No description available.

During the normal menstrual cycle, the human endometrium undergoes extensive tissue remodelling under the influence of ovarian-derived hormones. The endometrium has well defined stromal and epithelial compartments with the former containing both a well-developed vasculature as well as a diverse population of immune cells. Mast cells (MCs) are long-lived tissue resident immune cells characterised by the presence of granules containing proteases. Mast cells have been detected in the human uterus but little is known about their regulation or the impact of steroids on their differentiation status. Recently MCs have been implicated as key players in physiological and pathological pain pathways but little is known about their role in endometrial pathologies. Endometriosis is a chronic incurable condition characterized by the presence of endometrial tissue outside the uterine cavity: women with endometriosis can suffer from a debilitating range of symptoms including chronic pain. Whilst the aetiology of endometriosis is uncertain, close proximity between MCs and nerves has implicated them in aberrant activation of pain pathways. The aims of the current project were: 1. To determine the spatial and temporal location of uterine MCs and to explore their phenotype including expression of steroid receptors. 2. To explore the activation status of MCs in women with endometriosis and/or pain, 3. To explore the use of cells and mice as models to investigate the phenotype of mast cells and their regulation by steroids. Mast cell proteases tryptase and chymase were detected by RTPCR and immunohistochemistry in “full thickness” (uterine lumen to endometrial-myometrial junction) biopsies from women undergoing hysterectomy. In agreement with previous findings MCs were most abundant in the myometrium. Uterine MCs were predominantly of the classical MC subtypes: tryptasepos/chymaseneg and tryptasepos/chymasepos but a rare third subtype was also identified as tryptaseneg/chymasepos. Mast cell activation/degranulation was cycle stage dependent and for the first time their steroid receptor phenotype was identified as ERαneg/ERβpos/GRpos, suggesting potential regulation by the uterine steroid microenvironment. Studies on tissue samples from women with endometriosis revealed MCs with an altered activation status in the pelvic peritoneal wall, compared to controls, which showed an intense diffuse immunoexpression of chymase suggestive of MC activation and release of this protease during normal physiology of the peritoneum. Surprisingly, analysis of peritoneal fluids from controls, women with pain but no endometriosis, and pain with endometriosis did not detect differences in numbers of MCs or concentrations of tryptase or chymase. Analysis of peritoneal biopsies also provided the first evidence for a striking increase in immunoexpression of PAR-2, a protease-activated receptor, in women suffering from chronic pelvic pain and/or endometriosis which may provide a mechanism by which mast cell derived factors may alter pain pathways. Studies in a mouse model of endometriosis identified MCs within endometria-llike lesions and offer a platform for future studies. In vitro explorations using MCs derived from peripheral blood precursors and HMC-1, a cell line derived from a patient with MC leukaemia confirmed expression of ERβ but did not support previous studies claiming cells were ERαpos. In summary, this study has provided novel insights into the phenotype of endometrial mast cells in the normal cycling endometrium and contrasted them with those in women with endometriosis and pelvic pain. This is the first study to identify MCs as ERβpos. Further studies are required to determine whether inhibition of PAR- 2 might offer a therapeutic target in women with chronic pelvic pain.

