Avaliação do papel da proteína anti-inflamatória Anexina A1 na regulação do Inflamassoma NLRP3 e nas infecções por Candida Albicans e Candida Auris: estudo in vitro e caracterização do perfil lipidômico
Data
2020-07-17
Orientadores
Gil, Cristiane Damas 
Tipo
Tese de doutorado
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ISSN da Revista
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Objetivo: Avaliar o papel da AnxA1 na regulação do inflamassoma NLRP3 e na infecção por C. albicans e C. auris em macrófagos e neutrófilos isolados, bem como avaliar o perfil dos lipídios liberados por essas células após a ativação do NLRP3 e nas infecções por Candida spp. Metodologia: Camundongos machos C57BL/6 selvagens (WT) e nocautes para AnxA1 (AnxA1-/- ) receberam injeção intraperitoneal de solução de amido a 1,5% ou carragenina a 0,3% para obtenção de macrófagos e neutrófilos do lavado peritoneal, respectivamente. Essas células foram estimuladas com lipopolissacarídeo (LPS), (por 3 horas), seguida da administração dos agonistas de NLRP3, nigericina (1 hora) ou ATP (30 minutos). Também foi verificado a ação do peptídeo mimético da AnxA1 (Ac2-26) em neutrófilos ativados pelo NLRP3. Para verificar a resposta de neutrófilos em infecções fúngicas, bem como a relação da AnxA1 nestas condições, estas células foram cocultivadas com as cepas de C. albicans e C. auris por 4 horas. Resultados: Nos macrófagos evidenciou-se que a falta de AnxA1 exacerba a liberação de IL-1β após a indução da ativação do inflamassoma NLRP3, bem como acarreta mudanças no perfil lipídico, tendo as ceramidas como os principais lipídios mediadores. A expressão de NLRP3 foi maior em macrófagos AnxA1-/- tratados com nigericina e pontos de colocalização foram verificados entre o inflamassoma NLRP3 e AnxA1, evidenciando a participação dessa proteína na regulação do NLRP3. Em neutrófilos, a AnxA1 endógena demonstrou ser importante para a ativação do NLRP3, uma vez que a liberação de IL-1β foi inferior quando comparado aos WT. A adição do peptídeo Ac2-26 reduziu a liberação de IL-1β pela via de ativação do inflamassoma NLRP3 em neutrófilos WT, mas não reverteu a liberação desta citocina em neutrófilos AnxA1-/- . Potenciais biomarcadores lipídicos relacionados ao estresse e ativação celular, incluindo esfingolipídios específicos e glicerofosfolipídios, foram identificados no sobrenadante dos neutrófilos de ambos genótipos após ativação por nigericina. O tratamento com Ac2-26 aumentou a concentração de fosfatidilserinas e fosfocolinas oxidadas, biomarcadores lipídicos relacionados à via da resolução inflamatória. Neutrófilos AnxA1-/- cocultivados com C. albicans ou C. auris apresentam maior expressão de COX-2 e ativação das MAPKs em relação aos WT. A falta de AnxA1 promoveu diferença no perfil lipídico, tendo maior proeminência de glicerofosfolipídios, tanto em neutrófilos co-infectados quanto no grupo AnxA1-/- controle. Conclusão: A AnxA1 desempenha diferentes papeis na sinalização do inflamassoma NLRP3 com base em sua localização intra ou extracelular e tipo celular. Além disso, regula a ativação dos neutrófilos pela infecção por Candida spp., evidenciando seu potencial imunomodulador em doenças inflamatórias e infecciosas.
