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Participação do Nlrp12 na diferenciação de linfócitos Th17 e no desenvolvimento da artrite experimental / Role of Nlrp12 on Th17 differentiation and experimental arthritis developmentDouglas da Silva Prado 28 January 2016 (has links)
A Artrite reumatoide é uma doença autoimune que acomete cerca de 1% da população mundial adulta, sendo caracterizada pela participação de linfócitos Th17 no seu desenvolvimento. Na busca por novos alvos terapêuticos e pela compreensão da fisiopatologia, se destacam os inflamassomas que são plataformas proteicas, caracterizados pela produção de citocinas pró-inflamatórias por células do sistema imune inato. De forma interessante, foi demonstrado que linfócitos T CD4 também expressam alguns sensores dessas plataformas, como o Nlrp12. Adicionalmente, este sensor é responsável pela modulação negativa do NF-?B, demonstrando outra característica atípica em relação aos outros inflamassomas. Nesse sentido, foi avaliada a participação do Nlrp12 no desenvolvimento da artrite experimental e na diferenciação linfócitos Th17. Foi verificado nesse estudo que o Nlrp12 é regulado positivamente durante o desenvolvimento da artrite experimental, sendo um modulador negativo desse processo. Isso se deve a uma redução na resposta inflamatória inata do modelo e pela modulação negativa na resposta Th17. Nesse sentido, o controle da resposta Th17 e o desenvolvimento da artrite experimental ocorre por um mecanismo dependente da fosforilação do fator de transcrição Stat3, que é crítico na diferenciação de linfócitos Th17. Desta forma, este estudo demonstra uma nova função para o sensor Nlrp12 no desenvolvimento da artrite experimental, por modular a resposta imune adaptativa de forma direta nos linfócitos T CD4 / Rheumatoid Arthritis is an autoimmune disease that occurs in approximately 1% of the adult population worldwide, with critical role of Th17 cells in your development. In the search for new therapeutic targets and the understanding of the pathophysiology, there are inflammasomes which are protein platforms, characterized through pro-inflammatory cytokines production by innate immune system cells. Interestingly, it was demonstrated that CD4 T cells express some inflammasome sensors, as Nlrp12. Additionally, this sensor is responsible for downregulation of NF-?B, showing another atypical feature in relation to other inflammasomes. Thereby, it was evaluated the role of Nlrp12 on experimental arthritis development and Th17 differentiation. It was found in this study that Nlrp12 is upregulated during experimental arthritis development, working as negative regulator of this process. Thus, Nlrp12 downregulates innate inflammatory response from experimental model and Th17 response. Therefore, experimental arthritis development and Th17 differentiation control occurs in a Stat3 phosphorylation dependente-manne, which is a critical transcription factor on Th17 differentiation. Thus, this study demonstrates a new function for Nlrp12 on experimental arthritis development, by directly to modulate adaptive immune response in CD4 cells
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Participação do Nlrp12 na diferenciação de linfócitos Th17 e no desenvolvimento da artrite experimental / Role of Nlrp12 on Th17 differentiation and experimental arthritis developmentPrado, Douglas da Silva 28 January 2016 (has links)
A Artrite reumatoide é uma doença autoimune que acomete cerca de 1% da população mundial adulta, sendo caracterizada pela participação de linfócitos Th17 no seu desenvolvimento. Na busca por novos alvos terapêuticos e pela compreensão da fisiopatologia, se destacam os inflamassomas que são plataformas proteicas, caracterizados pela produção de citocinas pró-inflamatórias por células do sistema imune inato. De forma interessante, foi demonstrado que linfócitos T CD4 também expressam alguns sensores dessas plataformas, como o Nlrp12. Adicionalmente, este sensor é responsável pela modulação negativa do NF-?B, demonstrando outra característica atípica em relação aos outros inflamassomas. Nesse sentido, foi avaliada a participação do Nlrp12 no desenvolvimento da artrite experimental e na diferenciação linfócitos Th17. Foi verificado nesse estudo que o Nlrp12 é regulado positivamente durante o desenvolvimento da artrite experimental, sendo um modulador negativo desse processo. Isso se deve a uma redução na resposta inflamatória inata do modelo e pela modulação negativa na resposta Th17. Nesse sentido, o controle da resposta Th17 e o desenvolvimento da artrite experimental