Pesquisas recentes investigando o receptor ativado por protease do tipo 2 (PAR-2) sugerem uma associação entre este receptor e a inflamação periodontal. Além disso, é sabido que a gingipaína, protease bacteriana secretada por um importante periodontopatógeno, Porphyromonas gingivalis (Pg), tem a capacidade de ativar o PAR-2. Ademais, um estudo anterior do grupo, verificou que quanto mais profunda a bolsa periodontal, maior era a expressão do receptor PAR-2. No entanto, não é sabido se a expressão de PAR-2 é proporcional a severidade de doença periodontal e a quantidade de gingipaína expressa na bolsa periodontal. Desta forma, o presente estudo, verificou no fluido gengival a correlação entre a expressão gênica de PAR-2 (Real Time-PCR) com os parâmetros clínicos periodontais, e a expressão gênica da protease gingipaína em pacientes com periodontite crônica severa e moderada, antes e após o tratamento periodontal não-cirúrgico. A expressão de PAR-2 e da protease gingipaína foi estatisticamente maior nos pacientes do grupo periodontite crônica severa (PS) em comparação com os pacientes do grupo periodontite crônica modera (PM) e controle (C). Além disso, o tratamento periodontal levou à redução significativa (p<0.05) da expressão de PAR-2 nos pacientes com periodontite crônica moderada. Em conclusão, dentro dos limites do presente estudo, nós demonstramos que a severidade da doença periodontal e a expressão de gingipaína influenciaram a expressão de PAR-2 no fluido gengival de pacientes com periodontite crônica. / Recent studies investigating the protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. In addition, it is known that gingipain, a bacterial protease secreted by an important periodontopathogen, Porphyromonas gingivalis (Pg), has the ability to activate PAR- 2. Furthermore, a previous study from our group found that the deeper the periodontal pocket, the higher the expression of the PAR-2 receptor. However, it is not known whether the expression of PAR-2 is associated to the severity of periodontal disease and the amount of gingipain expressed in the periodontal pocket. Thus, the present study verified, in the gingival fluid, the correlation between the PAR-2 gene expression (Real Time-PCR) with the clinical periodontal parameters, and the gene expression of the gingipain protease in patients with moderate and severe chronic periodontitis before and after non-surgical periodontal treatment. PAR-2 expression and gingipain protease were statistically more expressed in patients of the severe chronic periodontitis group (PS) compared with those in the moderate chronic periodontitis group (PM) and control group (C). Furthermore, periodontal treatment led to a significant reduction (p <0.05) in the expression of PAR-2 in patients with moderate chronic periodontitis. In conclusion, within the limits of the present study, we demonstrated that the severity of periodontal disease and the expression of gingipain influenced the PAR-2 expression in the gingival fluid of patients with chronic periodontitis.

Pesquisas recentes investigando o receptor ativado por protease do tipo 2 (PAR-2) sugerem uma associação entre este receptor e a inflamação periodontal. Além disso, é sabido que a gingipaína, protease bacteriana secretada por um importante periodontopatógeno, Porphyromonas gingivalis (Pg), tem a capacidade de ativar o PAR-2. Ademais, um estudo anterior do grupo, verificou que quanto mais profunda a bolsa periodontal, maior era a expressão do receptor PAR-2. No entanto, não é sabido se a expressão de PAR-2 é proporcional a severidade de doença periodontal e a quantidade de gingipaína expressa na bolsa periodontal. Desta forma, o presente estudo, verificou no fluido gengival a correlação entre a expressão gênica de PAR-2 (Real Time-PCR) com os parâmetros clínicos periodontais, e a expressão gênica da protease gingipaína em pacientes com periodontite crônica severa e moderada, antes e após o tratamento periodontal não-cirúrgico. A expressão de PAR-2 e da protease gingipaína foi estatisticamente maior nos pacientes do grupo periodontite crônica severa (PS) em comparação com os pacientes do grupo periodontite crônica modera (PM) e controle (C). Além disso, o tratamento periodontal levou à redução significativa (p<0.05) da expressão de PAR-2 nos pacientes com periodontite crônica moderada. Em conclusão, dentro dos limites do presente estudo, nós demonstramos que a severidade da doença periodontal e a expressão de gingipaína influenciaram a expressão de PAR-2 no fluido gengival de pacientes com periodontite crônica. / Recent studies investigating the protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. In addition, it is known that gingipain, a bacterial protease secreted by an important periodontopathogen, Porphyromonas gingivalis (Pg), has the ability to activate PAR- 2. Furthermore, a previous study from our group found that the deeper the periodontal pocket, the higher the expression of the PAR-2 receptor. However, it is not known whether the expression of PAR-2 is associated to the severity of periodontal disease and the amount of gingipain expressed in the periodontal pocket. Thus, the present study verified, in the gingival fluid, the correlation between the PAR-2 gene expression (Real Time-PCR) with the clinical periodontal parameters, and the gene expression of the gingipain protease in patients with moderate and severe chronic periodontitis before and after non-surgical periodontal treatment. PAR-2 expression and gingipain protease were statistically more expressed in patients of the severe chronic periodontitis group (PS) compared with those in the moderate chronic periodontitis group (PM) and control group (C). Furthermore, periodontal treatment led to a significant reduction (p <0.05) in the expression of PAR-2 in patients with moderate chronic periodontitis. In conclusion, within the limits of the present study, we demonstrated that the severity of periodontal disease and the expression of gingipain influenced the PAR-2 expression in the gingival fluid of patients with chronic periodontitis.

