Papoutsopoulou Stamatia, Morris Lorna, Bayliff Andrew, Mair Thomas, England Hazel, Stagi Massimiliano, Bergey François, Alam Mohammad Tauqeer, Sheibani-Tezerji Raheleh, Rosenstiel Philip, Müller Werner, Martins Dos Santos Vitor A P, Campbell Barry J
Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK.
Department of Biochemistry and Biotechnology, School of Health Sciences, University of Thessaly, 413 34 Larissa, Greece.
Biomedicines. 2022 Mar 24;10(4):757. doi: 10.3390/biomedicines10040757.
The NFκB transcription factors are major regulators of innate immune responses, and NFκB signal pathway dysregulation is linked to inflammatory disease. Here, we utilised bone marrow-derived macrophages from the p65-DsRedxp/IκBα-eGFP transgenic strain to study the functional implication of xenogeneic (human) RelA(p65) protein introduced into the mouse genome. Confocal imaging showed that human RelA is expressed in the cells and can translocate to the nucleus following activation of Toll-like receptor 4. RNA sequencing of lipid A-stimulated macrophages, revealed that human RelA impacts on murine gene transcription, affecting both non-NFκB and NFκB target genes, including immediate-early and late response genes, e.g., and . Validation experiments on NFκB targets revealed markedly reduced mRNA levels, but similar kinetic profiles in transgenic cells compared to wild-type. Enrichment pathway analysis of differentially expressed genes revealed interferon and cytokine signaling were affected. These immune response pathways were also affected in macrophages treated with tumor necrosis factor. Data suggests that the presence of xenogeneic RelA protein likely has inhibitory activity, altering specific transcriptional profiles of key molecules involved in immune responses. It is therefore essential that this information be taken into consideration when designing and interpreting future experiments using this transgenic strain.
NFκB转录因子是先天免疫反应的主要调节因子，NFκB信号通路失调与炎症性疾病相关。在此，我们利用来自p65-DsRedxp/IκBα-eGFP转基因品系的骨髓来源巨噬细胞，研究引入小鼠基因组的异种（人类）RelA(p65)蛋白的功能意义。共聚焦成像显示，人类RelA在细胞中表达，并且在Toll样受体4激活后可转运至细胞核。对脂多糖刺激的巨噬细胞进行RNA测序，结果显示人类RelA影响小鼠基因转录，影响非NFκB和NFκB靶基因，包括即刻早期和晚期反应基因，例如 和 。对NFκB靶标的验证实验显示，转基因细胞中的mRNA水平显著降低，但与野生型相比具有相似的动力学特征。对差异表达基因的富集通路分析显示，干扰素和细胞因子信号传导受到影响。在用肿瘤坏死因子处理的巨噬细胞中，这些免疫反应通路也受到影响。数据表明，异种RelA蛋白的存在可能具有抑制活性，改变参与免疫反应的关键分子的特定转录谱。因此，在设计和解释使用该转基因品系的未来实验时，必须考虑到这些信息。