ocorre por um mecanismo dependente da fosforilação do fator de transcrição Stat3, que é crítico na diferenciação de linfócitos Th17. Desta forma, este estudo demonstra uma nova função para o sensor Nlrp12 no desenvolvimento da artrite experimental, por modular a resposta imune adaptativa de forma direta nos linfócitos T CD4 / Rheumatoid Arthritis is an autoimmune disease that occurs in approximately 1% of the adult population worldwide, with critical role of Th17 cells in your development. In the search for new therapeutic targets and the understanding of the pathophysiology, there are inflammasomes which are protein platforms, characterized through pro-inflammatory cytokines production by innate immune system cells. Interestingly, it was demonstrated that CD4 T cells express some inflammasome sensors, as Nlrp12. Additionally, this sensor is responsible for downregulation of NF-?B, showing another atypical feature in relation to other inflammasomes. Thereby, it was evaluated the role of Nlrp12 on experimental arthritis development and Th17 differentiation. It was found in this study that Nlrp12 is upregulated during experimental arthritis development, working as negative regulator of this process. Thus, Nlrp12 downregulates innate inflammatory response from experimental model and Th17 response. Therefore, experimental arthritis development and Th17 differentiation control occurs in a Stat3 phosphorylation dependente-manne, which is a critical transcription factor on Th17 differentiation. Thus, this study demonstrates a new function for Nlrp12 on experimental arthritis development, by directly to modulate adaptive immune response in CD4 cells
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The Nod-like receptor, Nlrp12, plays an anti-inflammatory role in experimental autoimmune encephalomyelitis / Le récepteur Nod-like, Nlrp12, joue un rôle anti-inflammatoire dans l’encéphalopathie expérimentale autoimmuneMohammadi Mahvelati, Tara January 2017 (has links)
Abstract : Multiple Sclerosis (MS) is an organ-specific autoimmune disease characterized by the presence of demyelinating plaques throughout the central nervous system (CNS) as a result of an abnormal inflammatory response. During MS, activated microglia can play the role of antigen presenting cells and can, therefore, skew T cell responses towards a pro-inflammatory phenotype. Once activated, microglia upregulate the expression of pro-inflammatory molecules. In addition to microglial responses during MS, astrocytes are also implicated in the development of MS lesions. Upon injury and nearby neuronal death, astrocytes undergo astrogliosis. To date, several molecular pathways were identified as targets for therapeutic interventions for MS such as NF-kB & Nlrs. Nlrs are regulatory proteins of the immune system capable of regulating both innate and adaptive responses. Nlrp12 is a pyrin-containing intracellular protein, largely expressed in cells of myeloid origin. Nlrp12 plays an important role in immune inflammatory responses by negatively regulating the NF-κB pathway and modulatory roles, such as dendritic cell migration. The focus of this study was to evaluate the hypothesis where Nlrp12 plays an anti-inflammatory role in Experimental Autoimmune Encephalomyelitis (EAE), a well-characterized mouse model to study MS. Over a course of 9 weeks, Nlrp12 KO mice demonstrated increased severity in disease levels compared to WT mice. In both genotypes, the disease was observed to peak around the 3rd week post immunization A significant increase in Nlrp12 mRNA was observed in diseased WT compared to healthy WT mice. A significant increase in the expression of CCR5, COX-2, and IL-1 β in Nlrp12 KO mice relative to WT mice, was observed. Interestingly, no differences in the percentage of gliosis was seen at 3 weeks post injection in both genotypes, however after 9 weeks of diseases, in Nlrp12 KO mice we observed a significant increase in the percentage of reactive gliosis compared to WT mice. A significant activation of NF-kB-dependent inflammation was seen in primary microglial cell cultures from Nlrp12 KO relative to WT following LPS stimulation. Moreover, supernatants of analysis for the level of nitrates with Griess reagent and with ELISA for TNFα and IL-6, demonstrated an increase in the pro-inflammatory phenotype from microglia from Nlrp12 KO mice compared to WT mice. These results suggest a critical role of Nlrp12 in suppressing inflammation during the development of the disease given that in its absence, we observed an increase in the inflammatory response. / La sclérose en plaques est une maladie auto-immune déclenchée par une réaction
