Influência de haplótipos no gene interleucina 8 em células com função imune
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Data
2017-09-22
Autores
Pigossi, Suzane Cristina
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Universidade Estadual Paulista (Unesp)
Resumo
Estudos realizados por nosso grupo investigaram polimorfismos no gene Interleucina 8 (IL8) em 500 indivíduos com e sem periodontite e identificaram que o haplótipo ATC/TTC conferiu 2 vezes maior suscetibilidade à doença periodontal crônica (DP) em comparação com o haplótipo ATT/TTC. Estudos clínicos revelaram que indivíduos portadores do haplótipo no gene IL8 associado à DP, mesmo com baixo desafio microbiano, desenvolviam periodontite de maneira mais exacerbada quando comparados aos indivíduos portadores do haplótipo não associado à DP. No entanto, a funcionalidade biológica desses haplótipos no gene IL8 ainda não foi investigada por meio de controlados ensaios in vitro. A proposta deste estudo é avaliar a funcionalidade dos haplótipos no gene IL8 em células imunes, incluindo a investigação por meio da regulação da expressão do gene repórter presente em plasmídeos recombinantes construídos artificialmente (constructos). Foi coletado sangue periférico de pacientes que carregam cada haplótipo e estimulados com mediadores inflamatórios e bactérias periodontopatogênicas para se avaliar a expressão gênica (mRNA, RT-qPCR), proteínas secretadas (Multiplex), perfil fenotípico celular (Citometria de fluxo), fagocitose e produção de espécies reativas de oxigênio (ROS) por macrófagos (Citometria de fluxo) e potencial de migração de linfócitos e neutrófilos. Constructos contendo cada haplótipo foram transfectados em linfócitos T humanos (células JM) e a expressão do gene repórter GFP foi avaliada utilizando citometria de fluxo. Os indivíduos portadores do haplótipo suscetível (ATC/TTC) apresentaram maiores níveis de IL-8, TNF-α (mRNA e proteína), IL-1β, IL-2R, IFN-γ, MIP-1α, MIP-1β e RANTES (proteína). Polarização significativamente maior para o fenótipo Th1/M1 foi observada para o haplótipo 15 suscetível enquanto que o haplótipo não suscetível (ATT/TTC) foi associado a uma resposta atenuada aos periodontopatógenos, com inclinação para o fenótipo Treg. Uma porcentagem mais elevada de macrófagos produtores de ROS tende a ocorrer em indivíduos portadores do haplótipo suscetível, assim como maior migração de neutrófilos após estímulo com IL-8 nesses indivíduos. Em relação à análise dos constructos contendo cada haplótipo isoladamente, foi observado que a presença do alelo T na região promotora foi a condição mais relevante no direcionamento da atividade transcricional do promotor de IL8. Em conclusão, os haplótipos no gene IL8 mostraram-se funcionais para a regulação da resposta imune frente aos periodontopatógenos e aos estímulos inflamatórios.
Studies by this group investigated polymorphisms in the Interleukin 8 (IL8) gene in 500 individuals with and without periodontitis and identified that the ATC/TTC haplotype conferred 2-fold increased susceptibility to periodontal disease (PD) compared to the ATT/TTC haplotype. Clinical studies showed that individuals with IL8 haplotype associated with PD, even with low microbial challenge, responded in a more exacerbated manner when compared to individuals with not susceptible haplotype. However, the biological functionality of these haplotypes in the IL8 gene was not investigated by in vitro controlled assays. . The purpose of this study is to evaluate the functionality of haplotypes in the IL8 gene in immune cells including the investigantion of gene reporter expression present in the recombinant plasmids constructed artificially (constructs). Peripheral blood was collected from patients from each haplotype and stimulated with inflammatory mediators and periodontopathogenic bacteria to evaluate the gene expression (mRNA, RT-qPCR), secreted proteins (Multiplex), cellular phenotype profile (flow cytometry), phagocytosis and reactive oxygen species (ROS) by macrophages (flow cytometry) and migration potential of lymphocytes and neutrophils. Constructs containing each haplotype were transfected into Human lymphocytes (JM cells) and expression of the GFP reporter gene was evaluated using flow cytometry. The individuals with the susceptible haplotype (ATC / TTC) had higher levels of IL-8, TNF- α (mRNA and protein), IL-1β, IL-2R, IFN-γ, MIP-1α, MIP-1β and RANTES (protein). Significantly greater polarization for the Th1 / M1 phenotype was observed for the susceptible haplotype whereas the non-susceptible haplotype (ATT/TTC) was associated with an attenuated response to periodontopathogens, inclined to the Treg 17 phenotype. A higher percentage of macrophages producing ROS tend to occur in individuals carrying the susceptible haplotype, as well as increased migration of neutrophils following IL-8 stimulation in these individuals. Regarding the analysis of the haplotypes alone, it was observed that the presence of the T allele in the promoter region is the most relevant condition in directing the transcriptional activity of the IL8 promoter. In conclusion, the haplotypes in the IL8 gene were functional for the regulation of immune response to periodontopathogens and to inflammatory stimuli.
Studies by this group investigated polymorphisms in the Interleukin 8 (IL8) gene in 500 individuals with and without periodontitis and identified that the ATC/TTC haplotype conferred 2-fold increased susceptibility to periodontal disease (PD) compared to the ATT/TTC haplotype. Clinical studies showed that individuals with IL8 haplotype associated with PD, even with low microbial challenge, responded in a more exacerbated manner when compared to individuals with not susceptible haplotype. However, the biological functionality of these haplotypes in the IL8 gene was not investigated by in vitro controlled assays. . The purpose of this study is to evaluate the functionality of haplotypes in the IL8 gene in immune cells including the investigantion of gene reporter expression present in the recombinant plasmids constructed artificially (constructs). Peripheral blood was collected from patients from each haplotype and stimulated with inflammatory mediators and periodontopathogenic bacteria to evaluate the gene expression (mRNA, RT-qPCR), secreted proteins (Multiplex), cellular phenotype profile (flow cytometry), phagocytosis and reactive oxygen species (ROS) by macrophages (flow cytometry) and migration potential of lymphocytes and neutrophils. Constructs containing each haplotype were transfected into Human lymphocytes (JM cells) and expression of the GFP reporter gene was evaluated using flow cytometry. The individuals with the susceptible haplotype (ATC / TTC) had higher levels of IL-8, TNF- α (mRNA and protein), IL-1β, IL-2R, IFN-γ, MIP-1α, MIP-1β and RANTES (protein). Significantly greater polarization for the Th1 / M1 phenotype was observed for the susceptible haplotype whereas the non-susceptible haplotype (ATT/TTC) was associated with an attenuated response to periodontopathogens, inclined to the Treg 17 phenotype. A higher percentage of macrophages producing ROS tend to occur in individuals carrying the susceptible haplotype, as well as increased migration of neutrophils following IL-8 stimulation in these individuals. Regarding the analysis of the haplotypes alone, it was observed that the presence of the T allele in the promoter region is the most relevant condition in directing the transcriptional activity of the IL8 promoter. In conclusion, the haplotypes in the IL8 gene were functional for the regulation of immune response to periodontopathogens and to inflammatory stimuli.
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Palavras-chave
Expressão gênica, Polimorfismo genético, Doenças periodontais, Gene expression, Genetic polymorphism, Periodontal diseases