Estudo da participação de microRNAs na regulação da resposta imune inata de macrófagos murinos à infecção por Paracoccidioides brasiliensis

During host-pathogen interactions both organisms go through a wide genetic reprogramming, a process believed to be crucial in the establishment of the infection. Amongst all the host’s genes showing this altered expression are the ones encoding for microRNAs (miRNAs), small regulatory RNA molecules acting on many biological processes, including regulation of both innate and adaptive immune responses. Although the importance of microRNAs in the immune response to bacteria and viruses by mammalian hosts has been reported by many groups, little is known about their participation in fungal infections. In this context, we aimed at analyzing the role of microRNAs in the innate immune response of murine hosts to infection by Paracoccidioides brasiliensis, the etiological agent of Paracoccidioidomycosis (PCM) – the most prevalent systemic mycosis in Latin America. Initial infection assays performed with peritoneal macrophages from resistant (A/J) and susceptible (B10.a) mouse strains and yeast cells from the virulent isolate Pb18 of P. brasiliensis showed an increase of the levels of five miRNAs: miR-125b, miR-132, miR-146a, miR-155 e miR455, indicating a participation of these molecules on the regulation of the response to P. brasiliensis. However, the similar patterns of miRNA induction on both strains possibly suggests that the miRNAs studied do not have a determinant role in the establishment of the distinct inflammatory responses presented by the two strains in the early stages of interaction with the fungus. Therefore, we chose to perform the following analyses just with the A/J strain. miR-155 was selected for a more complete characterization of its biogenesis and target transcripts regulation based on its key role in regulating many aspects of the immune response and the high levels of differential accumulation in response to P. brasiliensis observed by us. The accumulation of the precursor pri-miR155 observed suggests an influence of the transcription of the gene MIR155HG on the increase in the mature miRNA levels. The changes in the miR-155-3p to miR-155-5p ratio following P. brasiliensis infection may indicate an altered stability degree and turnover rate of the 3p strand. The assessment of the levels of miR-155-5p target mRNAs showed varying results. While upregulation of TNFα and downregulation of SHIP1 are in accordance with the expected effects of miR-155-5p, the high levels of SOCS1 and SOCS3, negatively regulated by this miRNA, reflect the difficulties in establishing direct parallels between the levels of a miRNA and its targets in a context of broad genetic reprogramming. Finally, we performed infection assays using BMDM from TLR4 or dectin-1 knockout mice to assess the role of these receptors in the regulation of miR-155 levels. Results show that in the absence of the receptors miR-155 accumulates in even higher levels, pointing the signaling by TLR4 and dectin-1 as negative regulators, directly or indirectly, of miR-155 in respose to P. brasiliensis. In order to allow the study of the functional role of miRNAs in the immune response, we tested a model for miRNA silencing by in vitro transfection of miRNA inhibitors (antimiR). Preliminary results using fluorescent-labelled antimiR and inhibitors for a control miRNA showed a high transfection efficiency in primary macrophages and proper effect on silencing the control miRNA let-7. In conclusion, the results presented are of great significance for suggesting, for the first time, the involvement of miRNAs in the regulation of the innate immune response to Paracoccidioides brasiliensis. LISTA DE ABREVIATURAS E SIGLAS BMDM – Macrófagos derivados de medula óssea cDNA – DNA complementar Ct – Threshold cycle dNTP – deoxirribonucleotídeos fosfato EGTA – ácido etilenoglicol tetracético FADD Fas-Associated protein with Death Domain HGMA2 High-mobility group AT-hook 2