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Abstract
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.
Highlights
The uterus is an organ with a complex immune repertoire, required to maintain an immune-tolerant environment during the development of a semi-allogenic fetus while retaining its ability to detect and respond to infectious agents if required
IL-1β protein levels were quantified by ELISA and found to be significantly elevated in animals diagnosed with cytological endometritis and purulent vaginal discharge (PVD) at both time points (Figure 1A)
nod-like receptor protein 3 (NLRP3) demonstrated the highest level of expression (Figure 1B) and so the NLRP3 inflammasome became the focus of our subsequent investigations into the IL-1β regulation in the endometrium
Summary
The uterus is an organ with a complex immune repertoire, required to maintain an immune-tolerant environment during the development of a semi-allogenic fetus while retaining its ability to detect and respond to infectious agents if required. Parturition is quickly followed by bacterial contamination of the uterus and tissue involution, resulting in a heightened inflammatory environment within the uterus post-partum, driven by local microbial associated molecular patterns (MAMPs) and damage associated molecular patterns (DAMPs). This has been described as a normal physiological inflammatory event, required for adequate tissue remodeling and restoration of homeostasis in the uterus in preparation for future conception [1]. The molecular and physiological events surrounding the switch from a healthy inflammatory state in the uterus to that of a pathological phenotype remain poorly understood. IL-1β levels were recently shown to be significantly higher in cervical-vaginal mucus samples from cows with endometritis when compared to healthy cows 7 and 21 DPP [5]
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