José Marcos Sanches1, Rebeca D Correia-Silva1, Gustavo H B Duarte2, Anna Maria A P Fernandes3, Salvador Sánchez-Vinces3, Patrícia O Carvalho3, Sonia M Oliani1,4, Karina R Bortoluci5, Vanessa Moreira6, Cristiane D Gil1,4. 1. Programa de Pós-Graduação em Biologia Estrutural e Funcional, Universidade Federal de São Paulo, São Paulo 04023-900, Brazil. 2. Instituto de Química, Universidade Estadual de Campinas, Campinas 13083-862, São Paulo, Brazil. 3. Laboratório de Pesquisa Multidisciplinar, Universidade São Francisco, Bragança Paulista 12916-900, São Paulo, Brazil. 4. Programa de Pós-Graduação em Biociências, Universidade Estadual Paulista (UNESP), Instituto de Biociências Letras e Ciências Exatas, São José do Rio Preto 15054-000, São Paulo, Brazil. 5. Departamento de Ciências Biológicas e Centro de Terapia Celular e Molecular, Universidade Federal de São Paulo, São Paulo 04044-010, Brazil. 6. Departamento de Farmacologia, Universidade Federal de São Paulo, São Paulo 04044-020, Brazil.
Abstract
This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1β production by WT neutrophils after nigericin and ATP stimulation. However, IL-1β release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1β release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1β production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.
This study evaluated the role of endogenous and exogenous annexin A1 (pan class="Gene">AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1β production by WT neutrophils after nigericin and ATP stimulation. However, IL-1β release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1β release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1β production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.
Entities:
Keywords:
ATP; Ac2-26; inflammation; lipidomics; mass spectrometry; nigericin
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