Role of GPER in the expression and secretion of pro-inflammatory cytokines and calcium signaling in bone marrow-derived macrophages

Abstract

Macrophages play an important role in mediating inflammation in numerous disease states by secreting pro-inflammatory cytokines including IL-1β and IL-18. Macrophages are driven into the M1-proinflammatory phenotype upon detection of pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs). As macrophages are polarized into the M1 phenotype, they upregulate the expression of pro-inflammatory cytokines and components of the NLRP3 inflammasome. An additional activating stimulus is required to induce the assembly of the NLRP3 inflammasome, which facilitates the activation of pro-caspase 1 and the subsequent conversion of the pro-forms of both IL-18 and IL-1β into their mature forms for their secretion through gasdermin D membrane pores. A canonical activator of the NLRP3 inflammasome is extracellular ATP. ATP activation of the P2X7 receptor induces potassium efflux and calcium influx, both of which are necessary for NLRP3 inflammasome activation. Previous studies suggest that the G protein-coupled receptor (GPER) agonist G-1 reduced inflammation in animal disease models. However, the GPER-dependent effect of G-1 on pro-inflammatory cytokine expression and NLRP3 inflammasome activation in macrophages is unknown. In the current study, we utilized bone marrow-derived macrophages (BMDMs) from either WT or GPER-/- mice to test the hypothesis that G-1 reduces inflammatory cytokine production and NLRP3 inflammasome expression in a GPER-dependent manner. We found that the treatment of WT BMDMs with 100 nM G-1 for 24 hrs inhibited LPS-induced NLRP3, pro-IL-1β, IL-6, CCL5 and expression, and IL-1β secretion. Importantly, G-1 had no effect on GPER-/- BMDMs. To begin to determine if GPER modulates NLRP3 inflammasome activation, we characterized ATP-induced Ca2+ signals in both WT and GPER BMDMs with and without LPS pretreatment. No differences were observed between unprimed WT and GPER-/- BMDMs with respect to basal Ca2+ level, ATP-induced intracellular Ca2+ release, and subsequent bulk Ca2+ signal upon reestablishment of extracellular Ca2+. However, priming with LPS significantly increased ATP-induced intracellular Ca2+ release in both WT and GPER-/- BMDMs. Compared to WT cells, LPS-primed GPER-/- BMDMs exhibit significantly higher basal Ca2+ yet significantly lower ATP-induced Ca2+ release. Consistently, 15-minute treatment with 100 nM G-1 agonist strongly reduces ATP-induced Ca2+ release in WT BMDMs but had no effect in GPER-/- BMDMs. These results suggest a role for GPER in mediating Ca2+ uptake into IP3-sensitive intracellular compartments in BMDMs during LPS priming. In keeping with the known role for mobilization of intracellular Ca2+ in ATP-induced secretion of pro-inflammatory cytokines, preliminary data suggests that short-term G-1 pretreatment reduced IL-1β secretion induced by ATP in LPS-primed BMDMs. Overall, these data indicate a role for GPER in the priming and activation of the NLRP3 inflammasome. This research was supported by Des Moines University IOER Research & Grant Award #03-22-10. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.