La cellule musculaire lisse (CML) a un rôle pivot dans la physiopathologie de l’asthme. Dans ce travail de thèse nous avons pu mettre en avant l’implication du récepteur de type 2 activé par les protéases (PAR-2) dans une composante majeure du remodelage bronchique : la prolifération musculaire lisse. Dans le premier travail, nous avons montré une augmentation de l’expression du PAR-2 au niveau des CML bronchiques d’asthmatiques in vitro. La réponse calcique est dépendante du niveau d’expression du récepteur, mais n’influence pas la réponse proliférante. La stimulation répétée du PAR-2 augmente la prolifération des seules CML d’asthmatiques, par un mécanisme dépendant de la voie ERK. Dans le second travail, nous avons montré que la production basale d’un épithélium reconstitué entraine une prolifération plus importante des CML d’asthmatiques comparée aux CML de témoins. Une augmentation supplémentaire de la prolifération des seules CML d’asthmatiques a été observée, après activation par le surnageant d’épithélium stimulé par des acariens de maison comparé au surnageant épithélial non stimulé. Ce mécanisme est dépendant du PAR-2 épithélial, qui induit la production de leucotriènes C4, sur des CML dont l’expression du récepteur (CysLTR1) est augmentée chez l’asthmatique. Ces résultats apportent de nouvelles connaissances dans le remodelage musculaire lisse bronchique de l’asthmatique et met en avant le PAR-2 comme cible thérapeutique potentielle. / Smooth muscle cells (SMC) play an important role in asthma pathophysiology. In this thesis, we have highlighted the involvement of protease activated receptor type-2 (PAR-2) in SMC proliferation, which is a major component of airway remodeling. In the first study, we have shown an increased expression of PAR-2 in asthmatic bronchial SMC in vitro. Calcium response is dependent on the expression level of PAR-2, which does not affect the proliferative response. Repeated stimulation of PAR-2 increases the proliferation of asthmatics SMC only, by an ERK-dependent mechanism. In the second study, we have demonstrated that the basal production of reconstituted epithelium leads to a greater proliferation of asthmatics SMC compared to controls. Increased proliferation of asthmatics SMC only was observed, after stimulation with supernatant of the epithelium stimulated by house dust mites (HDM) compared to unstimulated epithelial supernatant. This mechanism is epithelial PAR-2-dependent, which induces the production of leukotrienes C4, whose receptor expression (CysLTR1) is increased in asthmatics SMC. These results provide new insights into bronchial smooth muscle remodeling in asthma and highlights the PAR-2 as a potential therapeutic target.

Objetivo: o presente estudo prospectivo avaliou a presença de microrganismos periodontopatogênicos, os níveis salivares de arginase e de HBD-2 em indivíduos com gengivite e periodontite tendo como controle indivíduos periodontalmente saudáveis, correlacionando-os aos respectivos parâmetros clínicos. Também foi avaliada expressão gênica do PAR2 crevicular em indivíduos saudáveis e com periodontite. Método: Inicialmente, foram avaliados 89 indivíduos sem doenças sistêmicas, nunca fumantes, sendo 31 saudáveis (média de idade 25,06 5,97), 27 com gengivite (média de idade 33,22 12,09) e 31 com periodontite (média de idade 52,16 11,54), todos submetidos à terapia periodontal não cirúrgica. Coleta salivar para avaliação dos níveis de arginase (quantificada por meio de espectrofotometria) foi realizada no início do tratamento em todos os indivíduos, e naqueles com gengivite e periodontite respectivamente em 30 e 50 dias pós-tratamento. Avaliação clínica de profundidade de sondagem, perda de inserção clínica e índices de placa e gengival e microbiana (Campylobacter rectus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia, Treponema denticola e Prevotella intermedia) foram também avaliados nos mesmos tempos. Dentre os 89 indivíduos, amostras do fluido gengival foram coletadas em 10 indivíduos saudáveis e 10 com periodontite para mensuração da atividade do PAR2 crevicular por meio de RT-PCR. Como esta relação foi positiva, foi verificada a quantidade de HBD-2 salivar por meio de ELISA de todos os 89 indivíduos e sua relação com os parâmetros clínicos e microbiológicos. A significância de todas as relações e quantificações foi analisada por meio de testes estatísticos apropriados. Resultados: foi observada uma melhora estatisticamente significativa dos parâmetros clínicos e microbianos após o tratamento periodontal. A arginase salivar estava significativamente mais elevada nos indivíduos com periodontite em relação àqueles com