Inflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this
inflammation, resulting in
endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory
cytokine IL-1β early post-partum, in cows which subsequently develop
endometritis. As a result, targeting IL-1β expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1β expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with
endometritis at 21 DPP and cows that experienced a physiological level of
inflammation throughout involution. IL-1β was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1β
protein levels were significantly higher in animals that proceeded to develop
endometritis at 21 DPP. IL-1β production could be detected in
luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1β expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the
inflammasome activator
nigericin. Stromal fibroblast cells were particularly potent producers of IL-1β. Basolateral LPS stimulation of polarized epithelial cells induced IL1B
mRNA and a previously undescribed IL-1β
protein isoform, with preferential
protein secretion into the apical compartment. Key
inflammasome components [
nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like
protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (
MCC950) and pan-
caspase (
Z-VAD-FMK) inhibitors blocked IL-1β production, demonstrating its dependence on the NLRP3
inflammasome and on
caspase activity. Furthermore, caspase-4 specific
siRNA prevented IL-1β production, confirming that
inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of
inflammasome assembly and activation has critical relevance for our understanding of
inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including
endometritis in cattle.