Molecular Mechanisms involved in NAIP/NLRC4 inflammasome Activation

Author(s)

Laura Migliari Branco

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2019-08-15

Examining board members

Karina Ramalho Bortoluci; Reinaldo Salomão; Ivarne Luis dos Santos Tersariol; Marco Aurelio Ramirez Vinolo; Ricardo Weinlich

• Advisor: Karina Ramalho Bortoluci

Abstract

Inflammasomes are intracellular molecular complexes formed after detection of sterile or infectious stimuli that trigger caspase-1 activation, leading to the release of IL-1&#223, 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-1&#223 by a pathway not completely established. In the present work, we describe that lysosomal cathepsins and Ca2&#43 influx from this organelle contribute to the secretion of IL-1&#223 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-1&#223 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-1&#223 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-1&#223 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-1&#223 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. (AU)

• FAPESP's process: 15/09029-7 - Interaction between NLRP3 and NLRC4 inflamassomes in macrophages activation
• Grantee: Laura Migliari Branco
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)