Interleukin (IL)-1beta is a powerful pro-inflammatory cytokine with important roles in directing both innate and adaptive immunity. As a result, its production is tightly controlled, with the synthesis of an inactive form (pro-IL-1beta) and the requirement of a second signal. This induces the formation of the inflammasome, a macromolecular complex which mediates the maturation of IL-1beta into the bioactive cytokine. Given its significance, it is important to identify mechanisms of IL-1beta induction and modulation. Firstly, we describe serum amyloid A (SAA), an acute phase protein with immunomodulatory properties, as a novel inducer of IL-1beta. Using cells from genetically modified mice, the molecular mechanisms responsible were dissected, demonstrating the receptors TLR2 and NLRP3 as required for this effect. By instilling SAA into mice, we also show that SAA is able to induce IL-1beta production in vivo. Invariant natural killer T (iNKT) cells have also been shown to be important modulators of immunity, mediating both pro- and anti-inflammatory responses. iNKT cells are non-conventional T lymphocytes which recognise glycolipid in the context of CD1d, with the ability to interact with immature antigen presenting cells in an autoreactive manner. We link the regulatory ability of iNKT cells with IL-1beta production, showing that a low activation signal leads to the induction of an IL-13-dominated cytokine profile, as well as weak engagement of the CD40-CD40L pathway. We show for the first time that through these mechanisms, iNKT cells are able to dampen the secretion of IL-1beta upon subsequent stimulation of dendritic cells. We hypothesise that this effect of iNKT cells is important in controlling inflammatory responses in vivo, and demonstrate exacerbated IL-1beta production and inflammation during influenza virus infection of iNKT cell-deficient animals. This novel anti-inflammatory property of iNKT cells may be harnessed in the therapeutic intervention of inflammatory disorders.

O uso de álcool está aumentando em nossa sociedade e permanece associado a inúmeros problemas sociais, econômicos e de saúde. De fato, o álcool e os problemas de saúde associados a ele exercem um impacto importante na prática médica e representam um dos maiores desafios da saúde pública. O consumo de álcool na sociedade contemporânea é geralmente aceito positivamente, dificultando o reconhecimento de certos padrões de consumo como doença. O alcoolismo é um transtorno recidivante crônico caracterizado pela ingestão compulsiva de quantidades excessivas de etanol, perda de controle em sua ingestão, comportamento inadequado e a presença de um estado emocional negativo. O consumo de quantidades nocivas de álcool resulta em danos físicos e psicológicos e o vicio é um transtorno psiquiátrico que afeta as funções executivas, causando perda de interesse em outros alvos do prazer e comportamento compulsivo de busca por drogas. O álcool interage com vários sistemas neurológicos. O presente trabalho analisou a resposta imunoinflamatória no tecido cerebral de camundongos CRAMP knockout (KO) jovens e tipo selvagem (WT) submetidos ao modelo de intoxicação alcoólica, com o objetivo de investigar o impacto de CRAMP na dependência alcoólica no adolescente. O CRAMP é um peptídeo antimicrobiano com efeitos pleotrópicos e, até onde sabemos, seu papel nunca foi investigado nesse sentido. Também analisamos a secreção de vários neuropeptídeos, proteínas e citocinas. Nossos resultados mostraram uma diferença significativa na ingestão de etanol entre os animais comparados CRAMP KO e WT, o que foi relacionado a um aumento nos níveis cerebelares de IL-1beta. Concluimos que os pepitídeos antimicrobianos podem ter um papel importante na resposta imunoinflamatória que ocorre durante o etilismo agudo / The use of alcohol is increasing in our society and remains associated with countless social, economic and health problems. In fact, alcohol and the health issues associated to its abuse exert an important impact on medical practice and represent one of the biggest challenges of public health. The consumption of alcohol in contemporary society is generally accepted positively, making certain patterns of consumption very difficult to be recognized as a disease. Alcoholism is a chronic relapsing disorder characterized by compulsive ingestion of excessive amounts of ethanol, loss of control in its intake, inappropriated behavior and the presence of a negative emotional state. The consumption of harmful amounts of alcohol results in physical and or psychological damage and addiction is a psychiatric disorder that affects the executive functions, causing loss of interest in other aspects of life and a compulsive behavior. Alcohol interacts with several neurologic systems. The present work analyzed the immuno-inflammatory response in the brain tissue of young CRAMP knockout (KO) and wild-type (WT) mice submitted to a model of alcohol intoxication, in order to investigate the impact of CRAMP in teenager alcohol addiction. CRAMP is an antimicrobial peptide with pleotropic effects and, as far as we know, its role had never been investigation in this regard. We also analysed the secretion of several neuropeptides, proteins and cytokines. Our results showed a significant difference in ethanol intake when CRAMP KO and WT animals were compared, which was related to an increase in the cerebellar levels of IL-1beta. We conclude that antimicrobial peptides may play an important role in the immunoinflammatory response that occurs during acute alcoholism

