N-Acylhomoserine lactones are potent neutrophil chemoattractants that act via calcium mobilization and actin remodeling

In gram-negative bacteria, cell-cell communication based on HSL QS molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior. Using a Transwell migration assay, we found that human primary neutrophils are strongly stimulated by 3O-C(12)-HSL and -C(10)-HSL but not C(4)-HSL in a concentration-dependent manner. Moreover, 3O-C(12)-HSL and -C(10)-HSL activate PLCγ1 but not -γ2, mobilize intracellular calcium, and up-regulate IP(3)R. These changes were paralleled by F-actin accumulation, primarily in the leading edge of neutrophils, as evidenced by phalloidin staining and confocal microscopy. F- and G-actin isolation and quantification by immunoblotting revealed that the F/G-actin ratio was increased significantly after treatment with all three HSLs. Furthemore, 3O-C(12)-HSL- and 3O-C(10)-HSL treatment resulted in phosphorylation of Rac1 and Cdc42. In contrast, C(4)-HSL had negligible influence on the phosphorylation status of PLC and Rac1/Cdc42 and failed to attract neutrophils and induce calcium release. The calcium inhibitor thapsigargin, which blocks ER calcium uptake, strongly prevented neutrophil migration toward 3O-C(12)-HSL and -C(10)-HSL. These findings show that the bacterial QS molecules 3O-C(12)-HSL and -C(10)-HSL may attract human neutrophils to the sites of bacterial infection and developing biofilms. Indeed, recognition of HSL QS signals by neutrophils may play a critical role in their recruitment during infections.