Mutações na via do NF-kB e sua interface com o CD40L na doença de Behçet: modelo in vitro para compreensão do fenótipo autoinflamatório
Data
2024-07-04
Autores
Aires, Patricia Pontes 
Orientadores
Perazzio, Sandro Félix
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: atualmente a doença de Behçet (DB) tem sido classificada dentro do grupo das síndromes autoinflamatórias poligênicas. Mutações pontuais isoladas nos genes TNFAIP3 e NEMO foram associadas a fenótipos similares à DB, chamando a atenção para a importância da via do NF-κB na patogênese da doença. Ademais, o sCD40L, presente em altas concentrações plasmáticas na DB, é capaz de induzir à explosão respiratória via PI3K/NF-κB. Entretanto, pouco se sabe sobre a influência das mutações citadas no sistema imunitário ou na sinalização mediada pelo sCD40L. Ainda, o impacto de mutações da via não canônica, incluindo o gene NFKB2, na interação com a via canônica e, potencialmente, na patogênese de doenças autoinflamatórias, ainda não foi plenamente elucidado.
Objetivos: analisar o comportamento funcional das células HL-60, THP-1, Daudi e Jurkat, influenciado pela transfecção de componentes gênicos da via do NF-κB, na presença ou ausência de mutações pontuais, com enfoque na ativação pelo sCD40L. Métodos: células THP-1, HL-60, Daudi e Jurkat foram transfectadas com plasmídeos marcados com proteína fluorescente carreando os genes TNFAIP3, NEMO e NFKB2-p52 e suas respectivas variantes L227X, D406V e E418X. A influência da transfecção na expressão proteica dos componentes da via do NF-κB foi avaliada por citometria de fluxo. As linhagens celulares foram ainda avaliadas quanto às seguintes funções: capacidade proliferativa e resistência à indução de apoptose; capacidade fagocítica; metabolismo oxidativo; produção in vitro de TNF-, IL-1 e IL-6 e capacidade de formação de armadilhas extracelulares neutrofílicas (NET). Resultados: os resultados obtidos sugerem ser possível a determinação de um fenótipo celular específico para células portadoras de mutações na via do NF-kB, abrindo espaço para a avaliação funcional de pacientes com suspeita clínica de DB monogênica e outras patologias associadas à via do NF-kB. Em relação à transfecção da variante TNFAIP3 L227X, a expressão proteica de A20 foi reduzida em células THP-1 e HL-60, sugerindo um potencial efeito dominante-negativo da variante em fagócitos. A transfecção com a variante L227X levou a aumento da capacidade proliferativa, da produção de espécies reativas de oxigênio (ROS, reactive oxygen species), da produção in vitro de IL-6 e da liberação de armadilhas extracelulares de neutrófilos (NET, neutrophil extracellular traps). Células HL-60 transfectadas com TNFAIP3 L227X também apresentaram maior produção de IL-1β quando estimuladas com sCD40L. A capacidade fagocítica de células THP-1 foi reduzida após transfecção com TNFAIP3 L227X. A transfecção de NEMO D406V em células THP-1 levou à redução na capacidade proliferativa, resistência à apoptose e capacidade fagocítica; por outro lado, as células THP-1 transfectadas com NEMO D406V apresentaram maior produção de IL-6 e TNF-α. Células Jurkat transfectadas com NEMO D406V apresentaram maior capacidade proliferativa em estado basal, que não persistiu após estímulo; a resistência à apoptose foi menor. Células Daudi transfectadas com NEMO D406V apresentaram aumento da capacidade proliferativa e resistência à apoptose; a produção in vitro de IL-1β estava reduzida. As células HL-60 transfectadas com NEMO D406V apresentaram redução da fagocitose em estado basal, menor produção de ROS após estímulo e maior produção de NET. Em relação à transfecção de NFKB2-p52 E418X, as células THP-1 apresentaram redução na proliferação, resistência à apoptose, capacidade fagocítica e produção de ROS; a produção de IL-1β e IL-6 estava aumentada. Células Jurkat transfectadas com NFKB2-p52 E418X apresentaram menor resistência à apoptose e maior produção de IL-6, sob estímulo de sCD40L. Células Daudi transfectadas com NFKB2-p52 E418X apresentaram aumento na capacidade proliferativa, porém menor resistência à apoptose induzida por UV. Por fim, células HL-60 transfectadas com NFKB2-p52 E418X apresentaram redução na capacidade fagocítica e na produção de ROS e aumento na produção de NET após estímulo com sCD40L. Conclusões: o impacto das diferentes variantes varia de acordo com o tipo celular analisado, o que pode influenciar na diversidade de fenótipos encontrados em associação a cada mutação. A transfecção com TNFAIP3 L227X levou a uma redução na capacidade fagocítica e aumento na explosão respiratória de células THP-1, sugerindo que essa variante pode polarizar macrófagos para o subtipo M1. Além disso, a hiperprodução de IL-6 sugere a tendência a um fenótipo Th17, já previamente relatado na doença de Behçet, reforçando as semelhanças fisiopatológicas entre a haploinsuficiência de A20 e a DB. Já a transfecção com NEMO D406V induziu um fenótipo de exaustão parcial dos fagócitos, com comprometimento de funções celulares básicas, mas com aumento na produção de citocinas pró-inflamatórias. Efeito similar foi visto nas células transfectadas com NKFB2-p52 E418X, que também tiveram comprometimento das funções e sobrevivência celular, com maior produção de citocinas pró-inflamatórias em relação às células não transfectadas.
