Global Agromedicine Research Center, Obihiro University of Agricultural and Veterinary Medicine, Obihiro, Japan; Laboratory of Biotechnology and Animal Reproduction, Federal University of Santa Maria, Santa Maria, Brazil.
Global Agromedicine Research Center, Obihiro University of Agricultural and Veterinary Medicine, Obihiro, Japan; Department of Theriogenology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt.
Biochem Biophys Res Commun. 2021 May 14;553:37-43. doi: 10.1016/j.bbrc.2021.03.037. Epub 2021 Mar 22.
Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.
此前，我们报道过多枚第 7 天（D7）牛胚胎在子宫内的存在会诱导循环中性粒细胞（PMN）中全身免疫反应，但具体机制尚不清楚。因此，本研究旨在探究 D7 牛胚胎对 PMN 免疫反应的直接影响，以及这些 PMN 是否可以进一步放大并将胚胎信号传递给另一个 PMN 群体。PMN 直接用胚胎培养液（ECM）或干扰素 tau（IFNT）（10ng/ml）刺激，然后通过实时 PCR 评估 mRNA 表达，通过流式细胞术进行表型分析。为了测试 PMN 是否可以将胚胎信号传递给新的 PMN 群体，用 ECM 或 IFNT 刺激的 PMN 进行彻底洗涤和稀释，以去除任何培养基成分，然后再次在新鲜培养基中孵育 3 小时，收集培养上清液作为 PMN 条件培养基（CM），刺激新的 PMN 群体。与 ECM 类似，IFNT 直接刺激 PMN 中 IFN（IFNA、IFNG）、干扰素刺激基因（ISG；OAS1、ISG15、MX1）、STAT1、TGFB 和 IL8 的表达，并下调 TNFA。流式细胞术分析表明，IFNT 刺激 PMN 中与妊娠相关的表型标志物 CD16 和精氨酸酶 1（ARG1）的表达。最重要的是，PMN CM 诱导新鲜 PMN 中 ISG 和 STAT1 mRNA 的表达。由于 IFNT 直接上调 PMN 中 IFNA 的表达，因此进一步测试了 IFNA 对 PMN 免疫反应的影响。IFNA 刺激 PMN，特别是在低水平（1pg/ml）下，诱导类似于 PMN CM 诱导的 IFNT 样免疫反应。总之，这些发现表明，D7 牛胚胎主要通过 IFNT 诱导 PMN 中直接抗炎反应，上调 ISG 的表达。此外，PMN 可以通过细胞间通讯机制放大并将胚胎信号传递给新的 PMN 群体，该机制可能部分由 IFNA 介导。这种新的免疫相互作用可能有助于牛胚胎耐受和妊娠建立。
