 3 h-intervals, in contact mode and in 2 points for lesion. In experiment 1, 50 Wistar rats were used to determine the parameters of LLLT able to influence the dynamics of production proinflammatory cytokines (TNF-, IL-1 and IL-6) and antiinflammatory cytokine (IL- 10). Cytokines were measured by ELISA in the brain and blood of animals, 6 and 24 hours after injury. The experimental groups were: control (non-irradiated) and 4 irradiated groups [3 J/cm2 and 5 J/cm2 for each wavelength (n = 10 per group)]. For experiments 2 and 3 40 rats (20 non-irradiated - controls and 20 irradiated).were used. The LLLT was performed only with the parameter of laser irradiation on the infrared and the energy density of 3 J/cm2. In these experiments, the process of repair of cryogenic injury in the CNS was followed in 6 h, 1, 7 and 14 days after the last irradiation (n = 5 per group and time trial). In experiment 2 a morphometric analysis of the injured area of the CNS was done. In experiment 3 the distribution of inflammatory cells (lymphocytes, leukocytes and macrophages) was analyzed. Data from each experiment were compared statistically by analysis of variance (ANOVA) or Kruskal-Wallis followed by tests of Tukey or Dunn, respectively ( = 5%). Cryogenic trauma was able to create focal lesions in the cortex represented by necrosis, edema, hemorrhage and inflammatory infiltrate. The most striking findings were: in experiment 1 with the laser irradiation in the infrared and 3 J/cm 2, TNF- and IL-6 remained at the same levels at 6 and 24 h, while in the control there was a significant increase. Experiment 2: untreated lesions showed greater tissue loss than irradiated lesions in 6 h. Experiment 3: lesions irradiated had fewer leukocytes and lymphocytes in the first 24 h than control lesions. The amount of macrophages was similar in both groups. Conclusions: Considering the experimental conditions of this study it was concluded that LLLT has effects in the processes of inflammation and repair by decreasing the concentration of proinflammatory cytokines (TNF- and IL-6) in blood and holding the IL-1 in the brain. Additionally, decreases the initial tissue loss after cryogenic injury and the initial infiltration of leukocytes and lymphocytes T.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Chagas disease, caused by the protozoan Trypanosoma cruzi, is ranked as the most serious parasitic disease in Latin America and has become a worldwide problem. A bulk of studies substantiates that Th1-associated cytokines are essential elements in early resistance against the parasite and are associated with the development of the chronic cardiac form. Although cytokines have a key role in the immune response against T. cruzi, little is known about IL-3, IL-7 and IL-9 in this context. Then the aim of this study was to analyze the role of IL-3, IL-7 and IL-9 in the acute phase of T. cruzi experimental infection. For this purpose, parameters indicative of improvement in clinical status of the animals both infected and treated as just treated were studied, such as: morbidity, mortality and histopathology. The amount of cardiac mast cells and the serum cytokines profile were also evaluated. Our data revealed that the treatment with IL-3, IL-7 or IL-9 did not alter the clinical parameters analyzed or the amount of cardiac mast cells in mice infected with T. cruzi. However the treatment with IL-3 decreased the cardiac T. cruzi-induced inflammation and the treatment with IL-7 increased serum levels of IL-5 in infected animals. In addition, the treatment with IL-9 increased the serum levels of Th1 cytokine profile (IL-2, IFN-y and TNF-α) and decreased cardiac fibrosis in infected animals, suggesting a possible protective role of this cytokine in this context. Taken together, our results underline the importance of these cytokines in modulation of T. cruzi infection. Since studies involving IL-3, IL-7 and IL-9 activity during Chagas disease are critical in understanding the parasite control process and the protective and/or harmful action of these cytokines in the host. In addition, understanding of the immunological mechanisms mediated by these cytokines that are involved in disease development may contribute to the establishment of new therapeutic interventions to prevent Chagas disease and treat their symptomatic forms. / A Doença de Chagas, causada