The interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10. IL-10 is an anti-inflammatory cytokine with immunoregulatory function during the pathogenesis of many inflammatory disorders in livestock, including Johne’s disease (JD). JD is a chronic enteritis in cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is responsible for significant economic losses to the dairy industry. Several candidate genes including IL10RA have been found to be associated with JD. The aim of this study was to better understand the functional significance of IL10RA in the context of immune stimulation with MAP cell wall lysate. An IL10RA knock out (KO) bovine mammary epithelial cell (MAC-T) line was generated using the CRISPR/cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) gene editing system. These IL10RA KO cells were stimulated with the immune stimulant MAP lysate +/− IL-10, or with LPS as a positive control. In comparison to unedited cells, relative quantification of immune-related genes after stimulation revealed that knocking out IL10RA resulted in upregulation of pro-inflammatory cytokine gene expression (TNFA, IL1A, IL1B and IL6) and downregulation of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of pro-inflammatory cytokine signaling. At the protein level knocking out IL10RA also resulted in upregulation of inflammatory cytokines - TNF-α and IL-6 and chemokines - IL-8, CCL2 and CCL4, relative to unedited cells. The findings of this study illustrate the broad and significant effects of knocking out the IL10RA gene in enhancing pro-inflammatory cytokine expression and further support the immunoregulatory role of IL10RA in eliciting an anti-inflammatory response as well as its potential functional involvement during the immune response associated with JD.

中文翻译：

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牛白细胞分枝杆菌亚种对牛白细胞介素10受体α的功能分析。使用CRISPR / Cas9的副结核病裂解液

白细胞介素10受体α（IL10RA）基因编码与细胞因子IL-10结合的IL-10受体的α链。IL-10是一种抗炎细胞因子，在牲畜的许多炎性疾病（包括约翰尼氏病（JD））的发病机理中均具有免疫调节功能。JD是由鸟分枝杆菌亚种引起的牛慢性肠炎。副结核病（MAP），对乳制品行业造成重大经济损失。已经发现包括IL10RA在内的几种候选基因与JD相关。这项研究的目的是为了更好地了解IL10RA在MAP细胞壁裂解液免疫刺激下的功能意义。使用CRISPR / cas9（聚簇有规律间隔的短回文重复序列/ CRISPR相关蛋白9）基因编辑系统生成IL10RA敲除（KO）牛乳腺上皮细胞（MAC-T）系。这些IL10RA KO细胞用免疫刺激剂MAP裂解液+/- IL-10或LPS作为阳性对照刺激。与未经编辑的细胞相比，刺激后免疫相关基因的相对定量显示，敲除IL10RA会导致促炎性细胞因子基因表达（TNFA，IL1A，IL1B和IL6）上调，而细胞因子信号传导抑制因子3（SOCS3）的下调。 ，是促炎性细胞因子信号转导的负调节剂。在蛋白质水平，敲除IL10RA还会导致炎症细胞因子-TNF-α和IL-6和趋化因子-IL-8，CCL2和CCL4的上调，相对于未编辑的单元格 这项研究的结果表明，敲除IL10RA基因在增强促炎细胞因子表达方面具有广泛而显着的作用，并进一步支持IL10RA在引发抗炎反应中的免疫调节作用及其在免疫反应过程中的潜在功能参与与JD相关。