inflammatoire anormale et caractérisée par la dégradation de myéline au niveau du système
nerveux central. Durant la sclérose en plaques, la microglie promeut l’expression de
molécules pro-inflammatoires et joue le rôle de cellules présentatrices d’antigènes pour
forcer les cellules T à adopter un phénotype pro-inflammatoire. Outre les réponses
associées à la microglie, les astrocytes sont aussi impliqués dans le développement des
lésions. Jusqu’à présent, plusieurs voies moléculaires ont été identifiées comme cibles pour
des interventions thérapeutiques tel que les voies de NF-kB et Nlrs. Les récepteurs Nlrs
sont des protéines régulatrices du système immunitaire inné et adaptatif. Nlrp12 joue un
rôle important dans les réponses inflammatoires immunes en régulant négativement la voie
NF-kB et la migration de cellules dendritiques. L’objectif de cette étude est d’étudier
l’hypothèse dans laquelle Nlrp12 joue un rôle anti-inflammatoire dans l’encéphalopathie
expérimentale autoimmune (EAE), un modèle murin de la sclérose en plaques. Durant 9
semaines, des souris n’exprimant pas Nlrp12 ont démontré un état sévère de la maladie
comparativement aux souris de type sauvage (WT). Dans les deux types de génotypes, la
maladie était observée à son maximum autour de la 3ème semaine après immunisation. Une
augmentation significative de l’expression d’ARNm de Nlrp12 était observée dans les
souris contrôles malades comparativement aux souris saines. Une augmentation
significative de l’expression de Ccr5, COX-2 ainsi qu’IL-1β était détectée dans les souris
Nlrp12 KO par rapport aux souris WT. De plus, aucune différence dans le pourcentage de
gliose était observée dans les deux génotypes à 3 semaines post-injection. Par contre, le
pourcentage de gliose activée augmentait dans les souris Nlrp12 KO après 9 semaines de
maladie. Nous avons remarqué une activation prononcée de l’inflammation dépendante de
NF-kB dans des cultures cellulaires primaires de microglie provenant de souris Nlrp12 KO
soumise à une stimulation au LPS. Finalement, la quantification des niveaux de nitrates et
des cytokines TNFα et IL-6 traduisait une signature pro-inflammatoire de la microglie des
souris Nlrp12 KO comparativement aux souris WT. Ces résultats suggèrent un rôle antiinflammatoire
de Nlrp12 durant le développement de la sclérose en plaques considérant la
réponse inflammatoire accrue en absence de Nlrp12.
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The role of AIM2 and NLRP12 in the innate immune response to Francisella tularensisUlland, Tyler Kent 01 December 2014 (has links)
The innate immune response to pathogens by the host is dependent upon the interplay of both pathogen and host intrinsic factors. Nucleotide-binding domain leucine-rich repeat containing (NLR) and pyrin and HIN200 domain containing (PYHIN) proteins are intracellular sensors of damage-associated and pathogen-associated molecular patterns. The studies presented here focus on the PYHIN molecule, AIM2, and the NLR, NLRP12, and the importance of bacteria- and host-associated proteins in the coordination of the innate immune response to the Gram-negative pathogen Fracisella tularensis. We have found that several genes expressed by F. tularensis encode for proteins that, when disrupted, cause the bacteria to trigger hyper- or hypoactivation of the AIM2 inflammasome. Bacteria with a mutation in FTL_0724, which hyperactivates the AIM2 inflammasome, are highly attenuated in a mouse model of infection, and induce robust caspase-1 processing and secretion of IL-1β by bone marrow derived macrophages (BMDMs). In contrast the hypoactivating mutant of F. tularensis, FTL0699, reduces IL-1β secretion by BMDMs and remains virulent in in vivo models of infection.
We have also investigated the role of host-expressed NLRs in F. tularensis infection models. We have found that NLRP12 is important in the coordination of the innate immune response to F. tularensis through the modulation of CXCL1 production. We hypothesized that decreased CXCL1 production by Nlrp12-deficient mice was in turn responsible for the diminished recruitment of neutrophils in to the lungs of Nlrp12-deficient mice following intranasal challenge with F. tularensis. Nlrp12-deficient mice were found to be highly susceptible to infection with F. tularensis, and succumbed to infection at a much higher rate than wild-type mice. Taken together these data demonstrate that a number of pathogen and host factors can play critical roles in the outcome F. tularensis infections.