Introduction: Behçet’s disease (BD) is currently classified in the group of polygenic autoinflammatory diseases. Isolated point mutations in the TNFAIP3 and NEMO genes have been associated with a Behçet-like phenotype, highlighting the importance of the NF-kB pathway in the pathogenesis of the disease. Additionally, sCD40L, present in high plasma concentrations in BD, has been reported to induce respiratory burst through the PI3K/NF-kB pathway. However, little is known about the influence of the mutations in TNFAIP3 or NEMO on the immune system or on sCD40L-mediated signaling. Moreover, the impact of mutations in the non-canonical pathway, including the NFKB2 gene, on its interaction with the canonical pathway and potentially on the pathogenesis of autoinflammatory diseases, has not yet been fully elucidated. Objectives: to assess the functional behavior of HL-60, THP-1, Daudi and Jurkat cells, under the influence of the transfection of different genes involved in the NF-kB pathway, both wild-type and with specific point mutations, with an emphasis on sCD40L activation. Methods: HL-60, THP-1, Daudi and Jurkat cells were transfected with green fluorescent protein-tagged plasmids containing either TNFAIP3, NEMO or NFKB2-p52 or their respective variants L227X, D406V and E418X. The impact of the transfected plasmids on the expression of the respective protein was assessed using flow cytometry. Each lineage was also assessed for the following: proliferation rates, resistance to apoptosis, phagocytic capacity, respiratory burst, in vitro cytokine production and neutrophil extracellular traps (NET). Results: the results obtained suggest that mutations in NF-kB lead to specific cellular phenotypes, paving the way to future functional assays in patients with suspected monogenic BD and other NF-kB-related diseases. The transfection of TNFAIP3 L227X led to reduced A20 protein expression in THP-1 and HL-60, suggesting a possible negative-dominant effect of this variant on phagocytes. Transfection with TNFAIP3 L227X led to an increase in proliferation rates, respiratory burst, IL-6 in vitro production and NET release. HL-60 cells transfected with TNFAIP3 L227X also showed increased IL-1β levels, when stimulated with sCD40L. THP-1 transfected with TNFAIP3 L227X cells showed decreased phagocytosis rates. THP-1 cells transfected with NEMO D406V showed a reduction in proliferation rates, resistance to apoptosis and phagocytic capacity; on the other hand, these cells showed increased levels of IL-6 and TNF-α. Jurkat cells transfected with NEMO D406V had increased baseline proliferation rates, which didn’t persist upon stimulation. Daudi cells transfected with NEMO D406V showed increased proliferation rates and resistance to apoptosis; in vitro production of IL-1β was decreased. HL-60 cells transfected with NEMO D406V showed a decrease in phagocytosis and respiratory burst, while NET release was increased. The transfection of NFKB2-p52 E418X in THP-1 cells led to a decrease in proliferation rates, resistance to apoptosis, phagocytosis, and respiratory burst; there was an increase in IL-1β and IL-6 in vitro production. Jurkat cells transfected with NKFB2-p52 E418X showed decreased resistance to apoptosis and increased production of IL-6, upon stimulation with sCD40L. Daudi cells transfected with NFKB2-p52 E418X showed increased proliferation rates, but decreased resistance to apoptosis. Finally, HL-60 cells transfected with NFKB2-p52 E418X had reduced phagocytic capacity and respiratory burst, while NET release was increased upon stimulation with sCD40L. Conclusions: The impact of the variants can be different, according to the type of cell analyzed, which may influence the diversity of phenotypes associated with each mutation. Transfection with TNFAIP3 L227X led to a reduction in phagocytic capacity and an increase in respiratory burst of THP-1 cells, suggesting that this variant may polarize macrophages towards the M1 subtype. Additionally, the hyperproduction of IL-6 suggests a tendency towards a Th17 phenotype, which has been previously reported in Behçet's disease, reinforcing the pathophysiological similarities between A20 haploinsufficiency and BD. In contrast, transfection with NEMO D406V induced a phenotype of partial phagocyte exhaustion, with impairment of basic cellular functions, but with increased production of pro-inflammatory cytokines. A similar effect was observed in cells transfected with NFKB2-p52 E418X, which also exhibited compromised cellular functions, impaired survival, and higher production of pro-inflammatory cytokines compared to non-transfected cells.
