The role of NLRP3–PGE2 axis in mediating interactions between bovine endometrial myofibroblasts and M1 macrophages

The bovine endometrium undergoes dynamic structural and functional changes during the estrous cycle, driven by coordinated interactions between stromal and immune cells. Among the endometrial stromal populations, (myo)fibroblasts and macrophages play key roles in tissue remodeling, inflammation, and repair. However, their direct cellular communication and underlying molecular mechanisms are not fully understood. In this study, we investigated the bidirectional crosstalk between bovine endometrial myofibroblasts and M1 macrophages. Treatment of endometrial myofibroblasts with M1 macrophage-conditioned medium (M1ø CM) increased mRNA expression and protein abundance of fibroblast growth factor 2 (FGF-2) and matrix metalloproteinase 9 (MMP-9), while reducing mRNA expression and protein abundance of connective tissue growth factor (CTGF). No change was observed in the protein abundance of vascular endothelial growth factor (VEGF). Conversely, treatment of M1 macrophages with myofibroblast-conditioned medium (MFbCM) upregulated mRNA expression and secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Both conditioned media increased Nod-like receptor pyrin domain-containing protein 3 (NLRP3) mRNA expression and protein abundance in the interacting cell populations. Pharmacological inhibition of NLRP3 with MCC950 (10-5 M) suppressed M1ø CM-induced FGF-2 and MMP-9 mRNA expression and protein abundance in myofibroblasts, and reduced MFbCM-induced IL-6 and TNF-α mRNA expression and secretion in M1 macrophages. Furthermore, prostaglandin E2 (PGE2) was identified as a downstream mediator of NLRP3 signaling, regulating macrophage-myofibroblast communication. Collectively, these findings reveal a pivotal role for the NLRP3-PGE2 axis in mediating macrophage-stromal crosstalk, providing new insight into inflammatory and reparative dynamics within the bovine endometrium.