RESUMO
miRNAs are important immune regulators in the control of the CD4â¯+â¯T cells phenotype. miR-326 regulates the differentiation towards Th17 cells and the inhibition of miR-155 is associated with low levels of Treg cells. However, miRNAs expression and transcription factors associated with these lymphocyte subsets in obesity-induced adipose tissue inflammation is still unknown. The aim of this work was to identify Th17 cells in subcutaneous adipose tissue (SAT), proinflammatory cytokine production and their association with the miRNAs and transcription factors involved. We collected SAT samples obtained by lipoaspiration from individuals with normal weight, overweight and obesity. We obtained the stromal vascular fractions and then a Ficoll gradient was performed to obtain adipose tissue mononuclear cells (ATMC). Th17 cells were evaluated by flow cytometry and the expression of miR-326, miR-155, RORC2 and FOXP3 by qRT-PCR. We also analyzed cytokines from the supernatants of the ATMC culture and measured the FOXP3 methylation percentage by bisulfite conversion by PCR. According to the results, the frequency of Th17 cells and RORC2 expression was higher in individuals with obesity and associated with miR-326 expression. The ATMC from this group secreted a proinflammatory cytokine profile by in vitro assay. In contrast, lower levels of mRNA FOXP3 expression was detected in ATMC from individuals with obesity that correlated with methylation percentage of FOXP3 gene but no association with miR-155 was detected. Our results suggested that miR-326 participates in the polarization towards Th17 promoting the inflammatory state in the obesity-induced adipose tissue.
Assuntos
Tecido Adiposo/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Obesidade/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Adulto , Diferenciação Celular , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Humanos , Ativação Linfocitária , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Obesidade/genética , Adulto JovemRESUMO
The BLR-1 and BLR-2 proteins of Trichoderma atroviride are the Neurospora crassa homologs of white collar-1 and -2, two transcription factors involved in the regulation of genes by blue light. BLR-1 and BLR-2 are essential for photoinduction of phr-1, a photolyase-encoding gene whose promoter exhibits sequences similar to well-characterized light regulatory elements of Neurospora, including the albino proximal element and the light response element (LRE). However, despite the fact that this gene has been extensively used as a blue light induction marker in Trichoderma, the function of these putative regulatory elements has not been proved. The described LRE core in N. crassa comprises two close but variably spaced GATA boxes to which a WC-1/-2 complex binds transiently upon application of a light stimulus. Using 5' serial deletions of the phr-1 promoter, as well as point mutations of putative LREs, we were able to delimit an ~ 50 bp long region mediating the transcriptional response to blue light. The identified light-responsive region contained five CGATB motifs, three of them displaying opposite polarity to canonical WCC binding sites. Chromatin immunoprecipitation experiments showed that the BLR-2 protein binds along the phr-1 promoter in darkness, whereas the application of a blue light pulse results in decreased BLR-2 binding to the promoter. Our results suggest that BLR-2 and probably BLR-1 are located on the phr-1 promoter in darkness ready to perform their function as transcriptional complex in response to light.