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Papel do receptor NLRP12 na modulação da reabsorção óssea durante a progressão da lesão periapical experimental / Role of NLRP12 on bone resorption during the progression of a periapical lesion modelTaira, Thaise Mayumi 29 June 2017 (has links)
O receptor NLRP12 é um receptor intracelular que está envolvido no reconhecimento de PAMPs e DAMPs durante uma infecção. Foi visto que durante a osteoclastogênese, há uma diminuição da transcrição de NLRP12, e que este receptor inibe a reabsorção óssea através da supressão da via alternativa de NF-κB, exercendo um papel protetor sobre o tecido ósseo. Além disso, foi observado que a deficiência de NLRP12 em monócitos sob o estímulo de RANKL levou a maior estabilização de NIK e maior translocação de RelB para o núcleo, aumentando a formação dos osteoclastos in vitro. Portanto, o objetivo deste estudo foi avaliar o papel de NLRP12 no desenvolvimento e progressão da lesão periapical induzida em camundongos. Para isso, foi induzida a lesão periapical dos primeiros molares inferiores dos camundongos fêmeas C57/Bl6 (WT) e camundongos deficientes para o receptor NLRP12 (Nlrp12-/-). Após 14 e 21 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos; expressão gênica de marcadores osteoclastogênicos por qPCR; contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); avaliação enzimática das MMPs por Zimografia. Os linfonodos cervicais foram submetidos à análise da expressão dos fatores de transcrição T-bet, RORγt, GATA-3 e Foxp-3 por qPCR. Aos 14 dias, na análise histomorfométrica os animais Nlrp12-/- apresentaram uma maior lesão periapical quando comparados aos animais WT, associado com o aumento da expressão de Trap, Catepsina K e MMP-9 em amostras de hemi-mandíbulas com lesão. Além disso, o número de células TRAP positivas foi significantemente maior em Nlrp12-/- com lesão quando comparado com seu controle, enquanto no grupo WT com e sem lesão foram semelhantes. Assim como foi observado o maior aumento dos osteoclastos presentes no local da lesão dos animais Nlrp12-/-, estes também se mostraram com maior atividade gelatinolítica de MMP-9 e MMP-2 em relação ao seu controle, diferente dos animais WT que não apresentaram diferença entre os grupos controle e com lesão. Ainda nesse período, foi observado nas amostras de linfonodos cervicais que, no grupo Nlrp12-/- houve uma tendência à maior expressão de RORγt, seguidos de menor expressão de T-bet quando comparados com o grupo WT. Aos 21 dias, os animais WT e Nlrp12-/- apresentaram lesões periapicais de tamanhos semelhantes. Além disso, somente o grupo Nlrp12-/- com lesão apresentou um aumento significativo da expressão de Trap em relação ao seu controle e a expressão de Catepsina K foi semelhante em ambos os grupos. Neste período houve um aumento na quantidade de células TRAP positivas em ambos os grupos com lesão quando comparados com seus respectivos controles, entretanto também não houve diferença entre os animais WT e Nlrp12-/-. A atividade de MMP-9 e MMP-2 foram semelhantes entre os animais WT e Nlrp12-/- e entre seus respectivos controles aos 21 dias. Nossos dados sugerem que a deficiência de NLRP12 levou a uma maior perda óssea aos 14 dias de lesão periapical e que isso ocorre via modulação da formação e atividade dos osteoclastos. Portanto, o NLRP12 inibe a osteoclastogênese e a atividade dos osteoclastos durante a fase inicial da lesão periapical, retardando o desenvolvimento da doença. / NLRP12 is an intracellular sensor that recongnizes PAMPS and DAMPs during infeccion. It was seen that NLRP12 transcription is down-regulated during osteoclastogenesis, and NLRP12 protect bone via suppression of alternative NF-κB-induced osteoclastogenesis. It has been shown that NLRP12 deficiency in monocytes under RANKL stimuli exhibited more stabilization of NIK and nuclear translocation of RelB, increasing osteoclasts formation in vitro. Therefore, the aim of this study was to evaluate the role of NLRP12 in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and NLRP12 knockout (KO) mice. Samples were collected at 14 and 21 days of the lesion for the analyses. Jaw samples with lesion and control area were subjected to periapical lesions area determination by histological sections; osteoclastogenic markers expression by q-PCR; count of osteoclasts submitted to the enzyme assay for TRAP; evaluation of MMPs activity by Zymography. The expression of T cells markers´ transcription factors was evaluated in lymph nodes by q-PCR. Fourteen days after periapical lesion induction, histological analysis revealed that NLRP12KO mice exhibited higher area of periapical lesion compared to WT