The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF.
The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF.
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DOI：
10.4049/jimmunol.1200202
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发表时间：
2012-05-01
期刊：
影响因子：
--
通讯作者：
Medvedev AE
中科院分区：
文献类型：
--
作者：
Figueroa L;Xiong Y;Song C;Piao W;Vogel SN;Medvedev AE
Asp299Gly （D299G）和Thr399Ile (T399I) TLR4多态性在较小程度上与Gram阴性脓毒症和其他感染性疾病有关，但它们影响TLR4信号传导的机制尚不清楚。在这项研究中，我们确定了D299G和T399I多态性对TLR4表达、与髓样分化因子2 （MD2）的相互作用、LPS结合以及LPS介导的MyD88-和TIR结构域适配器激活诱导IFN-β (TRIF)信号通路的影响。将人胚胎肾293/CD14/MD2转染物与野生型（WT）或突变型黄色荧光蛋白（YFP）标记的TLR4变体互补，结果显示TLR4总表达量、TLR4- md -2相互作用和LPS结合相当。使用抗TLR4 Ab进行的FACS分析显示，D299G TLR4的细胞表面水平只有微小的变化。转染D299G TLR4的细胞表现出lps诱导的p38和TANK结合激酶-1磷酸化、NF-κB和IFN调节因子3的激活以及IL-8和IFN-β mRNA的诱导受损，而T399I TLR4未引起统计学上的显著抑制。与WT TLR4相比，在TLR4−/−小鼠巨噬细胞中，D299G突变体的表达未能诱导lps介导的TNF-α和IFN-β mRNA的表达。共免疫沉淀显示，MyD88和TRIF与D299G TLR4的lps驱动相互作用减弱，而WT TLR4则表现出强大的适配器招募。因此，D299G多态性影响了MyD88和TRIF对TLR4的招募，但不影响TLR4的表达、TLR4- md -2的相互作用或LPS的结合，这表明它干扰了TLR4的二聚化和细胞内对接平台的组装，从而招募适配器。
Asp299Gly (D299G) and, to a lesser extent, Thr399Ile (T399I) TLR4 polymorphisms have been associated with Gram negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and TIR domain-containing adapter inducing IFN-β (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein (YFP)-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4-MD-2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK binding kinase-1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-β mRNA, while T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4−/− mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β mRNA. Co-immunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD-2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.
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