pelo protozoário Trypanosoma cruzi, é classificada como a doença parasitária mais grave da América Latina e tornou-se um problema mundial. A maior parte dos estudos confirma que as citocinas de perfil Th1 são elementos essenciais na resistência precoce contra o parasita e estão associadas ao desenvolvimento da forma cardíaca crônica. Embora as citocinas tenham um papel chave na resposta imune contra T. cruzi, pouco se sabe a respeito da função de IL-3, IL-7 e IL-9 neste contexto. Assim, o objetivo deste estudo foi analisar o papel destas citocinas na fase aguda da infecção experimental com T. cruzi. Para isto, parâmetros indicativos de melhora clínica dos animais tanto infectados e tratados como apenas tratados foram estudados, tais como: morbidade, mortalidade e histopatologia. A quantidade de mastócitos cardíacos e o perfil de citocinas séricas também foram avaliados. Os dados revelaram que o tratamento com IL-3, IL-7 ou IL-9 não alterou os parâmetros clínicos analisados nem a quantidade de mastócitos cardíacos nos camundongos infectados com T. cruzi. No entanto, o tratamento com IL-3 diminuiu a inflamação cardíaca T. cruzi-induzida e o tratamento com IL-7 aumentou os níveis séricos de IL-5 nos animais infectados. Além disso, o tratamento com IL-9 aumentou os níveis séricos das citocinas de perfil Th1 (IL-2, IFN-y e TNF-α) e diminuiu a fibrose cardíaca nos animais infectados, sugerindo um possível papel protetor desta citocina neste contexto. Em suma, os nossos resultados demonstram a importância destas citocinas na modulação da infecção por T. cruzi. Já que estudos envolvendo a atividade de IL-3, IL-7 e IL-9 durante a doença de Chagas são fundamentais na compreensão sobre o processo de controle parasitário e da ação protetora e/ou prejudicial dessas citocinas no hospedeiro. Além disso, o entendimento dos mecanismos imunológicos mediados por estas citocinas que estão envolvidos no desenvolvimento da doença pode contribuir para o estabelecimento de novas intervenções terapêuticas a fim de prevenir a doença de Chagas e tratar suas formas sintomáticas / Doutor em Imunologia e Parasitologia Aplicadas

A lesão granulomatosa é caracterizada como um agregado compacto de fagócitos maduros formado em resposta à um estímulo persistente. Os mediadores pró-inflamatórios da família da IL-1, ao promoverem a ativação da imunidade inata e o remodelamento tecidual descontrolados, geram a fisiopatologia da paracoccidioidomicose, doença pulmonar granulomatosa causada pelo fungo P. brasiliensis. A principal via inflamatória envolvida na secreção de IL-1?, IL-18 e IL-1? é a ativação dos inflammasomas, complexos protéicos conhecidos pela sua capacidade de ativar proteoliticamente a enzima caspase-1. Neste estudo abordamos os mecanismos subjacentes às vias canônica e não canônica do inflamassoma, avaliando a importância funcional das caspases 1, 11 e 8 na resistência do hospedeiro durante a infecção por P. brasiliensis. Demonstramos que a resposta imunológica protetora mediada pelo IFN-?, após a liberação de IL-18 pela via da caspase-1, é o principal mecanismo responsável pelo controle fúngico quando o dano celular desencadeado pelo fungo é reconhecido pelo inflamassoma de NLRP3. No entanto, apesar do papel importante da caspase-1 na maturação da IL-1?, a falta de caspase-1 apenas reduz parcialmente os níveis de IL-1? durante a infecção por P. brasiliensis. Ao contrário do esperado, a deficiência de caspase-11 não prejudicou a produção de IL-1?, mas em vez disso, preveniu, na célula, a morte por piroptose e a secreção IL-1?, citocina importante para restringir o crescimento fúngico através da síntese de IL-17. Por fim, observamos que a ativação de caspase-8 pela sinalização de dectina-1 / Syk, além de mediar a maturação da IL-1?, que acontece de maneira independente de caspase-1 e 11, é necessária também para o funcionamento eficiente da via canônica de caspase-1, demonstrando uma rede interligada entre as vias canônica de caspse-1 e não canônica de caspase-8 para coordenar o processamento da IL-1?. Em conjunto, nossos resultados mostram contribuições distintas das vias canônica e não canônica do inflamassoma na produção de citocinas da família da IL-1, enfatizam a versatilidade desta plataforma em recrutar várias proteínas efetoras para adequar a resposta imunológica antifúngica e evidenciam a complexidade envolvida nas interações