Objective: To evaluate the role of the anti-inflammatory protein ANXA1 in the regulation of the NLRP3 inflammasome and in infection by C. albicans and C. auris in isolated macrophages and neutrophils, as well as to evaluate the profile of the lipids released after the activation of NLRP3 and in coinfection by Candida spp. Methodology: Male C57BL/6 wild (WT) mice and knockouts for AnxA1 (AnxA1-/- ) received intraperitoneal injection of 1.5% starch solution or 0.3% carrageenan to obtain macrophages and neutrophils from the peritoneal lavage, respectively. These cells were stimulated with lipopolysaccharide (LPS, 3 hours), followed by the administration of NLRP3 agonists, nigericin (1 hour) or ATP (30 minutes). AnxA1 mimetic peptide (Ac2-26) action on NLRP3 activated neutrophils was also verified. To check the response of neutrophils in fungal infections, as well as the relationship of AnxA1 in these conditions, these cells were incubated with the strains of C. albicans and C. auris for 4 hours. Results: In macrophages, it was evidenced that the lack of AnxA1 exacerbates the release of IL-1β after inducing the activation of the NLRP3 inflammasome, as well as causing changes in the lipid profile, with ceramides as the main mediating lipids. The expression of NLRP3 was higher in AnxA1-/- macrophages treated with nigericin and colocalization points were verified between the inflammasome NLRP3 and AnxA1, showing the participation of this protein in the regulation of NLRP3. In neutrophils, endogenous AnxA1 proved to be important for the activation of NLRP3, since the release of IL-1β was lower when compared to WT. The addition of Ac2-26 reduced the release of IL-1β by the NLRP3 inflammasome activation pathway in WT neutrophils, but did not reverse the release of this cytokine in AnxA1-/- neutrophils. Potential lipid biomarkers related to cell stress and activation, including sphingolipids and glycerophospholipids, were identified in supernatant of neutrophils from both genotypes after nigericin treatment. Treatment with Ac2-26 increased the concentration of phosphatidylserines and oxidized phosphocholines, lipid biomarkers related to the inflammatory pathway. AnxA1-/- neutrophils cocultured with C. albicans or C. auris show higher COX-2 expression and MAPKs performance compared to WT. The lack of AnxA1 promotes a difference in the lipid profile, with greater prominence of glycerophospholipids, either in coinfected neutrophils or in AnxA1-/- control group. Conclusion: The AnxA1 plays different roles in the signaling of the NLRP3 inflammasome based on its intra or extracellular location and cell type. In addition, it regulates the activation of neutrophils by Candida spp. infection, highlighting its immunomodulatory potential in inflammatory and infectious diseases.
Objective: To evaluate the role of the anti-inflammatory protein ANXA1 in the regulation of the NLRP3 inflammasome and in infection by C. albicans and C. auris in isolated macrophages and neutrophils, as well as to evaluate the profile of the lipids released after the activation of NLRP3 and in coinfection by Candida spp. Methodology: Male C57BL/6 wild (WT) mice and knockouts for AnxA1 (AnxA1-/- ) received intraperitoneal injection of 1.5% starch solution or 0.3% carrageenan to obtain macrophages and neutrophils from the peritoneal lavage, respectively. These cells were stimulated with lipopolysaccharide (LPS, 3 hours), followed by the administration of NLRP3 agonists, nigericin (1 hour) or ATP (30 minutes). AnxA1 mimetic peptide (Ac2-26) action on NLRP3 activated neutrophils was also verified. To check the response of neutrophils in fungal infections, as well as the relationship of AnxA1 in these conditions, these cells were incubated with the strains of C. albicans and C. auris for 4 hours. Results: In macrophages, it was evidenced that the lack of AnxA1 exacerbates the release of IL-1β after inducing the activation of the NLRP3 inflammasome, as well as causing changes in the lipid profile, with ceramides as the main mediating lipids. The expression of NLRP3 was higher in AnxA1-/- macrophages treated with nigericin and colocalization points were verified between the inflammasome NLRP3 and AnxA1, showing the participation of this protein in the regulation of NLRP3. In neutrophils, endogenous AnxA1 proved to be important for the activation of NLRP3, since the release of IL-1β was lower when compared to WT. The addition of Ac2-26 reduced the release of IL-1β by the NLRP3 inflammasome activation pathway in WT neutrophils, but did not reverse the release of this cytokine in AnxA1-/- neutrophils. Potential lipid biomarkers related to cell stress and activation, including sphingolipids and glycerophospholipids, were identified in supernatant of neutrophils from both genotypes after nigericin treatment. Treatment with Ac2-26 increased the concentration of phosphatidylserines and oxidized phosphocholines, lipid biomarkers related to the inflammatory pathway. AnxA1-/- neutrophils cocultured with C. albicans or C. auris show higher COX-2 expression and MAPKs performance compared to WT. The lack of AnxA1 promotes a difference in the lipid profile, with greater prominence of glycerophospholipids, either in coinfected neutrophils or in AnxA1-/- control group. Conclusion: The AnxA1 plays different roles in the signaling of the NLRP3 inflammasome based on its intra or extracellular location and cell type. In addition, it regulates the activation of neutrophils by Candida spp. infection, highlighting its immunomodulatory potential in inflammatory and infectious diseases.
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Citação
SANCHES JUNIOR, José Marcos. Avaliação do papel da proteína anti-inflamatória Anexina A1 na regulação do Inflamassoma NLRP3 e nas infecções por Candida Albicans e Candida Auris: estudo in vitro e caracterização do perfil lipidômico. São Paulo, 2020. 101 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2020.