Introduction: Behçet’s disease (BD) is currently classified in the group of polygenic autoinflammatory diseases. Isolated point mutations in the TNFAIP3 and NEMO genes have been associated with a Behçet-like phenotype, highlighting the importance of the NF-kB pathway in the pathogenesis of the disease. Additionally, sCD40L, present in high plasma concentrations in BD, has been reported to induce respiratory burst through the PI3K/NF-kB pathway. However, little is known about the influence of the mutations in TNFAIP3 or NEMO on the immune system or on sCD40L-mediated signaling. Moreover, the impact of mutations in the non-canonical pathway, including the NFKB2 gene, on its interaction with the canonical pathway and potentially on the pathogenesis of autoinflammatory diseases, has not yet been fully elucidated. Objectives: to assess the functional behavior of HL-60, THP-1, Daudi and Jurkat cells, under the influence of the transfection of different genes involved in the NF-kB pathway, both wild-type and with specific point mutations, with an emphasis on sCD40L activation. Methods: HL-60, THP-1, Daudi and Jurkat cells were transfected with green fluorescent protein-tagged plasmids containing either TNFAIP3, NEMO or NFKB2-p52 or their respective variants L227X, D406V and E418X. The impact of the transfected plasmids on the expression of the respective protein was assessed using flow cytometry. Each lineage was also assessed for the following: proliferation rates, resistance to apoptosis, phagocytic capacity, respiratory burst, in vitro cytokine production and neutrophil extracellular traps (NET). Results: the results obtained suggest that mutations in NF-kB lead to specific cellular phenotypes, paving the way to future functional assays in patients with suspected monogenic BD and other NF-kB-related diseases. The transfection of TNFAIP3 L227X led to reduced A20 protein expression in THP-1 and HL-60, suggesting a possible negative-dominant effect of this variant on phagocytes. Transfection with TNFAIP3 L227X led to an increase in proliferation rates, respiratory burst, IL-6 in vitro production and NET release. HL-60 cells transfected with TNFAIP3 L227X also showed increased IL-1β levels, when stimulated with sCD40L. THP-1 transfected with TNFAIP3 L227X cells showed decreased phagocytosis rates. THP-1 cells transfected with NEMO D406V showed a reduction in proliferation rates, resistance to apoptosis and phagocytic capacity; on the other hand, these cells showed increased levels of IL-6 and TNF-α. Jurkat cells transfected with NEMO D406V had increased baseline proliferation rates, which didn’t persist upon stimulation. Daudi cells transfected with NEMO D406V showed increased proliferation rates and resistance to apoptosis; in vitro production of IL-1β was decreased. HL-60 cells transfected with NEMO D406V showed a decrease in phagocytosis and respiratory burst, while NET release was increased. The transfection of NFKB2-p52 E418X in THP-1 cells led to a decrease in proliferation rates, resistance to apoptosis, phagocytosis, and respiratory burst; there was an increase in IL-1β and IL-6 in vitro production. Jurkat cells transfected with NKFB2-p52 E418X showed decreased resistance to apoptosis and increased production of IL-6, upon stimulation with sCD40L. Daudi cells transfected with NFKB2-p52 E418X showed increased proliferation rates, but decreased resistance to apoptosis. Finally, HL-60 cells transfected with NFKB2-p52 E418X had reduced phagocytic capacity and respiratory burst, while NET release was increased upon stimulation with sCD40L. Conclusions: The impact of the variants can be different, according to the type of cell analyzed, which may influence the diversity of phenotypes associated with each mutation. Transfection with TNFAIP3 L227X led to a reduction in phagocytic capacity and an increase in respiratory burst of THP-1 cells, suggesting that this variant may polarize macrophages towards the M1 subtype. Additionally, the hyperproduction of IL-6 suggests a tendency towards a Th17 phenotype, which has been previously reported in Behçet's disease, reinforcing the pathophysiological similarities between A20 haploinsufficiency and BD. In contrast, transfection with NEMO D406V induced a phenotype of partial phagocyte exhaustion, with impairment of basic cellular functions, but with increased production of pro-inflammatory cytokines. A similar effect was observed in cells transfected with NFKB2-p52 E418X, which also exhibited compromised cellular functions, impaired survival, and higher production of pro-inflammatory cytokines compared to non-transfected cells.
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Citação
AIRES, Patricia Pontes. Mutações na via do NF-kB e sua interface com o CD40L na doença de Behçet: modelo in vitro para compreensão do fenótipo autoinflamatório. 2024. 220 f. Tese (Doutorado em Pediatria) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024