O uso de álcool está aumentando em nossa sociedade e permanece associado a inúmeros problemas sociais, econômicos e de saúde. De fato, o álcool e os problemas de saúde associados a ele exercem um impacto importante na prática médica e representam um dos maiores desafios da saúde pública. O consumo de álcool na sociedade contemporânea é geralmente aceito positivamente, dificultando o reconhecimento de certos padrões de consumo como doença. O alcoolismo é um transtorno recidivante crônico caracterizado pela ingestão compulsiva de quantidades excessivas de etanol, perda de controle em sua ingestão, comportamento inadequado e a presença de um estado emocional negativo. O consumo de quantidades nocivas de álcool resulta em danos físicos e psicológicos e o vicio é um transtorno psiquiátrico que afeta as funções executivas, causando perda de interesse em outros alvos do prazer e comportamento compulsivo de busca por drogas. O álcool interage com vários sistemas neurológicos. O presente trabalho analisou a resposta imunoinflamatória no tecido cerebral de camundongos CRAMP knockout (KO) jovens e tipo selvagem (WT) submetidos ao modelo de intoxicação alcoólica, com o objetivo de investigar o impacto de CRAMP na dependência alcoólica no adolescente. O CRAMP é um peptídeo antimicrobiano com efeitos pleotrópicos e, até onde sabemos, seu papel nunca foi investigado nesse sentido. Também analisamos a secreção de vários neuropeptídeos, proteínas e citocinas. Nossos resultados mostraram uma diferença significativa na ingestão de etanol entre os animais comparados CRAMP KO e WT, o que foi relacionado a um aumento nos níveis cerebelares de IL-1beta. Concluimos que os pepitídeos antimicrobianos podem ter um papel importante na resposta imunoinflamatória que ocorre durante o etilismo agudo / The use of alcohol is increasing in our society and remains associated with countless social, economic and health problems. In fact, alcohol and the health issues associated to its abuse exert an important impact on medical practice and represent one of the biggest challenges of public health. The consumption of alcohol in contemporary society is generally accepted positively, making certain patterns of consumption very difficult to be recognized as a disease. Alcoholism is a chronic relapsing disorder characterized by compulsive ingestion of excessive amounts of ethanol, loss of control in its intake, inappropriated behavior and the presence of a negative emotional state. The consumption of harmful amounts of alcohol results in physical and or psychological damage and addiction is a psychiatric disorder that affects the executive functions, causing loss of interest in other aspects of life and a compulsive behavior. Alcohol interacts with several neurologic systems. The present work analyzed the immuno-inflammatory response in the brain tissue of young CRAMP knockout (KO) and wild-type (WT) mice submitted to a model of alcohol intoxication, in order to investigate the impact of CRAMP in teenager alcohol addiction. CRAMP is an antimicrobial peptide with pleotropic effects and, as far as we know, its role had never been investigation in this regard. We also analysed the secretion of several neuropeptides, proteins and cytokines. Our results showed a significant difference in ethanol intake when CRAMP KO and WT animals were compared, which was related to an increase in the cerebellar levels of IL-1beta. We conclude that antimicrobial peptides may play an important role in the immunoinflammatory response that occurs during acute alcoholism

Immunopathological disorders are no longer defined by dysregulated T-helper (Th) type 1/ Th2 responses but account for modulatory cell types such as regulatory and Th17 cells. The newly defined Th17 subset is an effector memory subtype which regulates mucosal host defense responses. A distinctive feature of interleukin (IL)-17 is its ability to invoke neutrophilic responses and to synergize cytokine responses in proximal structural cells. This effect is most evident for proinflammatory cytokines and neutrophil-mobilizing chemokines which are under the regulatory control of the canonical, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. The uniqueness of the IL-17A receptor (IL-17RA) signal transduction pathway however has been a limiting factor in uncovering