Folate receptor beta drives NLRP3 inflammasome activation and pyroptosis in macrophages independent of folate binding.

Folate receptor beta (FRβ), encoded by FOLR2, is selectively expressed in monocytes and macrophages, yet its function in innate immune signaling remains poorly defined. Here, we identify FRβ as a novel regulator of NLRP3 inflammasome activation and pyroptosis in human THP-1 macrophages. Using CRISPR/Cas9-mediated gene deletion, we show that loss of FOLR2 severely impaired caspase-1 activation, gasdermin D cleavage, and IL-1β release in response to multiple NLRP3 stimuli, without altering pro-IL-1β induction. These defects were not rescued by exogenous folate and were independent of extracellular folate concentrations. Mechanistically, FOLR2/FRβ appears to potentiate potassium efflux and the expression of multiple potassium channel-encoding genes. Single-cell RNA sequencing revealed broad transcriptional repression in FRβ-deficient macrophages, including genes involved in inflammasome signaling and ion transport. Genome-wide methylation profiling showed increased CpG hypermethylation in FOLR2-deficient cells, consistent with reduced transcriptional activity. Our findings indicate that FRβ promotes NLRP3 activation in a folate-independent manner potentially by regulating DNA methylation, gene transcription, and K+ efflux in macrophages. These findings uncover a previously unrecognized immunoregulatory function for FRβ, positioning it as a potential modulator of macrophage-driven inflammation in contexts such as host defense, autoimmunity, and tissue-specific immune responses at the tumor and maternal-fetal interface.