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Doctoral Thesis
DOI
https://doi.org/10.11606/T.42.2019.tde-16122019-120656
Document
Author
Full name
Laura Migliari Branco
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Bortoluci, Karina Ramalho (President)
Salomão, Reinaldo
Tersariol, Ivarne Luis dos Santos
Vinolo, Marco Aurelio Ramirez
Weinlich, Ricardo
Title in Portuguese
Mecanismos moleculares envolvidos na ativação do inflamassoma NAIP/NLRC4
Keywords in Portuguese
Catepsina
Flagelina
IL-1ß
Lisossomo
NLRC4
Abstract in Portuguese
Inflamassomas sao complexos moleculares intracelulares formados apos a deteccao de estimulos estereis ou infecciosos que desencadeiam a ativacao da caspase-1, levando a liberacao de IL-1ß, IL-18 e a morte celular por piroptose. Dentre os inflamassomas estudados, destacam-se os formados pelas proteinas NLRP3 e NLRC4. O inflamassoma NLRP3 atua como um sensor da homeostase celular e ativado apos a geracao de disturbios citosolicos gerados por estimulos derivados de patogenos ou do hospedeiro. Ja os inflamassomas formados por NAIP/NLRC4 são classicamente associados com a deteccao de proteinas de T3SS/T4SS e flagelina de bacterias patogenicas que alcancam o citoplasma. Contudo, diversos estudos sugerem que essas plataformas moleculares podem conter mais de um NLR e que vias citoplasmaticas acessorias sao capazes de amplificar a resposta mediada pelos inflamassomas. Nesse sentido, nosso grupo demonstrou que a estimulacao de macrofagos com flagelina derivada de B. subtilis desencadeia uma via lisossomal que contribui para a secrecao de IL-1ß por uma via nao completamente estabelecida. No presente trabalho, descrevemos que as catepsinas lisossomais e o influxo de Ca2+ dessa organela contribuem para a secrecao de IL-1ß em resposta a flagelina de S. thyphimurium entregue ao citoplasma diretamente ou com o auxilio de vesiculas de transfeccao, de maneira independente da inducao de morte celular. Embora esteja estabelecido na literatura que o dano lisossomal e uma via comum para a ativacao de NLRP3 em resposta a materiais particulados e que esse sensor coopera com NLRC4 para induzir respostas mais potentes frente a infeccao com S. thyphimurium, nossos dados indicam que a via lisossomal desencadeada pela flagelina amplifica a secreção de IL-1ß de maneira independente de NLRP3. Por meio de inibicao farmacologica e delecao genetica, verificamos que a catepsina B apresenta um papel importante na modulacao do inflamassoma em resposta a flagelina. Importante, a inibicao das catepsinas nao influencia a inducao de pro-IL-1ß e producao de TNF-a, descartando o papel dessa protease na fase de priming do inflamassoma. Em contrapartida, as catepsinas parecem regular a clivagem/liberacao da IL-1ß ativa. Surpreendentemente, a atuacao das catepsinas na maturacao/secrecao de IL-1ß parece acontecer apos ou independentemente da formacao do complexo inflamassoma, uma vez que a inibição das mesmas nao influencia a agregacao de specks de ASC e nem a maturacao de caspase-1. Esses dados mostram que as catepsinas lisossomais regulam as respostas de NAIP/NLRC4 por mecanismos distintos aos descritos para o inflamassoma NLRP3, sugerindo uma atuacao dessas proteases em processos não convencionais de clivagem/liberacao de IL-1ß. Em conjunto, os resultados obtidos indicam que o inflamassoma NLRC4 e modulado por sinais adicionais a interação entre ligante-receptor. Considerando o emergente papel de NLRC4 em doenças inflamatorias estereis, a via lisossomal descrita pode contribuir para a inflamação exacerbada decorrente da ativacao aberrantede NLRC4 nessas patologias, representando um possivel alvo de intervencao terapeutica.
Title in English
Molecular Mechanisms involved in NAIP/NLRC4 inflammasome Activation
Keywords in English
Cathepsin
Flagellin
IL-1ß
Lysosome
NLRC4
Abstract in English
Inflammasomes are intracellular molecular complexes formed after detection of sterile or infectious stimuli that trigger caspase-1 activation, leading to the release of IL-1ß, IL-18 and cell death by pyroptosis. Among the described inflammasomes, the ones formed by NLRP3 and NLRC4 proteins are the best characterized. NLRP3 inflammasome acts as a sensor of cellular homeostasis and is activated after the generation of cytosolic disturbs generated by pathogen or host-derived stimuli. In contrast, the inflammasomes formed by NAIP/NLRC4 are classically associated with the detection of T3SS/T4SS proteins and flagellin from pathogenic bacteria that reach the cytoplasm. However, several studies suggest that these molecular platforms may contain more than one NLR and that accessory cytoplasmic pathways are capable of amplify inflammasome-mediated responses. In this regard, our group demonstrated that macrophage stimulation with flagellin derived from B. subtilis triggers a lysosomal pathway that contributes to the secretion of IL-1ß by a pathway not completely established. In the present work, we describe that lysosomal cathepsins and Ca2+ influx from this organelle contribute to the secretion of IL-1ß in response to flagellin from S. thyphimurium delivered to the cytoplasm directly or using transfection vesicles, independently of the induction of cell death. Although it is established in the literature that lysosomal damage is a common pathway to promote NLRP3 activation and that this sensor cooperates with NLRC4 to induce more potent responses to S. thyphimurium infection, our data indicate that the lysosomal pathway triggered by flagellin amplifies secretion of IL-1ß independently of NLRP3. Through pharmacological inhibition and genetic ablation, we found that cathepsin B plays na important role on inflammasome modulation in response to flagellin. Importantly, the inhibition of cathepsins does not influence the induction of PRO-IL-1ß and the production of TNF-a, ruling out the role of this protease in the priming phase of the inflammasome. Conversely, cathepsins appear to regulate cleavage/release of active IL-1. Surprisingly, the role of cathepsins on the maturation/secretion of IL-1ß appears to occur after or independently of the formation of the inflammasome complex, since the inhibition does not influence ASC speck aggregation or caspase-1 maturation. These data show that lysosomal cathepsins regulate NAIP/NLRC4 inflammatory responses by other mechanisms than those described for NLRP3 inflammasome, suggesting that these proteases act in non-conventional IL-1ß cleavage/release processes. Taken together, the results obtained indicate that the NLRC4 inflammasome is modulated by additional signals besides the interaction between ligand-receptor. Considering the emerging role of NLRC4 in sterile inflammatory diseases, the described lysosomal pathway may contribute to the exacerbated inflammation resulting from the NLRC4 aberrant activation in these pathologies, representing a possible target for therapeutic intervention.
 
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Release Date
2021-12-15
Publishing Date
2019-12-17
 
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