IL-17-mediated effector functions since the receptor bears little homology to other known receptors and contains a unique cytoplasmic consensus binding motif. Hence, the composition, dynamics and subunit interactions of the IL-17R complex have become an emerging area of research where novel recruitment motifs and adaptor proteins are actively being explored. Our study sought to uncover the signal transduction and molecular mechanisms mediating the initiation and amplification responses induced by IL-17. We hypothesize that (i) IL-17 represents a key cytokine which initiates inflammatory responses by acting on proximal structural cells to rapidly release neutrophil-mobilizing chemokines and myeloid growth factors and that (ii) IL-17 directly promotes survival responses of immune effector cells. Genomic analysis of stimulated human airway smooth muscle cells support the proinflammatory nature of IL-17 as NF-κB associated genes and chemokines were most significantly upregulated within 2 hours. However, IL-17 induced a modest fold increase in gene expression levels whereby only 4 genes achieved greater than 2 fold increases. This, along with the observation that IL-17 enhanced IL-1β-mediated CXCL8 expression via transcriptional promoter activation levels and post-transcriptional mRNA stabilization mechanisms suggests that IL-17 cooperatively functions with secondary cytokines to mediate inflammatory responses. Despite activating the p38-mitogen-activated protein kinase (MAPK) signaling pathway in peripheral blood neutrophils, IL-17 did not directly affect the apoptotic capacity of these cells but unexpectedly antagonized the survival response mediated by the granulocyte-macrophage colony stimulating factor (GM-CSF). Collectively, our results suggest that IL-17 is a potent synergistic cytokine which signals via the MAPK-NF-κB pathway to indirectly recruit neutrophils via CXC-chemokines produced by non-hematopoietic cells and that IL-17 may potentially dampen inflammatory responses by directly antagonizing inflammatory effector cells.

Immunopathological disorders are no longer defined by dysregulated T-helper (Th) type 1/ Th2 responses but account for modulatory cell types such as regulatory and Th17 cells. The newly defined Th17 subset is an effector memory subtype which regulates mucosal host defense responses. A distinctive feature of interleukin (IL)-17 is its ability to invoke neutrophilic responses and to synergize cytokine responses in proximal structural cells. This effect is most evident for proinflammatory cytokines and neutrophil-mobilizing chemokines which are under the regulatory control of the canonical, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. The uniqueness of the IL-17A receptor (IL-17RA) signal transduction pathway however has been a limiting factor in uncovering IL-17-mediated effector functions since the receptor bears little homology to other known receptors and contains a unique cytoplasmic consensus binding motif. Hence, the composition, dynamics and subunit interactions of the IL-17R complex have become an emerging area of research where novel recruitment motifs and adaptor proteins are actively being explored. Our study sought to uncover the signal transduction and molecular mechanisms mediating the initiation and amplification responses induced by IL-17. We hypothesize that (i) IL-17 represents a key cytokine which initiates inflammatory responses by acting on proximal structural cells to rapidly release neutrophil-mobilizing chemokines and myeloid growth factors and that (ii) IL-17 directly promotes survival responses of immune effector cells. Genomic analysis of stimulated human airway smooth muscle cells support the proinflammatory nature of IL-17 as NF-κB associated genes and chemokines were most significantly upregulated within 2 hours. However, IL-17 induced a modest fold increase in gene expression levels whereby only 4 genes achieved greater than 2 fold increases. This, along with the observation that IL-17 enhanced IL-1β-mediated CXCL8 expression via transcriptional promoter activation levels and post-transcriptional mRNA stabilization mechanisms suggests that IL-17 cooperatively functions with secondary cytokines to mediate inflammatory responses. Despite activating the p38-mitogen-activated protein kinase (MAPK) signaling pathway in peripheral blood neutrophils, IL-17 did not directly affect the apoptotic capacity of these cells but unexpectedly antagonized the survival response mediated by the granulocyte-macrophage colony stimulating factor (GM-CSF). Collectively, our results suggest that IL-17 is a potent synergistic cytokine which signals via the MAPK-NF-κB pathway to indirectly recruit neutrophils via CXC-chemokines produced by non-hematopoietic cells and that IL-17 may potentially dampen inflammatory responses by directly antagonizing inflammatory effector cells.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 5 uppsatser.

