Implicação da GTPase Rap2a na imunidade inata: expressão, ativação e seu papel em macrófagos ativados por receptores do tipo Toll (TLRs)

Abstract

Pathogen recognition is one of the essential functions of the innate immune system, performed by pattern recognition receptors (PRRs) in several cells, such as macrophages and neutrophils. The most studied PRRs are Toll-like receptors (TLRs). The cellular signaling triggered by TLRs leads to the activation of several transcription factors, which in turn regulate several genes related to the inflammatory response. The cellular signaling triggered by TLRs leads to the activation of several transcription factors, which in turn regulate several genes related to the inflammatory response. An important transcription factor in this context is NF-κB, which activation needs to be properly modulated, since its deregulation plays a fundamental role in several pathologies. There are few studies that relate Ras GTPases, proteins involved mainly in cell migration and proliferation, to TLR pathways and NF-κB activation. However, it has been demonstrated that Rap1 is a GTPase activated in the TLR4 pathway and that NF-κB activation is attenuated in macrophages with loss of Rap1 function. Rap1 and Rap2 proteins have a high similarity and although the involvement of Rap1 in TLR-mediated NF-κB activation has already been demonstrated, there are no data on Rap2 proteins in the same context. Furthermore, biochemical approaches have already shown that Rap2a is activated by RasGEF1b, a gene induced by TLR agonists. Thus, the main objective of the present study was to investigate the involvement of Rap2a in innate immune response mediated by Toll-like receptors (TLRs). First, we determined by RT-qPCR that Rap2a mRNA levels are altered in response to stimulation with TLR agonists in immortalized murine macrophages (RAW264.7) and murine bone marrow macrophages (BMDMs). By using western blot assays, we have found that the protein expression of Rap2a is also altered in RAW264.7 cells after stimulation with TLRs agonists. In an intriguing way, we observed that there is no direct correlation between the expression profile of the mRNA and the Rap2a protein after the TLRs agonists treatments. Next, we demonstrated by pull-down assays that Rap2a is activated after stimulation with LPS in RAW264.7 cells and BMDMs. Furthermore, we obtained results from BMDMs devoid of RasGEF1b, in which the activation of Rap2a is reduced compared to wild-type cells. The results described above suggest the implication of Rap2a in TLR triggered pathways. Thus, we performed experiments to obtain functional data for Rap2a in this context. Initially, we performed siRNA transfections in order to silence Rap2a in RAW264.7 macrophages. By reporter gene assays, we observed that xviii NF-кB activation is reduced in RAW264.7 cells transfected with siRNA-Rap2a when compared to those transfected with siRNA-control. In addition, RT-qPCR and ELISA analyzes showed that there is a decrease in CXCL1 and IL-6 production in murine macrophages transfected with siRNA-Rap2a. Furthermore, we evaluated the effect of Rap2a overexpression on the activation of NF-κB induced by several molecules such as agonists of TLRs and proteins downstream of the signaling pathway initiated in TLRs. Interestingly, the results show that when overexpressed, Rap2a also reduces the NF-κB activation induced by these molecules, except for the TLR3 agonist. Taken together, our results demonstrate that Rap2a is a GTPase implicated in the innate immune system in pathways triggered by TLRs in macrophages.