Dysregulated IL-1β signaling is implicated in the pathogenesis of auto-inflammatory and autoimmune disorders. However, how human monocytes – the main source of IL-1β in patients - activate the NLRP3 inflammasome and release IL-1β remains ambiguous. The ‘alternative’ NLRP3 inflammasome pathway has recently been proposed as an active secretion pathway of IL-1β primarily based on studies in the immortalized BLaER1 cell line. In this pathway, the NLRP3 inflammasome promotes secretion of IL-1β without inducing ASC speck formation and pyroptosis. However, immortalized cell lines may not fully recapitulate the mechanism operating in primary human monocytes, and rare events may not be captured reliably in bulk cell population studies. Given the importance of IL-1β in acute and chronic diseases, we propose to characterize IL-1β secretion at the single cell level in primary human monocytes. Our preliminary results suggest that TLR4 stimulation induces NLRP3-dependent IL-1β secretion concomitant with ASC speck assembly and cell death in a subpopulation of about 5% of LPS-stimulated human primary monocytes. The proposed studies will clarify the mechanisms that govern IL-1β secretion in primary human monocytes at the single cell level. This proposal thus has high potential to definitively settle a longstanding scientific debate, and reveal novel therapeutic strategies to therapeutically modulate IL-1β secretion levels in patients.