Sickle cell anemia (HbSS) is a mendelian genetic disorder caused by a point mutation (A/T) on hemoglobin’s beta globin gene, originating an abnormal hemoglobin called hemoglobin S (HbS). People with HbSS have more susceptibility to develop infections, inflammation, ischemia, episodes of pain and tissue necrosis, as well as changes in the stomatognathic system and its associated structures. The aim of this study was to evaluating socioeconomic, clinical, genetic and caries in children with HbSS in the State of Sergipe. This cross-sectional case-control study involved 210 volunteers, 70 HbSS composing the study group (SG) and 140 composing the group of control (CG). The SG was paired with CG on age and gender. Data collection included interviews with the parent/guardian, verification of medical records and clinical examination. Statistical analyzes were performed using SPSS version 20, considering a confidence interval of 95% (p <0.05). The mean age of the sample was 6.73 ± 2.62. The majority (74.3%) of the patient’s parents of SG had little schooling, and 45.7% are in the lower economic classes (D and E). In SG were observed coadjuvant systemic diseases and comorbidities of HbSS. Regarding dental caries, 29 (41.4%) of the volunteers of SG and 46 (36.9%) of the CG showed zero decay. The caries indices in permanent teeth showed no statistical difference between groups, although the decay component (C) performed statistically different between the groups, for teeth (p=0.028) and to surfaces (p=0.035). The frequencies of genotypes and alleles of SNPs rs1143641, rs1143633, rs1143634 showed no similarity to the European (CEU) and Sub-Saharan African (YRI) populations. The proinflammatory genotypes for SNP rs1143634 presented: additive [TC (32.8%) and CC (3.0%)], dominant [TC + CC (35.8%)] and recessive [CC (3.0%)]. With the data it can be concluded that SG showed lower socioeconomic profile compared to the CG. The descriptive analysis of systemic diseases and complications demonstrated harm to health and quality of life in the population with HbSS. The groups had a similar experience of tooth decay, no statistical difference between caries indices. The genetic profile frequencies of genotypes and alleles indicate ethnic mix of the population studied between CEU and YRI populations. The pro-inflammatory genotypes didn’t show high prevalence in SG and can’t evaluate their impact, this evoluation just can be possible with indreased sample size. The results demonstrate that the patients with HbSS have a health impairment arising because of the systemic and oral aspects, requiring prioritized monitoring and early monitoring. / A anemia falciforme (AF) é uma doença genética mendeliana causada por uma mutação originando uma hemoglobina anormal (HbS). Pessoas com AF apresentam susceptibilidade aumentada as infecções, inflamações, isquemias, episódios de dor e necroses teciduais, bem como alterações no sistema estomatognático e em suas estruturas anexas. O objetivo deste trabalho é analisar fatores socioeconômicos, clínicos, genéticos e de cárie dentária em crianças com anemia falciforme no Estado de Sergipe. Este estudo epidemiológico transversal controlado envolveu 210 voluntários, 70 indivíduos com AF compondo o grupo de estudo (GE) e 140 controles (GC). O GE foi pareado com o GC em idade e gênero. A coleta de dados incluiu entrevista com os responsáveis, verificação de prontuários e exame clínico odontológico. As análises estatísticas foram feitas no SPSS versão 20, considerando um intervalo de confiança de 95% (p<0,05). A média de idade da amostra foi 6,73±2,62. A maioria (74,3%) dos pais do GE possuía escolaridade baixa, além de 45,7% estarem nas classes econômicas mais baixas (D e E). No GE foram observadas doenças sistêmicas coadjuvantes a AF e comorbidades da AF. No que se refere a cárie dentária, 29 (41,4%) dos voluntários do GE e 46 (36,9%) do GC apresentaram cárie zero. Os índices de cárie na dentição permanente não demonstram diferença estatística entre os grupos, porém o componente cariado (C) apresentou diferença estatística entre os grupos, tanto para dentes (p=0,028) quanto para superfícies (p=0,035). As frequências dos genótipos e alelos dos SNPs rs1143641, rs1143633, rs1143634 não apresentaram semelhança com as populações africana subsaariana (YRI) ou europeia (CEU). Os genótipos pró-inflamatórios do SNP rs1143634 apresentou baixa prevalência no GE: modelo aditivo [TC (32,8%) e TT (3,0%)], recessivo [TT (3,0%)] e dominante [TC+TT (35,8%)]. Com os dados pode-se concluir que o GE demonstrou perfil socioeconômico mais baixo em comparação ao GC. A análise descritiva de doenças sistêmicas e complicações demonstra prejuízos à saúde e qualidade de vida nos indivíduos com AF. Os grupos apresentaram uma experiência de cárie dentária semelhante, sem diferença estatística entre os índices de cárie, porém o componente cariado com valor mais alto em pacientes com AF na dentição permanente. Com relação ao perfil genético, as frequências dos genótipos e alelos indicam miscigenação étnica da população estudada entre as populações YRI e CEU. Os genótipos pró-inflamatórios não apresentaram alta prevalência no GE e não se pode avaliar o seu impacto, sendo esta avaliação somente possível com número amostral aumentado. Os resultados demonstram que os pacientes com AF tem um prejuízo da saúde advindo do quadro sistêmico e oral, necessitando de acompanhamento priorizado e precoce.

