Requerimento de receptores scavenger na sinalização via TLR4 para a produção de óxido nítrico por macrófagos estimulados com LPS: papel na ativação de IRF-3 e STAT-1

Abstract

Nitric oxide (NO) is a versatile molecule with both toxic and regulatory effects. In macrophages, the antimicrobial role of NO is, to date, the most evident, and several lethal or inhibitory mechanisms for a number of microorganisms, due to both direct and indirect effects, have been described. The role of TLR4 in the LPS-induced transcription of iNOS is due to the initiation of a complex cellular signaling that leads to production of the cytokine IFN-β, synthesis of IRF-1 and activation of NF-κB and STAT-1, transcription factors required for iNOS synthesis. The uptake of a high concentration of LPS (1 μg/ml) is mediated by Scavenger Receptor A (SR-A) with participation of CD14 and this receptor acts in concert with TLR4 to mediated LPS inflammatory responses. The literature on signaling SR-A in macrophages is small and often contradictory. Mechanisms by which LPS promotes SR-A interaction with TLR4 and her pathways need to be better understood. Then, our main goal in this study is evaluate the effect of LPS stimulus on TLR4 signaling pathway associated with SR-A to NO production by peritoneal macrophages from C57BL/6 mice. Our experiments confirm that TLR4 knockout mice don’t make NO when stimulated with LPS only, but the addition of IFN-β to the LPS stimulus restores the ability of these macrophages to express iNOS and produce NO. These results suggest that LPS could stimulate macrophages by another receptor different from TLR4. The inhibition of SR-A using DSS and neutralizing antibody shows that SR-A associated with TLR4 is essential for the expression of iNOS and NO production. Our results reinforce that TLR4 and SR-A acts in concert activating mainly TLR4-TRIF pathway for the production of IFN-β and consequently stimulating the synthesis of STAT-1 and IRF-1, they all together activate the NO production by macrophages.