Comparative Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.
Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Grange, Ireland.
Front Immunol. 2019 Feb 5;10:102. doi: 10.3389/fimmu.2019.00102. eCollection 2019.
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 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.
产后子宫炎症是一种正常的生理现象，对于组织退化和修复至关重要。然而，在牛中，一些牛无法解决这种炎症，导致子宫内膜炎，从而降低了生育能力。早期的研究已经发现，在随后发展为子宫内膜炎的牛中，产后早期 IL-1β 的表达上调。因此，靶向 IL-1β 的表达可能是治疗这种疾病的一种新方法，但牛子宫内膜中导致 IL-1β 表达的调节机制尚不清楚。为了研究这一点，从产后 7 天和 21 天（DPP）被诊断为在 21 DPP 发生子宫内膜炎的牛和在退化过程中经历生理水平炎症的牛中获得子宫内膜组织样本。通过 qPCR、ELISA 和免疫组织化学测量 IL-1β。在随后在 21 DPP 发展为子宫内膜炎的动物中，7 DPP 的子宫内膜 IL-1β 蛋白水平显著更高。可以在腔上皮和腺上皮、下基质成纤维细胞以及浸润的免疫细胞中检测到 IL-1β 的产生。为了研究调节 IL-1β 表达的机制，用 LPS 和炎性体激活剂 Nigericin 刺激原代子宫内膜上皮细胞、基质成纤维细胞和 PBMCs。基质成纤维细胞是 IL-1β 的特别强的产生者。极化上皮细胞的基底外侧 LPS 刺激诱导 mRNA 和以前未描述的 IL-1β 蛋白同工型，具有优先的蛋白分泌到顶端隔室。在刺激后，子宫内膜细胞表达关键的炎性体成分[核苷酸结合寡聚化结构域样受体蛋白 3（NLRP3）、NIMA 相关激酶 7（NEK7）、凋亡斑点样蛋白包含 CARD（ASC）和 Gasdermin-D]。在存在 NLRP3 受体（MCC950）和泛半胱天冬酶（Z-VAD-FMK）抑制剂的情况下刺激子宫内膜细胞可阻断 IL-1β 的产生，表明其依赖于 NLRP3 炎性体和半胱天冬酶活性。此外， caspase-4 特异性 siRNA 可防止 IL-1β 的产生，证实炎性体在子宫内膜细胞中的激活依赖于 caspase-4 而不是 caspase-1，这与其他物种一致。鉴定炎性体组装和激活的组织和物种特异性对于我们理解炎症具有重要意义，并表明了新的治疗靶点，以增强包括牛子宫内膜炎在内的炎症性病理的消退。