gengivite, e nestes em relação aos saudáveis. O tratamento periodontal promoveu melhora dos indivíduos doentes, cujos parâmetros avaliados tornaram-se estatisticamente semelhantes aos dos saudáveis. Houve maior atividade do PAR2 nos 10 indivíduos com periodontite em relação aos saudáveis e, após o tratamento, houve uma redução estatisticamente significativa deste parâmetro. Por fim, foram observados níveis estatisticamente mais elevados de HBD-2 salivar nos indivíduos com periodontite comparados àqueles com gengivite e aos saudáveis. Não foi possível observar uma correlação entre HBD-2 salivar e os microrganismos analisados. Conclusões: com base nos resultados observados, podemos concluir que: a arginase salivar está significativamente aumentada nos indivíduos periodontalmente comprometidos em relação aos saudáveis; o tratamento periodontal promoveu melhora dos indivíduos doentes em relação aos parâmetros avaliados; indivíduos com periodontite têm maior expressão gênica do PAR2 do que aqueles saudáveis e o tratamento tornou esta expressão semelhante nos dois grupos; indivíduos com periodontite têm níveis estatisticamente mais significativos de HBD-2 salivar do que aqueles saudáveis e aqueles com gengivite; a saliva parece ser uma ferramenta útil para o diagnóstico periodontal e para o monitoramento da eficácia do tratamento periodontal. / Objectives: This prospective study evaluated the presence of periodontopathogenics microorganisms, as it also examined the salivary levels of arginase and HBD-2 from subjects with gingivitis and periodontitis and periodontally healthy subjects as controls, correlating them to relevant clinical parameters. The gene expression of PAR2 crevicular in healthy subjects and periodontitis was also assessed. Methods: Initially, 89 individuals without systemic diseases, who were never smokers, were evaluated. Out of the 89, 31 were healthy subjects (average age 25.06 5.97), 27 have gingivitis (average age 33.22 12.09) and 31 were with periodontitis (average age 52.16 11.54), all underwent nonsurgical periodontal therapy. Saliva was collected for assessing levels of arginase at baseline in all subjects (quantified by spectrophotometry), and repeated on those with gingivitis and periodontitis respectively at 30 and 50 days post treatment. Clinical evaluation of probing depth, clinical attachment loss, both plaque and gingival index as well as microbial evaluation (Campylobacter rectus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia, Prevotella intermedia and Treponema denticola) were also assessed at the same time. Among the 89 individuals, gingival fluid samples were collected from 10 healthy ones and from 10 with periodontitis to measure crevicular PAR2 activity by RT-PCR. As the results came out positive, the amount of HBD-2 salivary was tested by ELISA for all 89 subjects assessing its relationship with clinical and microbiological parameters. The significance of all relationships and quantifications were analyzed using appropriate statistical tests. Results: we observed a statistically significant improvement of clinical and microbial parameters after periodontal treatment. Salivary arginase was significantly higher in subjects with periodontitis than in those with gingivitis, and those with gingivitis had higher results than the healthy ones. Periodontal treatment promoted improvement of the non-healthy individuals whose parameters became statistically similar to the healthy ones. There was a greater PAR2 activity in 10 individuals with periodontal disease compared to healthy ones, and after treatment the results showed a statistically significant reduction in this parameter. Finally, we observed statistically higher levels of salivary HBD-2 in individuals with periodontitis compared to both those with gingivitis and those individuals in the healthy group. It has not been possible to observe a correlation between HBD-2 and salivary microorganisms analyzed. Conclusion: Based on the observed results, we can conclude that salivary arginase is significantly increased in periodontally compromised individuals relative to the healthy ones; periodontal treatment promoted improvement of individuals in relation to the assessed parameters; individuals with periodontitis have higher gene expression of PAR2 than those healthy and the periodontal treatment brought similar results to both groups; individuals with periodontitis have statistically more significant levels of salivary HBD-2 than those with healthy gums and gingivitis; and, finally, saliva, besides being useful for periodontal diagnosis, appears to be also helpful for monitoring efficacy of periodontal treatment.