group, which was associated with up-regulated mRNA expression of Trap, Cathepsin K and MMP-9 in the jaw samples. Moreover, the number of multinuclear TRAP-stained cells was significantly higher in the NLRP12KO with lesion group when compared to it control, whereas in the WT the number between the lesion and control groups were similar. Still, NLRP12KO showed higher MMP-9 and -2 gelatinolytic activity than it control, unlike WT mice that showed no difference between control and lesion group. In this period, NLRP12KO mice lymph nodes showed more RORγt expression than WT mice and less T-bet expression. At 21 days, WT and NLRP12KO presented periapical lesions of similar sizes. In addition, NLRP12KO group with lesion showed a significant increase in Trap expression when compared with their control, but the increase in Trap e Cathepsin K was similar in both groups. Additionally, there was an increase of multinuclear TRAP-stained cells in both lesion groups when compared with their respective controls; however, there was also no difference between WT and NLRP12KO mice. MMP-9 and -2 activity was similar between WT and NLRP12KO and with their respective controls at 21 days. Our results suggest that NLRP12 deficiency led to increased bone loss at 14 days of periapical lesion and it occurs due to increased osteoclasts formation and activity. Therefore, NLRP12 inhibits osteoclastogenesis and osteoclasts activity during the early stages of periapical lesion, slowing the development of the disease.
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Papel do receptor NLRP12 na modulação da reabsorção óssea durante a progressão da lesão periapical experimental / Role of NLRP12 on bone resorption during the progression of a periapical lesion modelThaise Mayumi Taira 29 June 2017 (has links)
O receptor NLRP12 é um receptor intracelular que está envolvido no reconhecimento de PAMPs e DAMPs durante uma infecção. Foi visto que durante a osteoclastogênese, há uma diminuição da transcrição de NLRP12, e que este receptor inibe a reabsorção óssea através da supressão da via alternativa de NF-κB, exercendo um papel protetor sobre o tecido ósseo. Além disso, foi observado que a deficiência de NLRP12 em monócitos sob o estímulo de RANKL levou a maior estabilização de NIK e maior translocação de RelB para o núcleo, aumentando a formação dos osteoclastos in vitro. Portanto, o objetivo deste estudo foi avaliar o papel de NLRP12 no desenvolvimento e progressão da lesão periapical induzida em camundongos. Para isso, foi induzida a lesão periapical dos primeiros molares inferiores dos camundongos fêmeas C57/Bl6 (WT) e camundongos deficientes para o receptor NLRP12 (Nlrp12-/-). Após 14 e 21 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos; expressão gênica de marcadores osteoclastogênicos por qPCR; contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); avaliação enzimática das MMPs por Zimografia. Os linfonodos cervicais foram submetidos à análise da expressão dos fatores de transcrição T-bet, RORγt, GATA-3 e Foxp-3 por qPCR. Aos 14 dias, na análise histomorfométrica os animais Nlrp12-/- apresentaram uma maior lesão periapical quando comparados aos animais WT, associado com o aumento da expressão de Trap, Catepsina K e MMP-9 em amostras de hemi-mandíbulas com lesão. Além disso, o número de células TRAP positivas foi significantemente maior em Nlrp12-/- com lesão quando comparado com seu controle, enquanto no grupo WT com e sem lesão foram semelhantes. Assim como foi observado o maior aumento dos osteoclastos presentes no local da lesão dos animais Nlrp12-/-, estes também se mostraram com maior atividade gelatinolítica de MMP-9 e MMP-2 em relação ao seu controle, diferente dos animais WT que não apresentaram diferença entre os grupos controle e com lesão. Ainda nesse período, foi observado nas amostras de linfonodos cervicais que, no grupo Nlrp12-/- houve uma tendência à maior expressão de RORγt, seguidos de menor expressão de T-bet quando comparados com o grupo WT. Aos 21 dias, os animais WT e Nlrp12-/- apresentaram lesões periapicais de tamanhos semelhantes. Além disso, somente o grupo Nlrp12-/- com lesão apresentou um aumento significativo da expressão de Trap em relação ao seu controle e a expressão de Catepsina K foi semelhante em ambos os grupos. Neste período houve um aumento na quantidade de células TRAP positivas em ambos os grupos com lesão quando comparados com seus respectivos controles, entretanto também não houve diferença entre os animais WT e Nlrp12-/-. A atividade de MMP-9 e MMP-2 foram semelhantes entre os animais WT e Nlrp12-/- e entre seus respectivos controles aos 21 dias. Nossos dados sugerem que a deficiência de