Broad Institute of MIT and Harvard, Cambridge, MA;
Broad Institute of MIT and Harvard, Cambridge, MA.
Immunohorizons. 2021 Oct 19;5(10):818-829. doi: 10.4049/immunohorizons.2100010.
In this study, we report that the TLR4 ligand, LPS, and TLR3 ligand polyinosinic:polycytidylic acid failed to activate IRF3 or STAT1 in bone marrow-derived macrophages (BMMs) isolated from two independently generated lines of Rosa26-integrated Cas9-expressing C57BL/6J (B6) mice. RNA-sequencing analysis reveals that hundreds to thousands of genes including IFN-stimulated genes were differentially expressed in BMMs from these Cas9 strains compared with B6 upon LPS stimulation. Furthermore, the NF-κB signaling axis and TRIF-mediated necroptosis were also strongly reduced in response to LPS and polyinosinic:polycytidylic acid. In contrast, there were no defects in the responses of BMMs to ligands of the RIG-I, STING, TLR2, TLR9, and IFN receptors. Defects in TLR3 and TLR4 signaling were observed in mice with the B6 but not 129 background, and when Cas9 was integrated at the Rosa26 but not H11 locus. However, integration at the Rosa26 site, CAG promoter-driven Cas9 or eGFP were not individually sufficient to cause the defect. Taken together, the results of this study suggest a putative TRIF-mediated defect in TLR-3/4 signaling in BMMs from commercially available and widely used B6-Cas9-expressing mice.
在这项研究中，我们报告称，TLR4 配体 LPS 和 TLR3 配体聚肌苷酸：聚胞苷酸未能激活来自两个独立生成的 Rosa26 整合 Cas9 表达 C57BL/6J（B6）小鼠系的骨髓来源巨噬细胞（BMM）中的 IRF3 或 STAT1。RNA 测序分析显示，与 LPS 刺激下的 B6 相比，来自这些 Cas9 株的 BMM 中有数百到数千个基因（包括 IFN 刺激基因）的表达存在差异。此外，NF-κB 信号轴和 TRIF 介导的坏死也强烈减少对 LPS 和聚肌苷酸：聚胞苷酸的反应。相比之下，BMM 对 RIG-I、STING、TLR2、TLR9 和 IFN 受体配体的反应没有缺陷。在具有 B6 但不是 129 背景的小鼠中观察到 TLR3 和 TLR4 信号转导的缺陷，并且当 Cas9 整合在 Rosa26 而不是 H11 基因座时也是如此。然而，整合到 Rosa26 位点、CAG 启动子驱动的 Cas9 或 eGFP 单独不足以引起缺陷。总之，这项研究的结果表明，商业上可获得和广泛使用的 B6-Cas9 表达小鼠的 BMM 中存在假定的 TRIF 介导的 TLR-3/4 信号转导缺陷。
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