Orientador: Carlos Amilcar Parada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T20:52:22Z (GMT). No. of bitstreams: 1 Araldi_Dioneia_D.pdf: 2812057 bytes, checksum: ba0136e00ebc1ab271d4e1ed908bcd8c (MD5) Previous issue date: 2012 / Resumo: A liberação de Interleucina-1? (IL-1?) no tecido periférico estimula a síntese de Prostaglandinas (PGs), especialmente, da Prostaglandina-E2 (PGE2), que leva a sensibilização dos nociceptores aferentes primários induzindo a hiperalgesia inflamatória. Recentemente demonstramos que a IL-1? pode ativar diretamente o receptor de Interleucina-1 (IL-1R) do nociceptor aferente periférico e levar a liberação de PGE2 associada ao desenvolvimento da hiperalgesia. A IL-1? também é liberada no Gânglio da Raiz Dorsal (GRD), entretanto a função que a IL-1? desempenha no GRD para o desenvolvimento da hiperalgesia inflamatória ainda não está clara. Portanto, o objetivo deste estudo foi investigar se a liberação de IL-1? e a ativação do Receptor de Interleucina-1 Tipo I (IL-1RI) no GRD estão envolvidos no desenvolvimento da hiperalgesia inflamatória. A administração de IL-1Ra (antagonista natural de receptor IL- 1, 6 ?g) no GRD de ratos preveniu a hiperalgesia mecânica (avaliada por meio do von Frey Eletrônico) induzida pela administração intraplantar (i.pl) de Adjuvante Completo de Freund (CFA, 100 ?L), Carragenina (Cg, 100 ?g) ou IL-1? (0,5 pg), mas não pela administração i.pl de PGE2 (100 ng), avaliadas 3 horas após suas administrações. Além disso, a administração i.pl periférica de CFA ou Cg aumentaram as concentrações de IL-1? (avaliadas por ELISA) no GRD. O tratamento ganglionar (GRD-L5) com oligonucleotídeo (ODN) antisense contra IL-1RI (30 ?g/dia durante 4 dias) reduziu de maneira significativa a expressão de IL-1RI no GRD-L5 e a hiperalgesia mecânica induzida por CFA, Cg e IL-1?, mas não pela PGE2, administradas no tecido periférico da pata. Também verificamos a hipótese de que a prévia ativação do receptor neuronal, IL-1RI, no tecido periférico é importante para a liberação de IL-1? no GRD e para a subsequente hiperalgesia induzida por PGE2. A IL-1? (0,5 pg/pata) co-administrada com a dose sub-limiar de PGE2 (10 ng/pata) em patas traseiras tratadas com indometacina induziu uma proeminente hiperalgesia, que foi prevenida pelo prétratamento com ODN antisense contra IL-1RI ou IL-1Ra (6 ?g) administrados no GRD. Além disso, o IL-1Ra reduziu a expressão de COX-2 em células do GRD. Para confirmar a ativação do IL-1RI em células do GRD, administramos Cg ou CFA no tecido periférico o que levou ao aumentou da expressão de IRAK-1 e IRAK-4 em células do GRD. Os resultados deste estudo sugerem que o desenvolvimento da hiperalgesia inflamatória depende da ativação do receptor IL-1RI neuronal no tecido periférico que, em partes, induz a liberação de IL-1? no GRD e subsequente ativação da COX-2. Os dados aqui apresentados oferecem novas perpectivas sobre a participação das células do GRD nos mecanismos envolvidos na hiperalgesia inflamatória e revelam novos e interessantes alvos para o controle das hiperalgesias inflamatórias / Abstract: The release of Interleukin-1? (IL-1?) in the peripheral tissue stimulates the synthesis of Prostaglandins, specially, Prostaglandin-E2 (PGE2) that ultimately sensitize the peripheral afferent nociceptor inducing inflammatory hyperalgesia. We have recently demonstrated that IL-1? can directly activate IL-1R receptor of peripheral afferent nociceptor to induce release of PGE2 associated to development of hyperalgesia. IL-1? is also released in Dorsal Root Ganglion (DRG), however the role that IL-1? in DRG plays to development of inflammatory hyperalgesia is not yet elucidated. Therefore, the aim of this study was to investigate whether IL-1? released in the DRG and the activation of Interleukin-1 