TLR4 polymorphisms such as Asp299Gly and Thr399Ile related to Gram-negative sepsis have been reported to result in significantly blunted responsiveness to LPS. Our study group previously screened other TLR4 polymorphic variants by checking the NF-κB activation in comparison to wild type (WT) TLR4 in human embryonic kidney 293T cells. In this study, we found that the Lys694Arg (K694R) polymorphism reduced the activation of NF-κB, and the production of downstream inflammatory factors IL-1, TNF-α and IL-6, representing the K694R polymorphism, led to blunted responsiveness to LPS. Then, we examined the influence of the K694R polymorphism on total and cell-surface TLR4 expression by Western blotting and flow cytometry, respectively, but observed no differences between the K694R polymorphism and WT TLR4. We also used co-immunoprecipitation to determine the interaction of the K694R polymorphism and WT TLR4 with their co-receptor myeloid differentiation factor 2 (MD2) and their downstream signal adaptor MyD88. We found that K694R reduced the recruitment of MyD88 in TLR4 signalling but had no impact on the interaction with MD2.

据报道，与革兰氏阴性脓毒症相关的 TLR4 多态性如 Asp299Gly 和 Thr399Ile 导致对 LPS 的反应显着减弱。我们的研究小组之前通过检查人胚胎肾 293T 细胞中的野生型 (WT) TLR4 与野生型 (WT) TLR4 相比的 NF-κB 活化来筛选其他 TLR4 多态性变体。在这项研究中，我们发现 Lys694Arg (K694R) 多态性降低了 NF-κB 的激活，并且下游炎症因子 IL-1、TNF-α 和 IL-6 的产生，代表 K694R 多态性，导致对脂多糖。然后，我们分别通过蛋白质印迹和流式细胞术检查了 K694R 多态性对总和细胞表面 TLR4 表达的影响，但观察到 K694R 多态性和 WT TLR4 之间没有差异。我们还使用免疫共沉淀来确定 K694R 多态性和 WT TLR4 与它们的共受体骨髓分化因子 2 (MD2) 及其下游信号适配器 MyD88 的相互作用。我们发现 K694R 减少了 MyD88 在 TLR4 信号传导中的募集，但对与 MD2 的相互作用没有影响。