patógeno-hospedeiro. / Granuloma is a lesion characterized by a compact aggregate of mature phagocytes arising in response to a persistent stimulus. By driving uncontrolled innate immunity and tissue remodelling, IL-1 family pro-inflammatory mediators govern the pathophysiology of paracoccidioidomycosis, a granulomatous lung disorders caused by Paracocccidioides brasiliensis. A major inflammatory pathway involved in IL-1?, IL-18 e IL-1? secretion is the activation of inflammasomes, large multimolecular complexes best known for their ability to control activation of the proteolytic enzyme caspase-1. In this study we addressed the mechanisms that underlie canonical and non-canonical inflammasome pathways, assessing the functional importance of caspase-1, caspase-11 and caspase-8 in the regulation of inflammasome-mediated host resistance during P. brasiliensis infection. We found that IFN-?-mediated protective immune response following by caspase-1- dependent IL-18 release after is the key mechanism responsible for the fungal control after P. brasiliensis-induced cell damage recognition by NLRP3 canonical inflammasome pathway. Nonetheless, despite the important role of caspase-1 in the IL-1? maturation, the lack of caspase-1 only partially reduced IL-1? levels during P. brasiliensis infection. Unlike caspase-1, caspase-11 deficiency did not impair IL-1? production, cytokine strictly secreted by canonical caspase-1 inflammasome pathway. Instead, P. brasiliensis-triggered caspase-11 activation in an ill-defined manner leads to a rapid pore-mediated cell lysis and is required for IL-1? production during P. brasiliensis infection. IL-1?, in turn, is important for promoting the restriction of fungal growth trough IL-17-based inflammation. Finally, the caspase-8 induction by dectin-1/Syk signaling besides playing a role in mediating the caspase-1/11-independent IL-1? maturation is also required to efficient canonical caspase-1 inflammasome pathway, demonstrating a connected network between non-canonical caspase-8 and canonical caspase-1 inflammasome pathways to coordinate IL-1?. Taken together, our results revealed distincts contributions of both canonical and non-canonical inflammasome pathways in IL-1 family cytokine production and emphasizes the versatility of this platform to recruit several effector proteins to tailor the antifungal immune response, sheding new light on the complexity of this hostpathogen interaction.

La mucoviscidose est une pathologie pulmonaire causée par un dysfonctionnement du canal CFTR et caractérisée par un mucus visqueux, une susceptibilité accrue aux infections chroniques et une inflammation excessive. Une première partie de ma thèse a eu pour objectif d’étudier les mécanismes inflammatoires impliqués dans le développement de la pathologie. Nous avons plus particulièrement analysé le rôle de l’IL-1β et de l’IL-17 dans la réponse à l’infection par Pseudomonas aeruginosa, dans le modèle murin ΔF508 de mucoviscidose. La seconde partie de ma thèse a porté sur l’étude de la malaria pulmonaire et cérébrale, une complication létale de l’infection à P. falciparum. Nous avons mis en évidence l’importance de trois voies d’activation des lymphocytes T CD8+ cytotoxiques dans le développement de la neuropathologie induite par Plasmodium berghei ANKA chez la souris : la protéine PKC-θ, la sous-unité β2 du récepteur à l’IL-12 et le récepteur des IFN de type I, mais qui ne semblent pas impliquées dans l’inflammation pulmonaire associée. / Cystic fibrosis is a pulmonary pathology, caused by the CFTR channel dysfunction, and characterized by high mucus viscosity, increased sensitivity to chronic infections and excessive inflammation. The aim of my thesis was first to study the inflammatory mechanisms involved in this lung pathology. Indeed, we analyzed the role of IL-1β and IL-17 in response to Pseudomonas aeruginosa infection, in the ΔF508 mouse model of cystic fibrosis. In the second part of my thesis, I studied pulmonary and cerebral malaria, a lethal complication of P. falciparum infection. We showed the importance of three pathways implicated in cytotoxic CD8+ T lymphocytes activation during the Plasmodium berghei ANKA-induced neuropathology development in mice: PKC-θ protein, β2 subunit of IL-12 receptor and type I IFN receptor, which did not seem essential for the associated lung inflammation.