Receptor Type I (IL-1RI) is involved in the development of the inflammatory hyperalgesia. Administration of IL-1Ra (IL-1 receptor antagonist, 6 ?g) in the DRG of rats prevented the mechanical hyperalgesia (measured with Electronic von Frey) induced by intraplantar (i.pl) administration of Complete Freund's Adjuvant (CFA, 100 ?L), Carrageenan (Cg, 100 ?g) or IL- 1? (0.5 pg), but not by PGE2 (100 ng), measured 3 hours after their administrations. Also, peripheral i.pl administration of CFA or Cg induced an increase in IL-1? concentrations (measured by ELISA) in the DRG. Ganglionar (DRG-L5) treatment with oligonucleotides (ODN) antisense against IL-1RI (30 ?g/day for four days) reduced the expression of IL-1RI in the DRG-L5 and the mechanical hyperalgesia induced by CFA, Cg, and IL-1?, but not by PGE2 administered in peripheral tissue. We also verified the hypothesis that previous activation of neuronal IL-1RI in the peripheral tissue is important to the release of IL-1? in the DRG and to the subsequent PGE2-induced hyperalgesia. IL-1? (0.5 pg/paw) co-administrated with a sub-threshold dose of PGE2 (10 ng/paw), in hind paws treated with indomethacin, greatly induces hyperalgesia, which was prevented by pre-treatment with ODN antisense against IL-1RI or IL-1Ra (6 ?g) administrated in DRG. Also, IL-1Ra administrations reduced the COX-2 expression in DRG cells. To confirm IL-1RI activation in DRG cells, it was observed that IRAK-1 and IRAK-4 expression was increased in DRG neurons after administration of Cg or CFA in the peripheral tissue. These findings suggest that the development of inflammatory hyperalgesia depends on neuronal IL-RI activation in the peripheral tissue that, in turn, induces the release of IL- 1? in the DRG and subsequent COX-2 activation. These data provide new insights about the participation of DRG cells in the mechanisms underlying inflammatory hyperalgesia and reveal new interesting targets to control inflammatory hyperalgesia / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Au cours de ce travail, nous nous sommes attachés à étudier certaines réponses cellulaires secondaires à l’activation des récepteurs purinergiques P2X dans deux modèles différents, les macrophages péritonéaux et les cellules des glandes sous-maxillaires. Ces cellules contribuent à notre immunité innée, soit tournée vers l’intérieur (macrophages), soit vers l’extérieur (glandes sous-maxillaires).<p><p>Nous avons dans un premier temps confirmé par Western blot et par des dosages de la concentration intracellulaire de calcium ([Ca2+]i) que les deux types de cellules étudiés expriment des récepteurs P2X4 et P2X7 fonctionnels.<p><p>Nous nous sommes alors concentrés sur deux réponses impliquées dans la protection de l’hôte contre les agressions et l’élimination de pathogènes :la production d’espèces réactives de l’oxygène (ROS) ainsi que la sécrétion de la cytokine pro-inflammatoire interleukine-1beta (IL-1beta). Nos résultats montrent que la production de ROS en réponse à l’ATP extracellulaire est secondaire à l’activation d’une NADPH oxydase dans les deux types de cellules. Cette réponse est médiée par les récepteurs P2X7 ainsi que, dans les macrophages, par d’autres récepteurs purinergiques comme par exemple les récepteurs P2X4 et des récepteurs P2Y. Dans les glandes exocrines, contrairement aux macrophages, la protéine kinase C ainsi que ERK1/2 interviennent dans l’activation de la NADPH oxydase. <p><p>Par la suite nous avons comparé la régulation de l’expression et de la sécrétion d’IL-1beta par les macrophages et les glandes sous-maxillaires. Nous avons observé que l’IL-1beta est présente dans la salive collectée chez des souris injectées par de la pilocarpine. Des analyses par ELISA, RT-PCR et Western blot montrent que la cytokine est exprimée de manière