Background: Epididymitis is a major cause of inflammation-associated tissue damage that can impair epididymal function and contribute to male infertility. While infiltrating macrophages are known to drive inflammatory responses, the mechanisms linking immune activation to epithelial injury remain poorly understood. The NLRP3 inflammasome is a key regulator of innate immunity and can trigger pyroptosis, an inflammatory form of programmed cell death. This study investigated whether macrophage inflammasome activation contributes to pyroptotic cell during epididymal inflammation.
Methods: Immortalized mouse epididymal epithelial cells (mECap18) and murine macrophages (RAW 264.7) were stimulated with lipopolysaccharide (LPS) and canonical NLRP3 activators. Inflammasome activation was assessed by Western blot analysis of NLRP3, Caspase-1, IL-1β, and Gasdermin D, while IL-1β secretion was quantified by ELISA. To investigate macrophage-epithelial crosstalk, conditioned medium from inflammasome-activated RAW 264.7 cells was transferred to mECap18 cultures. Pyroptosis was assessed by Gasdermin D immunocytochemistry and lactate dehydrogenase (LDH) release assays.
Results: RAW 264.7 macrophages exhibited robust canonical NLRP3 inflammasome activation, including caspase-1 activation and IL-1β secretion. In contrast, mECap18 cells expressed inflammasome-associated proteins but did not release detectable IL-1β following stimulation. However, exposure to conditioned medium from inflammasome-activated macrophages induced pyroptotic cell death in mECap18 cells, evidenced by Gasdermin D activation and increased LDH release, supporting a role for macrophage-derived inflammatory signals in driving epithelial injury. Despite the absence of IL-1β secretion.
Conclusions: These findings support a model in which macrophage NLRP3 inflammasome activation promotes pyroptotic death of epididymal epithelial cells through paracrine inflammatory signalling. This macrophage-epithelial cell axis provides a potential mechanism linking innate immune activation to tissue damage during epididymitis and identifies the NLRP3-pyroptosis pathway as a promising therapeutic target to preserve epididymal function and male fertility.