NLRP12 levou a uma maior perda óssea aos 14 dias de lesão periapical e que isso ocorre via modulação da formação e atividade dos osteoclastos. Portanto, o NLRP12 inibe a osteoclastogênese e a atividade dos osteoclastos durante a fase inicial da lesão periapical, retardando o desenvolvimento da doença. / NLRP12 is an intracellular sensor that recongnizes PAMPS and DAMPs during infeccion. It was seen that NLRP12 transcription is down-regulated during osteoclastogenesis, and NLRP12 protect bone via suppression of alternative NF-κB-induced osteoclastogenesis. It has been shown that NLRP12 deficiency in monocytes under RANKL stimuli exhibited more stabilization of NIK and nuclear translocation of RelB, increasing osteoclasts formation in vitro. Therefore, the aim of this study was to evaluate the role of NLRP12 in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and NLRP12 knockout (KO) mice. Samples were collected at 14 and 21 days of the lesion for the analyses. Jaw samples with lesion and control area were subjected to periapical lesions area determination by histological sections; osteoclastogenic markers expression by q-PCR; count of osteoclasts submitted to the enzyme assay for TRAP; evaluation of MMPs activity by Zymography. The expression of T cells markers´ transcription factors was evaluated in lymph nodes by q-PCR. Fourteen days after periapical lesion induction, histological analysis revealed that NLRP12KO mice exhibited higher area of periapical lesion compared to WT group, which was associated with up-regulated mRNA expression of Trap, Cathepsin K and MMP-9 in the jaw samples. Moreover, the number of multinuclear TRAP-stained cells was significantly higher in the NLRP12KO with lesion group when compared to it control, whereas in the WT the number between the lesion and control groups were similar. Still, NLRP12KO showed higher MMP-9 and -2 gelatinolytic activity than it control, unlike WT mice that showed no difference between control and lesion group. In this period, NLRP12KO mice lymph nodes showed more RORγt expression than WT mice and less T-bet expression. At 21 days, WT and NLRP12KO presented periapical lesions of similar sizes. In addition, NLRP12KO group with lesion showed a significant increase in Trap expression when compared with their control, but the increase in Trap e Cathepsin K was similar in both groups. Additionally, there was an increase of multinuclear TRAP-stained cells in both lesion groups when compared with their respective controls; however, there was also no difference between WT and NLRP12KO mice. MMP-9 and -2 activity was similar between WT and NLRP12KO and with their respective controls at 21 days. Our results suggest that NLRP12 deficiency led to increased bone loss at 14 days of periapical lesion and it occurs due to increased osteoclasts formation and activity. Therefore, NLRP12 inhibits osteoclastogenesis and osteoclasts activity during the early stages of periapical lesion, slowing the development of the disease.
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Corrélation génotype – phénotype chez les patients pédiatriques porteurs de mutations de NLRP12Beaufils, Camille 08 1900 (has links)
Le syndrome périodique lié à NLRP12 (NLRP12-AD) est une maladie rare appartenant au groupe des maladies auto-inflammatoires systémiques héréditaires. Ces maladies sont causées par des anomalies du système immunitaire inné. Les cryopyrinopathies (CAPS) sont une famille de maladie auto-inflammatoire liées à des mutations gain de fonction du gène NLRP3. NLRP3 fait partie d’un composant central de l’inflammation, l’inflammasome. En 2007, Jéru et al. ont décrit un premier patient présentant des symptômes évocateurs d’un CAPS qui n’était pas porteur de mutation de NLRP3 mais présentait une mutation de NLRP12. Depuis, 33 patients pédiatriques atteints de NLRP12-AD ont été décrits dans la littérature. Les signes cliniques de la maladie sont variables et ne permettent pas d’établir de critères cliniques diagnostics fiables. La pathogénicité des mutations observées chez les patients est difficile à établir. Les patients atteints de NLRP12-AD représentent également un défi thérapeutique, un certain nombre d’entre eux ne répondant pas aux anti-IL1, un traitement pourtant efficace dans la plupart des inflammasomopathies. De nombreuses études se sont intéressées au rôle de NLPR12, qui posséderait à la fois des propriétés pro et anti-inflammatoires par sa capacité à inhiber les voies canoniques et non canoniques de NFkb mais aussi à former un inflammasome. Le rôle exact de NLRP12 reste controversé, avec des résultats différents selon les cellules ou les stimuli utilisés lors des expériences.
