INFLAMMASOME ACTIVATED IL-1β AND THE SWITCH TO PATHOLOGICAL INFLAMMATION IN THE BOVINE ENDOMETRIUM

Abstract

Inflammation of the postpartum uterus is a normal physiological event, crucial for tissue involution and repair. Immediately following parturition, the uterus experiences dysregulation of its microbiome and is exposed to inflammatory DAMPs as a result of tissue repair mechanisms. Endometrial epithelial cells and underlying stromal fibroblasts provide appropriate innate defence mechanisms to maintain the local microbiome, while also retaining the capacity to respond to pathogens when required. However, in the bovine, some cows fail to resolve this inflammation, resulting in a switch from this physiological inflammatory event to that of a pathological inflammatory phenotype, termed endometritis, which compromises fertility. Previous work from our group identified elevated levels of IL-1β expression in cows with uterine disease (Foley, Chapwanya et al. 2015, Adnane, Chapwanya et al. 2017). We hypothesized that inflammasome mediated cleavage of the inflammatory cytokine IL-1β is a key driver of pathological inflammation in endometritic cows. We began by establishing a primary culture model of endometrial cell populations. We processed bovine endometrial tissue from non-pregnant bovine uteri immediately post-mortem to obtain primary epithelial cells and stromal fibroblast populations, separated using differential size filtering. Cell transport and isolation conditions were optimised. Following expansion in culture, the purity of cell populations was confirmed using morphology and positive staining for cytokeratin and vimentin which identifies epithelial and stromal fibroblast populations, respectively. Using PCR, cDNA from both cell populations was negative for CD45, a marker of immune cells. On challenge with a bacterial PAMP (LPS), epithelial and stromal fibroblasts showed a marked increase in the expression of the inflammatory mediators IL8, IL6, S100A8 and S100A9, with both cell populations displaying distinct expression profiles. In order to investigate IL-1β as a possible driver of pathological inflammation, endometrial tissue samples were obtained at 7 and 21 days post-partum (DPP) from cows that developed endometritis at 21 DPP and cows that remained healthy throughout involution. IL-1β was measured by ELISA and immunohistochemistry. Seven DPP, endometrial IL-1β protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP with production by the epithelium (both luminal and glandular), underlying stromal fibroblasts as well as infiltrating immune cells. Stimulation of primary endometrial epithelial cells, stromal fibroblasts and PBMCs with LPS and the inflammasome activator nigericin induced high levels of IL-1β. Stromal fibroblast cells were particularly potent producers of IL-1β. Furthermore, 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. Stimulation of polarized epithelial cells following a calcium switch assay with IL-1β demonstrated the ability of this cytokine to delay the re-establishment of the epithelial barrier. Key inflammasome components (NLRP3, NEK7, 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. Interestingly, in contrast to inflammasome pathways detected in other species, expression of caspase-4 but not caspase-1 was detected. Caspase-4 specific siRNA prevented IL-1β production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent. In conclusion, this study has successfully established a reproducible primary culture model of bovine endometrial cell populations. This culture model was used to demonstrate inflammasome activity in endometrial epithelial cells and stromal fibroblasts. Species- and tissue-specific differences in bovine endometrial cell inflammasome assembly were also revealed. Identifying the mechanisms of inflammasome activation operating in the bovine has critical relevance for our understanding of inflammation and suggests new therapeutic targets for the resolution of inflammatory pathologies in the bovine, and in particular for the prevention of bovine endometritis.