Le déclin de la fonction respiratoire dans l’asthme est associé à une augmentation de la massedu muscle lisse bronchique. La cellule musculaire lisse (CML) bronchique joue un rôle central dans la physiopathologie de l'asthme. Elle participe à l'inflammation et constitue une composante importante du remodelage des voies aériennes. Récemment, le rôle de la chitinaseYKL-40 comme bio marqueur de ce remodelage dans l'asthme a été évoqué. Dans ce contexte, la question centrale ayant motivé une partie de ce travail de thèse, a été d'étudier les effets d'YKL-40 sur différentes propriétés des CML. Nous nous sommes également intéressés au récepteur activé par les protéases, de type 2 (PAR-2), potentiel récepteur d'YKL-40, mais aussi acteur principal de l’inflammation bronchique.Ce travail a permis d'établir qu'YKL-40 est plus qu'un simple bio marqueur dans l'asthme puisqu’elle altère les propriétés physiologiques de la CML, et semble jouer un rôle dans le remodelage musculaire lisse bronchique. Par ailleurs, ce travail a également permis de mettre en évidence une surexpression du PAR-2 dans les CML d’asthmatiques ainsi qu’une augmentation de son expression et de la prolifération cellulaire, en réponse à une stimulation chronique. Ce travail a enfin permis d’optimiser la technique de l’interférence ARN lentivirale dans les CML bronchiques humaines. / The decline in lung function in asthma is associated with an increased bronchial smooth muscle (BSM) mass. BSM cells play a central role in the pathophysiology of asthma. They are involved in inflammation and are an important component of airway remodelling. Recently, the role of the chitinase YKL-40 as biomarker of this remodelling in asthma has been evoked. In this context, the central question that motivated part of this thesis was to study the effects of YKL-40 on various properties of BSM cells. We also studied the protease activated receptor 2 (PAR-2), as a potential receptor of YKL-40, as well as an important actor of airway inflammation.This work has established that YKL-40 is more than just a biomarker for asthma since YKL-40 alters physiological properties of BSM cells and appears to play a role in BSM remodelling. Moreover, this work has also highlighted an overexpression of PAR-2 in asthmatic BSM cells, as well as an increase of both PAR-2 expression and BSM cell proliferation in response to chronic stimulation. This work has finally allowed us to optimize lentiviral RNA interference in human BSM cells.

Neste trabalho investigamos o efeito de doadores de H2S no prurido agudo mediado por PAR-2 em camundongos. A injeção i.d. do agonista PAR-2 SLIGRL-NH2, induziu prurido que não foi afetado pelo pré-tratamento com o antagonista H1 pirilamina. A coinjeção dos doadores de H2S GYY4137 (lento) ou NaHS (espontâneo) com SLIGRL-NH2 reduziu significativamente o prurido (P<0,05). A glibenclamida (bloqueador de canais KATP) e o SNP (doador de NO), mas não o ODQ (inibidor da sGC), evitaram estes efeitos. O antagonista TRPA1 HC-030031 reduziu significativamente o prurido induzido pelo SLIGRL-NH2 (P<0,05), mas o prurido induzido pelo agonista TPRA1 AITC não foi afetado por NaHS. Ensaios de Western blot mostraram que ambos PAR-2 e TRPA1 são expressos constitutivamente na pele de camundongos. Nossos dados mostram que o prurido secundário à ativação do PAR-2 pode ser reduzido por H2S, atuando via a abertura dos canais KATP e ativação da via NO-GMPc. Ademais, o receptor TRPA1 pode mediar o prurido induzido por SLIGRL-NH2, mas o H2S não interfere nesta via. / In this study we investigated the effect of H2S donors in PAR-2-mediated acute pruritus in mice. The i.d. injection of the PAR-2 agonist SLIGRL-NH2 induced itching that was unaffected by pre-treatment with the H1 antagonist pyrilamine. Co-injection of the H2S donors GYY4137 (slow) or NaHS (spontaneous) with SLIGRL-NH2 significantly reduced pruritis (P <0.05). Glibenclamide (a KATP channel blocker) and SNP (a NO donor), but not ODQ (a sGC inhibitor) prevented these effects. The TRPA1 antagonist HC-030031 significantly reduced SLIGRL-NH2-induced pruritus (P<0.05), but the pruritus induced by the TPRA1 agonist AITC was unaffected by NaHS. Western blot assays showed that both TRPA1 and PAR-2 are constitutively expressed in the mouse skin. Our data show that itching secondary to PAR-2 activation can be reduced by H2S which acts via the opening of KATP channels and activation of the NO-cGMP pathway. Furthermore, TRPA1 receptors may mediate SLIGRL-NH2-induced pruritus, however, H2S does not interfere with this pathway.