L’objectif de notre étude est de créer un modèle in vitro fiable et reproductible afin de mieux comprendre la physiopathologie de NLRP12. Il permettra ensuite d’étudier les mutations de NLRP12 retrouvées chez des patients présentant des symptômes de maladie auto-inflammatoire pour améliorer les performances et la fiabilité du diagnostic et proposer des traitements personnalisés aux patients concernés.
Nous avons créé un modèle de cellules THP1 NLRP12 KO en utilisant la technologie de CRISPR/Cas9 qui a permis d’induire une délétion homozygote à la jonction entre le 2ème intron et le 3ème exon de NLRP12, qui code pour le domaine fonctionnel de la protéine. Nos cellules KO semblent sécréter moins de cytokine pro-inflammatoire (IL-1β et TNFα) que les cellules WT, suggérant un rôle pro-inflammatoire de NLRP12 dans notre modèle. Nous avons par ailleurs décrit une cohorte nord-américaine de 17 patients porteurs de mutations de NLRP12 afin d’étudier leurs mutations et produit des vecteurs lentiviraux contenant ces mutations.
Nous prévoyons d’explorer le rôle de NLRP12 sur l’activation de la voie NFkB et la formation d’un inflammasome puis de transduire nos cellules KO avec les différentes mutations de nos patients et d’analyser leurs conséquences sur ces mêmes voies et sur la sécrétion cytokinique. / NLRP12-AD is part of a new group of rheumatics’ diseases: the systemic autoinflammatory diseases. Those diseases are caused by defect or dysfunction in the innate immune system. Cryopyrin-associated periodic syndromes (CAPS) is a family of systemic autoinflammatory disease initially linked to NLRP3 gain-of-function heterozygous mutations. NLRP3 is part of a key component of inflammation, the inflammasome. In 2007, Jeru et al. described the first patient with CAPS phenotype, but without NLRP3 mutations. This patient had NLRP12 mutations. Since then, 33 pediatric patients with NLRP12-AD have been published. There is no specific clinical presentation that allow homogenous diagnosis. Determination of the causality of the mutations remains tricky. Lastly, some patients have shown resistance to anti-IL1 treatment, a medication that is highly effective in other inflammasomopathies such as CAPS, a puzzling observation as to the role of NLRP12. NLRP12 has been described with both anti- and pro- inflammatory roles, by its ability to inhibit the canonical and non-canonical NFkB pathways, but also through its hypothetic capacity to form an inflammasome. Hence, the exact role of NLRP12 remains controversial and its role might be stimuli- or cell-dependant.
Our objective is to create a reliable and reproductible in vitro model to better understand the role of NLRP12. This model will then allow us to study NLRP12 mutations found in patients with auto-inflammatory symptoms. This will improve our diagnostic performance and help to offer to patients the most suitable therapy.
We created NLRP12 knockout THP-1 cells by using the CRPISP-Cas 9 gene editing technology. We were able to induce a homozygous deletion at the intron 2/exon 3 junction that encodes the protein functional domain. Our KO cells seems to secrete less pro-inflammatory cytokines (IL-1β and TNFα) than WT cells. This suggests a pro-inflammatory role of NLRP12. We described a North American cohort of 17 pediatric patients with NLRP12 mutations to study their mutations in our model and produced lentiviral vectors containing those mutations.
We planned to study the effects of NLRP12 on NFkB activation and on inflammasome formation. Then, we will transduce our KO cells with the patient’s mutations and compare their consequences on inflammation pathways and cytokine secretion.
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