Mechanisms of signaling talk between trichomonas vaginalis-derived LTB4 and BLT1 on human mast cells

Abstract

Trichomonas vaginalis is a sexually transmitted protozoan parasite that causes vaginitis and cervicitis in women. T. vaginalis itself secretes lipid mediator LTB4, which can activate immune cells. Mast cells are tissue residing immune cells that provoke tissue inflammation in allergic disease and during infection with parasites. In trichomoniasis, a large number of mast cells have been detected in vaginal smears and the vaginal wall. Although T. vaginalis-derived secretory products (TvSP) have been shown to induce migration and degranulation in mast cells, information regarding the signaling mechanisms involved in mast cell activation induced by LTB4 in the TvSP is limited. Herein, I investigated the signaling interation and mechanisms between T. vaginalis-derived lipid mediator LTB4 and its receptor BLT1 on HMC-1 cells during infection of T. vaginalis. First, we examined mast cell responses induced by TvSP. Next, we checked the signaling role of T. vaginalis-secreted lipid mediator LTB4 in mast cell activation. Finally, we examined the intracellular signaling mechanisms of mast cell responses induced by T. vaginalis-secreted LTB4. First, I found that TvSP induced migration and degranulation in HMC-1 cells. TvSP-induced degranulation occurred via exocytosis leading to up-regulation of surface CD63 expression. I also found that NOX2, PKC, PI3K and MAP kinases signaling, calcium influx and nitric oxide participate in TvSP-induced ROS generation, mast cell migration and exocytosis in HMC-1 cells. In addition, TvSP-induced mast cell responses were inhibited by G protein inhibitor, suggesting that G protein-coupled receptors may be involved in mast cell responses induced by TvSP. Secondly, I found that lipid mediator LTB4 in the TvSP plays an important role in provoking mast cell responses via BLT1-mediated signalings. T. vaginalis spontaneously secreted 700 pg/ml of LTB4 per 107 trichomonads. Interestingly, TvSP-induced NOX2 glycosylation, ROS generation, migration and exocytosis were significantly inhibited by pretreatment of TvSP with lipase, but not with heat or proteinase K. Moreover, 5-LO inhibitor-treated trichomonads showed reduced stimulatory effects of TvSP on NOX2 glycosylation, migration and exocytosis. Furthermore, pretreatment of HMC-1 cells with LTB4 receptor BLT1 antagonist or BLT1 siRNA prevented TvSP-induced ROS generation, NOX2 glycosylation, migration and exocytosis. Thirdly, I found that SNAP23-dependent surface trafficking of NOX2 and BLT1 is required for T. vaginalis-derived LTB4-induced mast cell responses. TvSP induced surface trafficking of BLT1 and NOX2, which were expressed in intracellular area, but not on the cell surface in resting HMC-1 cells. Stimulation with modified TvSP collected from trichomonads pretreated with 5-LO inhibitor failed to induce surface trafficking of NOX2 and BLT1. Surface trafficking of NOX2 and BLT1 induced by TvSP was inhibited by pretreatment of cells with BLT1 siRNA and NOX2 siRNA, respectively. Indeed, co-IP assay showed that there was a physical interaction between BLT1 and NOX2, suggesting that there may be a crosstalk between NOX2 and BLT1 in TvSP-stimulated HMC-1 cells. In addition, N-glycosylation of NOX2 induced by TvSP was found to be required for trafficking of NOX2 to the cell surface. I also found that t-SNARE SNAP23 were essential for TvSP-induced ROS generation, surface trafficking of NOX2 and BLT1, and exocytosis in HMC-1 cells. In conclusion, these results suggest that t-SNARE SNAP23-dependent surface trafficking of NOX2 and BLT1 are essential for ROS-dependent migration and exocytotic degranulation in human mast cells induced by T. vaginalis-secreted LTB4. These results can help to unveil the secret of parasitism at the signaling immunological dimension that delicate cross talk mechanism between parasite and host can govern deliberately tissue inflammatory responses.