Neste trabalho investigamos o efeito de doadores de H2S no prurido agudo mediado por PAR-2 em camundongos. A injeção i.d. do agonista PAR-2 SLIGRL-NH2, induziu prurido que não foi afetado pelo pré-tratamento com o antagonista H1 pirilamina. A coinjeção dos doadores de H2S GYY4137 (lento) ou NaHS (espontâneo) com SLIGRL-NH2 reduziu significativamente o prurido (P<0,05). A glibenclamida (bloqueador de canais KATP) e o SNP (doador de NO), mas não o ODQ (inibidor da sGC), evitaram estes efeitos. O antagonista TRPA1 HC-030031 reduziu significativamente o prurido induzido pelo SLIGRL-NH2 (P<0,05), mas o prurido induzido pelo agonista TPRA1 AITC não foi afetado por NaHS. Ensaios de Western blot mostraram que ambos PAR-2 e TRPA1 são expressos constitutivamente na pele de camundongos. Nossos dados mostram que o prurido secundário à ativação do PAR-2 pode ser reduzido por H2S, atuando via a abertura dos canais KATP e ativação da via NO-GMPc. Ademais, o receptor TRPA1 pode mediar o prurido induzido por SLIGRL-NH2, mas o H2S não interfere nesta via. / In this study we investigated the effect of H2S donors in PAR-2-mediated acute pruritus in mice. The i.d. injection of the PAR-2 agonist SLIGRL-NH2 induced itching that was unaffected by pre-treatment with the H1 antagonist pyrilamine. Co-injection of the H2S donors GYY4137 (slow) or NaHS (spontaneous) with SLIGRL-NH2 significantly reduced pruritis (P <0.05). Glibenclamide (a KATP channel blocker) and SNP (a NO donor), but not ODQ (a sGC inhibitor) prevented these effects. The TRPA1 antagonist HC-030031 significantly reduced SLIGRL-NH2-induced pruritus (P<0.05), but the pruritus induced by the TPRA1 agonist AITC was unaffected by NaHS. Western blot assays showed that both TRPA1 and PAR-2 are constitutively expressed in the mouse skin. Our data show that itching secondary to PAR-2 activation can be reduced by H2S which acts via the opening of KATP channels and activation of the NO-cGMP pathway. Furthermore, TRPA1 receptors may mediate SLIGRL-NH2-induced pruritus, however, H2S does not interfere with this pathway.

L’inflammation neurogène cutanée (INC) est une inflammation de la peau induite par l’activation des fibres nerveuses intra-épidermiques qui secrètent des neuropeptides tels que la substance P (SP). L’INC est impliquée dans des dermatoses inflammatoires prurigineuses comme le psoriasis, la dermatite atopique (DA) et le syndrome de Netherton (SN). Un nouveau concept émerge, suggérant que les kératinocytes sont également des acteurs majeurs de l’INC. Le récepteur activé par des protéases de type 2 (PAR-2) est fortement incriminé dans l’INC associée à ces dermatoses, ce qui permet de comprendre les voies du prurit non-histaminergique. Les enjeux thérapeutiques sont de taille puisqu’il n’existe actuellement aucun traitement efficace permettant la prise en charge spécifique du prurit histamino-indépendant au cours des dermatoses prurigineuses associées à l’INC.Bien que le rôle de PAR-2 dans la sécrétion de neuropeptides à partir des neurones sensoriels soit clairement établi, son implication dans la modulation de gènes pouvant contribuer à l’entretien ou l’amplification de l’INC reste méconnue. Le rôle inflammatoire de PAR-2 a également été démontré sur des kératinocytes cultivés en monocouche via la sécrétion de cytokines par des mécanismes dépendants du Ca2+. La surexpression de PAR-2 et la perte d’expression de certains canaux calciques impliqués dans sa réponse calcique dans les kératinocytes différenciés suggèrent des mécanismes d’action de PAR-2 différents pour ceux-ci. Dans le but d’étudier le rôle pro-inflammatoire de PAR-2 au cours des dermatoses prurigineuses, nous avons analysé l’effet de son activation sur des monocultures de neurones sensoriels issus de ganglions rachidiens dorsaux (GRD) de rat et de kératinocytes humains différenciés (DhPK), en criblant l’expression de médiateurs de l’inflammation. Pour approfondir, les voies calciques de PAR-2 sous-jacente à la modulation d’expression dans les kératinocytes différenciés, des expériences d’imagerie calcique ont été réalisées et différents antagonistes ont été utilisés