Transcriptional alterations in human dendritic cells and CD4+ t cells in response to Paracoccidioides brasiliensis

Abstract

The dimorphic fungus Paracoccidioides brasiliensis (P. brasiliensis) is the etiologic agent of paracoccidioidomycosis (PCM), a systemic mycosis that is endemic in Latin America. Studies aiming to characterize the immune response in PCM have turned to the understanding of the role of antigen-presenting cells since the fine regulation of this response is due, in large part, to the initial interaction between the fungus and dendritic cells (DCs) that act as a bridge between innate and adaptive immunity, setting the type of immune response to be developed. In this context, previous studies aimed to assess during the interaction of human DCs with P. brasiliensis was the release of lipid mediators such as prostaglandins and consequences of this release for the functions of these cells in response to the fungus. Contrary to expectation, we found that P. brasiliensis inhibits endogenous PGE2 production by DCs. We also show that the PRR involved in the interaction DC: fungus is mannose receptor whose binding result in signal transduction for inhibition of PGE2 production. These results quite intriguing demanded the continuation of studies in order to understand the consequences of inhibiting the production of prostaglandins on the response of DCs to the fungus. Accordingly, we hypothesized that inhibition of prostaglandin production by DCs, when meeting with the fungus, could be linked to the immunopathogenesis in paracoccidioidomycosis. This inhibition would result in a decrease in expression of the enzyme indoleamine 2, 3 dioxygenase (IDO) with consequent failure in the induction of regulatory T cells (TReg) and preferential induction of Th17 that for fungal infections may be associated with the development of a protective immune response is not fungal destruction and ineffective, despite the intense inflammatory infiltrate that was associated with tissue injury. To test our hypothesis, this project has been formulated with the aim of analyzing the global transcriptional profile of DCs in response to P. brasiliensis, with special attention to genes involved in the control of IDO and metabolism of arachidonic acid (phospholipase A2, COX 1 and COX 2). Will also be assessed the transcriptional profile of CD4+ lymphocytes co-cultured with DCs sensitized with P. brasiliensis. The transcriptional profile of the two cell populations will be performed using the technique RNAseq. In addition to this assessment, the expression of RNAs bound to certain molecules of greatest interest will be validated by RT-qPCR. In the DCs are valued expression of messenger RNA (mRNA) for the IDO and important enzymes for metabolism of prostaglandins such as phospholipase A2 (PLA2) and cyclooxygenase (COX-1 and COX-2). CD4+ cells are assessed for their transcriptional profile by the technique of RNA-seq and as to their phenotype by evaluating the expression of the major surface molecule CD4+ and detection of intracytoplasmic IFN-³, IL-4 and IL-17 by flow cytometry. Similarly to the proposed DCs, the expression of RNAs bound to proteins of greatest interest are validated by RT-qPCR as RNAs encoding transcription factors involved in the different subpopulations of T lymphocytes as Tbet, Gata-3, ROR³t and Foxp3. In addition, the expression will be evaluated mRNA for the cytokines IFN-³, IL-10, IL-4, IL-17 and TGF-². (AU)