Analyzing the biological significance in intracellular localization of Toll Like Receptors

Toll样受体（TLR）可检测多种微生物产物。TLR激活先天免疫应答并引发适应性免疫应答。TLR 4/MD-2是LPS的传感器，从细胞表面传递MyD 88依赖的信号，然后运输到内溶酶体并传递TRIF/TICAM-1依赖的信号。这两种信号被认为依赖于细胞表面TLR 4/MD-2。尽管TLR 4/MD-2也位于再循环内体、高尔基体或内质网中，但关于胞内TLR 4/MD-2在LPS应答中的作用知之甚少。我们在这里研究了巨噬细胞中的细胞内LPS传感。PRAT 4A（与TLR 4A相关的蛋白质）是一般伴侣蛋白gp 96的共伴侣蛋白，并且是TLR 4/MD-2的细胞表面表达所需的。在PRAT 4A缺陷型巯基乙酸盐诱导的腹膜巨噬细胞（P-Macs）和骨髓源性巨噬细胞（BM-Macs）上检测不到细胞表面TLR 4/MD-2。在PRAT 4A缺陷的P-Macs中，LPS反应全部消失，而在PRAT 4A缺陷的BM-Macs中，仍可检测到部分LPS反应。值得注意的是，PRAT 4A缺陷型BM-Mac中的LPS应答不一定依赖于TRIF/TICAM-1信号传导。PRAT 4A缺陷BM-Mac显示TRIF/TICAM-1依赖性趋化因子RANTES（CCL 5）和MyD 88依赖性趋化因子MCP-1（CCL 2）的产生未受损。此外，共刺激分子CD 40和CD 86的上调也没有改变。相反，TRIF/TICAM-1依赖的I型IFN的产生受到严重损害。这些结果表明，细胞内TLR 4/MD-2负责独特的一组LPS应答。

Toll-like receptor s(TLRs) sense a variety of microbial products. TLRs activate innate immune responses and prime adaptive immune responses. TLR4/MD-2, a sensor for LPS, delivers the MyD88-dependent signal from the cellsurface, then traffics to endolysosomes and delivers the TRIF/TICAM-1-dependent signal. Both signals are thought to be dependent on cell surface TLR4/MD-2. Although TLR4/MD-2 is located also in recycling endosomes, the Golgi apparatus or the endoplasmic reticulum, little is known abouta role for intracellular TLR4/MD-2 in LPS responses. We here studied intracellular LPS sensing in macrophages. PRAT4A (protein associated with TLR4 A) is a cochaperone for a general chaperone gp96 and required for cell surface expression of TLR4/MD-2. Cell surface TLR4/MD-2 was undetectable on PRAT4A deficient thioglycollate-elicited peritoneal macrophage cells (P-Macs) and bone marrow-derived macrophages (BM-Macs). LPS responses were all abolished in PRAT4A deficient P-Macs, whereas a part of LPS responses remained detectable in PRAT4A deficient BM-Macs. Of note, LPS responses in PRAT4A deficient BM-Macs were not necessarily dependent on TRIF/TICAM-1 signaling. PRAT4A deficient BM-Macs showed unimpaired production of both TRIF/TICAM-1-dependent chemokine RANTES (CCL5) and MyD88-dependent chemokine MCP-1 (CCL2). Moreover, up-regulation of co-stimulatory molecules, CD40 and CD86 was notaltered. In contrast, TRIF/TICAM-1-dependent production of type I IFN was profoundly impaired. These results demonstrate that intracellular TLR4/MD-2 is responsible for unique set of LPS responses.