Papel da Galectina-3 na regulação do inflamassoma NLRP3 na resposta alérgica: estudo em modelos in vivo e in vitro
Data
2024-12-09
Autores
Lourenço, Renata Pereira 
Orientadores
Gil, Cristiane Damas
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O NLRP3 é um receptor citoplasmático que, quando ativado, forma um inflamassoma crucial para a liberação de IL-1β e IL-18, participando da patogênese de muitas doenças, incluindo reações alérgicas. Estudos mostram que o eixo NLRP3/IL-1β desempenha um papel importante no desenvolvimento da conjuntivite alérgica (CA). Ainda, a expressão da proteína galectina-3 (Gal-3) está envolvida com a ativação do inflamassoma NLRP3 e a produção de IL-1β pelos macrófagos; no entanto, sua relação com a ativação do NLRP3 ainda não foi elucidada na resposta alérgica. Neste trabalho, avaliamos o papel da Gal-3 na resposta alérgica e sua possível relação com a regulação do inflamassoma NLRP3, utilizando modelo experimental de CA e ensaios com cultura de queratinócitos humanos estimulados com IL-4. Para indução da CA, camundongos BALB/c machos selvagens (WT) e nocautes para Gal-3 (Gal-3-/-) imunizados com ovalbumina (OVA) foram desafiados com colírios contendo OVA nos dias 14-16, e suas conjuntivas palpebrais foram avaliadas nas condições experimentais (n = 5 animais/grupo): SHAM (controle), que recebeu apenas salina estéril, e CA. Após 24 horas do último desafio com a OVA, as pálpebras foram coletadas para análises (CEUA 2125200323). A CA em camundongos WT e Gal-3-/- foi caracterizada por um aumento significativo no número de eosinófilos (68±10 e 73±9, respectivamente) na conjuntiva palpebral em comparação aos seus respectivos controles (p < 0,05). A proporção de mastócitos degranulados (~60%) foi maior na conjuntiva palpebral de animais Gal-3-/- CA em comparação com células intactas (~40%); enquanto no grupo WT CA, a proporção de células intactas (58%) foi maior que a de degranuladas (42%). A análise dos transcriptomas mostrou aumento dos transcritos de Il1β em mastócitos estimulados por anti-OVA-IgE (GSE96696) e eosinófilos estimulados por OVA (GSE57757). Nessas condições, apenas os mastócitos apresentaram aumento nos transcritos de Lgals3. A conjuntiva palpebral de camundongos WT CA mostrou um aumento significativo nos níveis de IL-1β, NLRP3 e p38 fosforilada em comparação aos grupos WT SHAM e Gal-3-/- CA. In vitro, a adição de Gal-3 recombinante a queratinócitos estimulados por IL-4 mostrou um aumento nos níveis de NLRP3 e ASC detectados por imunofluorescência, corroborando nossas observações in vivo. Houve, ainda, maior nível de coexpressão desses marcadores nos grupos tratados com Gal-3, em relação aos grupos controle e estimulado com IL-4. Os resultados do nosso estudo evidenciaram o papel da Gal-3 na patogênese da alergia ocular, especialmente na regulação da ativação de mastócitos e dos níveis endógenos de NLRP3, apontando esses marcadores como possíveis alvos terapêuticos para os processos alérgicos oculares.