pour analyser les acteurs impliqués.Dans le cadre d’un partenariat avec les laboratoires dermatologiques d’Uriage, nous avons testé les effets de l’eau thermale d’Uriage sur la modulation de gènes induite par PAR-2 dans les DhPK. Nous avons également utilisé une lignée de PC12 différenciables en neurones par le NGF afin de les utiliser comme alternatives des neurones sensoriels issus des GRD de rat pour l’étude de l’INC.L’ensemble des résultats obtenus au cours du criblage des gènes modules par PAR-2 confirme le rôle pro-inflammatoire de PAR-2 dans les neurones sensoriels de rat et dans les DhPK. La découverte d’une nouvelle voie calcique de PAR-2 dans les DhPK offre de nouvelles pistes thérapeutiques pour les dermatoses prurigineuses telles que le psoriasis, la DA et le NS. Les résultats obtenus avec l’eau thermale d’Uriage peuvent présenter une perspective thérapeutique pour les patients souffrants de dermatoses prurigineuses réfractaires aux traitements conventionnels. L’utilisation d’une lignée neuronale comme lesPC12 pour l’étude de l’INC serait une alternative utile dans le développement des tests cosmétiques avec les industriels pour notre laboratoire. / Cutaneous neurogenic inflammation (CNI) is an inflammation of the skin induced by the activation of intraepidermal nerve fibers that release neuropeptides such as substance P (SP). CNI is involved in pruritic inflammatory skin disorders such as psoriasis, atopic dermatitis (AD) and Netherton syndrome (NS). A new concept is growing, suggesting that keratinocytes could also trigger INC. The proteases activated receptor 2 (PAR-2) is strongly incriminated in CNI associated with these dermatoses, which allow to understand the histamine-independent itching pathways. The therapeutic stakes are high since there is currently no effective treatment allowing the specific management of histamine-independent pruritus during skin disorders associated with CNI.Although the role of PAR-2 in the secretion of neuropeptides from sensory neurons is clearly established, its involvement in the modulation of genes involved in the maintenance or amplification of CNI remains unknown. The inflammatory role of PAR-2 on keratinocytes has also been demonstrated through the production of cytokines in a Ca2+-dependent mechanisms. The overexpression of PAR-2 and the loss of ORAI1 expression, a calcium channel following keratinocytes differentiation suggest different signaling pathways downstream to PAR-2 activation between undifferentiated and differentiated keratinocytes.In order to study the pro-inflammatory role of PAR-2 during pruritic dermatoses, we analyzed the effect of its activation on rat primary sensory neurons from dorsal spinal ganglia (DRG) and on differentiated human primary keratinocytes (DhPK) by screening the expression of inflammatory mediators. To deepen the Ca2+ pathways underlying PAR-2-mediated inflammatory mediator modulation in DhPK, we performed Ca2+ imaging experiments and different antagonists were used to analyze the involvement of intracellular actors. In a partnership with the dermatological laboratories of Uriage, we tested the effects of Uriage thermal water on PAR-2-induced gene modulation in DhPK. We also used a PC12 cell line differentiable in neurons by the NGF in order to use them as alternatives of rat primary sensory neurons from DRG for the study of INC. We also used a PC12 cell line differentiable in neurons by the NGF use them as alternatives of rat primary sensory neurons from DRG for the study of INC.The results obtained during the screening of the PAR-2-modulated genes confirmed the proinflammatory role of PAR-2 in rat primary sensory neurons and in DhPK. The discovery of a new PAR-2-mediated Ca2+ pathway in DhPK offers new therapeutic pathways for pruritic dermatoses such as psoriasis, AD and NS. The results obtained with the thermal water of Uriage can present a therapeutic perspective for patients suffering from pruritic dermatoses refractory to conventional treatments. The use of a neuronal cell line as the PC12 for the study of INC would be an useful alternative in the development of cosmetic tests.