RESUMO
Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.
Assuntos
Inflamassomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , RNA Nuclear Pequeno/farmacologia , Animais , Complexo I de Transporte de Elétrons/metabolismo , Camundongos , Quinases Relacionadas a NIMA/metabolismo , Quinona Redutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor 7 Toll-Like/metabolismoRESUMO
Through its ability to control the proteolytic maturation and secretion of interleukin-1 family cytokines, the inflammasome occupies a central role in the activation of inflammation and also influences the shaping of adaptive immunity. Since it affects a multitude of different immune responses from autoinflammatory diseases to host defense, vaccine efficacy, and even cancer, it has become of interest to many researchers. Here, we describe a straightforward method for inflammasome assays in primary murine bone marrow--derived myeloid cells. The protocol encompasses cell handling, inflammasome activation and inhibition, as well as the detection of IL-1ß, caspase-1, and IL-1α by ELISA and Western blot.