NLRP3 is a cytoplasmic receptor that, when activated, forms an inflammasome crucial for the release of IL-1β and IL-18, contributing to the pathogenesis of many diseases, including allergic reactions. Studies show that the NLRP3/IL-1β axis plays an important role in the development of allergic conjunctivitis (AC). Furthermore, the expression of the protein galectin-3 (Gal-3) is involved with the activation of the NLRP3 inflammasome and the production of IL-1β by macrophages; however, its relationship with the NLRP3 activation in the allergic response has not yet been elucidated. In this study, we evaluated the role of Gal-3 in the allergic response and its possible relationship with the regulation of the NLRP3 inflammasome, using an experimental model of AC and assays with human keratinocyte cultures stimulated with IL-4. For AC induction, wild-type (WT) and Gal-3 knockout (Gal-3-/-) male BALB/c mice immunized with ovalbumin (OVA) were challenged with eye drops containing OVA on days 14-16, and their palpebral conjunctivae were evaluated under experimental conditions (n = 5 animals/group): SHAM (control), which received only sterile saline, and AC. After 24 hours of the last challenge with OVA, the eyelids were collected for analysis (CEUA 2125200323). AC in WT and Gal-3-/- mice was characterized by a significant increase in the number of eosinophils (68±10 and 73±9, respectively) in the palpebral conjunctiva compared to their respective controls (p < 0.05). The proportion of degranulated mast cells (~60%) was higher in the palpebral conjunctiva of Gal-3-/- AC animals compared to intact cells (~40%); while in the WT AC group, the proportion of intact cells (58%) was higher than that of degranulated cells (42%). Transcriptome analysis showed increased Il1β transcripts in mast cells stimulated with anti-OVA-IgE (GSE96696) and eosinophils stimulated with OVA (GSE57757). Under these conditions, only mast cells showed an increase in Lgals3 transcripts. The palpebral conjunctiva of WT AC mice showed a significant increase in IL-1β, NLRP3, and phosphorylated p38 levels compared to the WT SHAM and Gal-3-/- AC groups. In vitro, the addition of recombinant Gal-3 to IL-4-stimulated keratinocytes showed an increase in NLRP3 and ASC levels detected by immunofluorescence, corroborating our in vivo observations. Moreover, there was a higher level of co-expression of these markers in the groups treated with Gal-3, compared to the control and IL-4-stimulated groups. The results of our study highlighted the role of Gal-3 in the pathogenesis of ocular allergy, particularly in the regulation of mast cell activation and endogenous NLRP3 levels, pointing to these markers as possible therapeutic targets for ocular allergic processes.
NLRP3 is a cytoplasmic receptor that, when activated, forms an inflammasome crucial for the release of IL-1β and IL-18, contributing to the pathogenesis of many diseases, including allergic reactions. Studies show that the NLRP3/IL-1β axis plays an important role in the development of allergic conjunctivitis (AC). Furthermore, the expression of the protein galectin-3 (Gal-3) is involved with the activation of the NLRP3 inflammasome and the production of IL-1β by macrophages; however, its relationship with the NLRP3 activation in the allergic response has not yet been elucidated. In this study, we evaluated the role of Gal-3 in the allergic response and its possible relationship with the regulation of the NLRP3 inflammasome, using an experimental model of AC and assays with human keratinocyte cultures stimulated with IL-4. For AC induction, wild-type (WT) and Gal-3 knockout (Gal-3-/-) male BALB/c mice immunized with ovalbumin (OVA) were challenged with eye drops containing OVA on days 14-16, and their palpebral conjunctivae were evaluated under experimental conditions (n = 5 animals/group): SHAM (control), which received only sterile saline, and AC. After 24 hours of the last challenge with OVA, the eyelids were collected for analysis (CEUA 2125200323). AC in WT and Gal-3-/- mice was characterized by a significant increase in the number of eosinophils (68±10 and 73±9, respectively) in the palpebral conjunctiva compared to their respective controls (p < 0.05). The proportion of degranulated mast cells (~60%) was higher in the palpebral conjunctiva of Gal-3-/- AC animals compared to intact cells (~40%); while in the WT AC group, the proportion of intact cells (58%) was higher than that of degranulated cells (42%). Transcriptome analysis showed increased Il1β transcripts in mast cells stimulated with anti-OVA-IgE (GSE96696) and eosinophils stimulated with OVA (GSE57757). Under these conditions, only mast cells showed an increase in Lgals3 transcripts. The palpebral conjunctiva of WT AC mice showed a significant increase in IL-1β, NLRP3, and phosphorylated p38 levels compared to the WT SHAM and Gal-3-/- AC groups. In vitro, the addition of recombinant Gal-3 to IL-4-stimulated keratinocytes showed an increase in NLRP3 and ASC levels detected by immunofluorescence, corroborating our in vivo observations. Moreover, there was a higher level of co-expression of these markers in the groups treated with Gal-3, compared to the control and IL-4-stimulated groups. The results of our study highlighted the role of Gal-3 in the pathogenesis of ocular allergy, particularly in the regulation of mast cell activation and endogenous NLRP3 levels, pointing to these markers as possible therapeutic targets for ocular allergic processes.
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Citação
LOURENÇO, Renata Pereira. Papel da Galectina-3 na regulação do inflamassoma NLRP3 na resposta alérgica: estudo em modelos in vivo e in vitro. 2024. 70 f. Trabalho de Conclusão de Curso (Graduação em Biomedicina) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.