constitutive par les cellules acineuses et ductales des glandes sous-maxillaires, à un niveau plus élevé que dans les macrophages. Contrairement aux cellules phagocytaires, l’expression de la cytokine dans les cellules des glandes salivaires n’est pas augmentée suite à la stimulation par des lipopolysaccharides. De même, dans ces cellules l’ATP n’a pas provoqué la sécrétion d’IL-1beta malgré l’efflux de K+ secondaire à l’activation des récepteurs P2X7. <p><p>Dans une dernière série d’expériences nous avons évalué les effets du peptide antimicrobien CRAMP sur les macrophages murins. Le CRAMP a inhibé toutes les réponses secondaires à l’activation des récepteurs P2X7 (ouverture du canal cationique, formation de pore, production de ROS, libération d’IL-1beta, d’acide oléique et de lactate déshydrogénase). L’inhibition par le CRAMP de l’augmentation de la [Ca2+]i en réponse à l’ATP n’était pas médiée par les récepteurs aux peptides formylés car les agonistes de ces récepteurs n’ont pas bloqué cette augmentation. Le CRAMP n’a pas eu d’effet sur l’augmentation de la [Ca2+]i secondaire à l’activation des récepteurs P2X4 par une combinaison d’ATP et d’ivermectine.<p><p>Nos expériences ont révélé que les récepteurs P2X7 sont couplés à diverses voies de signalisation dans les macrophages et dans les glandes exocrines. Les voies activées diffèrent en fonction du type de cellules. Nous avons également conclu que les peptides antimicrobiens de la famille de cathélicidines ne sont pas des agonistes universels des récepteurs P2X7.<p><p><p><p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Sterile particles underlie the pathogenesis of numerous inflammatory diseases. These diseases can often become chronic and debilitating. Moreover, they are common, and include silicosis (silica), asbestosis (asbestos), gout (monosodium urate), atherosclerosis (cholesterol crystals), and Alzeihmer’s disease (amyloid Aβ). Central to the pathology of these diseases is a repeating cycle of particle-induced cell death and inflammation. Macrophages are the key cellular mediators thought to drive this process, as they are especially sensitive to particle-induced cell death and they are also the dominant producers of the cytokine responsible for much of this inflammation, IL-1β. In response to cytokines or microbial cues, IL-1β is synthesized in an inactive form (pro-IL-1β) and requires an additional signal to be secreted as an active cytokine. Although a multimolecular complex, called the NLRP3 inflammasome, controls the activation/secretion of IL-1β (and has been thought to also control cell death) in response to particles in vitro, the in vivo inflammatory response to particles occurs independently of inflammasomes. Therefore, I sought to better understand the mechanisms governing IL-1β production and cell death in response to particles, focusing specifically on the role of lysosomal cathepsin proteases. Inhibitor studies have suggested that one of these proteases, cathepsin B, plays a role in promoting inflammasome activation subsequent to particle-induced lysosomal damage, however genetic models of cathepsin B deficiency have argued otherwise. Through the use of inhibitors, state-of-the-art biochemical tools, and multi-cathepsin-deficient genetic models, I found that multiple redundant cathepsins promote pro-IL-1β synthesis as well as particle-induced NLRP3 activation and cell death. Importantly, I also found that particle-induced cell death does not depend on inflammasomes, suggesting that this may be why inflammasomes do not contribute to particle-induced inflammation in vivo. Therefore, my observations suggest that cathepsins may be multifaceted therapeutic targets involved in the two key pathological aspects of particle-induced inflammatory disease, IL-1β production and cell death.