IL-36 gamma is a proinflamatory cytokine which belongs to the IL-1 family of cytokines. It is expressed in the skin and by epithelial cells (ECs) lining lung and gut tissue. We used human 3-D organotypic cells, that recapitulate either in vivo human vaginal or cervical tissue, to explore the possible role of IL-36 gamma in host defense against pathogens in the human female reproductive tract (FRT). EC were exposed to compounds derived from virus or bacterial sources and induction and regulation of IL-36 gamma and its receptor was determined. Polyinosinic-polycytidylic acid (poly I:C), flagellin, and synthetic lipoprotein (FSL-1) significantly induced expression of IL-36 gamma in a dose-dependent manner, and appeared to be TLR-dependent. Recombinant IL-36 gamma treatment resulted in self amplification of IL-36 gamma and its receptor (IL-36R) via increased gene expression, and promoted other inflammatory signaling pathways. This is the first report to demonstrate that the IL-36 receptor and IL-36 gamma are present in the human FRT EC and that they are differentially induced by microbial products at this site. We conclude that IL-36 gamma is a driver for epithelial and immune activation following microbial insult and, as such, may play a critical role in host defense in the FRT.

IL-12 family cytokines IL-23 and IL-27 play an important role linking innate and adaptive immunity, and regulating T-cell responses. The production of IL-12, a structurally similar cytokine, is decreased in chronic HIV infection; therefore IL-23 and IL-27 may also be influenced by HIV infection. I hypothesized that HIV inhibits LPS-induced IL-23 and IL-27 production in human MDMs by suppressing the activation of signalling pathways regulating their expression. In vitro HIV-infection of MDMs did not have any effect on basal secretion of IL-23 or IL-27; however, HIV inhibited LPS-induced production of IL-12/23 p40 and IL-23 p19, and IL-27 EBI3 and IL-27 p28 mRNA expression, and IL-23, IL-12/23 p40 and IL-27 secretion. In order to evaluate the molecular mechanisms by which HIV inhibits IL-23 and IL-27 in LPS-stimulated MDMs, the signalling pathways regulating their expression were evaluated. The PI3K, p38 MAPK, and JNK MAPK pathways were found to positively regulate LPS-induced IL-27 secretion. Interestingly, in vitro HIV infection inhibited LPS-induced p38 and JNK MAPK activation in MDMs. In summary, I have shown that HIV inhibits IL-23 and IL-27 production in LPS-stimulated MDMs and that HIV may inhibit LPS-induced IL-27 production through the inhibition of p38 and JNK MAPK activation. It is currently unknown whether PKCs regulate LPS-induced IL-23 or IL-27 in human monocytes/macrophages. I demonstrated that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 secretion within THP-1 cells, primary monocytes, and MDMs. Classical PKCs were found to positively regulate LPS-induced IL-12/23 p40 and IL-27 p28 mRNA expression and IL-12/23 p40, IL-23, and IL-27 secretion in primary human monocytes. Similarly, the classical PKCs were found to positively regulate IL-27 p28 mRNA expression and IL-27 secretion in THP-1 cells. However, classical PKCs did not regulate LPS-induced IL-27 production in MDMs, or LPS-induced IL-23 production in THP-1 cells. Overall, this demonstrates that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 production in different myeloid cells.

Successful treatment of cancer with adoptive immunotherapy (AIT) is dependent on the ability to produce large numbers of tumor-specific, functional T cells. The purpose of this thesis is to explore ways in which T cell expansion could be augmented. We have focused on exploring alternate gamma chain cytokines as stimulators of T cell proliferation and differentiation in addition to investigating the potential usefulness of gemcitabine (GEM) in abrogating the immunosuppressive effects of myeloid derived suppressor cells (MDSCs). B16 melanoma sensitized draining lymph node cells that have been activated in vitro with bryostatin-1 and ionomycin (B/I) were expanded in either IL-7/15 or in IL-2. We found that IL-7/15 was superior to IL-2 in expanding T cells for AIT of pulmonary metastases. Expansion of antitumor T cells was also improved by suppressing accumulation of MDSCs in mice bearing 4T1 mammary carcinoma using GEM. GEM directly inhibits both 4T1 mammary carcinoma cells and MDSCs. Its inhibition of MDSCs rescued tolerant T cells, augmenting both expansion and response to tumor antigen.