Extracellular calcium elicits feedforward regulation of the Toll-like receptor-triggered innate immune response

Despite the expanding knowledge on feedback regulation of Toll-like receptor (TLR) signaling, the feedforward regulation of TLR signaling for the proper innate response to invading microbes is not fully understood. Here, we report that extracellular calcium can coordinate the activation of the small GTPases Ras and Ras-proximate-1 (Rap1) upon TLR stimulation which favors activation of macrophages through a feedforward mechanism. We show that different doses of TLR agonists can trigger different levels of cytokine production, which can be potentiated by extracellular calcium but are impaired by the chelating reagent ethylene glycol tetraacetic acid (EGTA) or by knockdown of stromal interaction molecule 1 (STIM1). Upon TLR engagement, GTP-bound Ras levels are increased and GTP-bound Rap1 is decreased, which can be reversed by EGTA-mediated removal of extracellular calcium. Furthermore, we demonstrate that Rap1 knockdown rescues the inhibitory effects of EGTA on the TLR-triggered innate response. Examination of the TLR signaling pathway reveals that extracellular calcium may regulate the TLR response via feedforward activation of the extracellular signal-regulated kinase signaling pathway. Our data suggest that an influx of extracellular calcium, mediated by STIM1-operated calcium channels, may transmit the information about the intensity of extracellular TLR stimuli to initiate innate responses at an appropriate level. Our study may provide mechanistic insight into the feedforward regulation of the TLR-triggered innate immune response.