Natsuko Tanimura

Roles for LPS-dependent interaction and relocation of TLR4 and TRAM in TRIF-signaling

Toll-like receptor 4 (TLR4) activates two distinct signaling pathways inducing production of proinflammatory cytokines or type I interferons (IFNs), respectively. MyD88 and TIRAP/Mal are essential adaptor molecules for the former but not for the latter pathway. In contrast, TRIF/TICAM-1 and TRAM/TICAM-2 are essential for both. TIRAP is a sorting adaptor molecule recruiting MyD88 to activated TLR4 in the plasma membrane. TRAM is thought to bridge between TLR4 and TRIF by physical association. Little is known, however, how TRAM interacts with TLR4 or with TRIF during LPS response. Here, we show that TRAM recruits TRIF to the plasma membrane. Moreover, LPS induces upregulation of TLR4-association with TRAM and their subsequent translocation into endosome/lysosome. The internalized signaling complex consisting of TLR4 and TRAM colocalizes with TRAF3, a signaling molecule downstream of TRIF, in endosome/lysosome. These results suggest that TLR4 activates TRIF-signaling in endosome/lysosome after relocation from the cell surface.

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe119 to Lys132 (Arg132 in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly59 was found to be a novel critical amino acid for LPS binding outside the region 119–132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe126 or Gly129 impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.

A protein associated with Toll-like receptor (TLR) 4 (PRAT4A) is required for TLR-dependent immune responses.

Immune cells express multiple Toll-like receptors (TLRs) that are concomitantly activated by a variety of pathogen products. Although there is presumably a need to coordinate the expression and function of TLRs in individual cells, little is known about the mechanisms governing this process. We show that a protein associated with TLR4 (PRAT4A) is required for multiple TLR responses. PRAT4A resides in the endoplasmic reticulum, and PRAT4A knockdown inhibited trafficking of TLR1 and TLR4 to the cell surface and ligand-induced trafficking of TLR9 to lysosomes. Other cell-surface molecules were expressed normally on immunocytes from PRAT4A−/− mice. There was impaired cytokine production to TLR ligands, except to the TLR3 ligand poly(I:C), and to whole bacteria. Activation of antigen-specific T helper type 1 responses were also defective. Moreover, PRAT4A−/− bone marrow chimeric mice were resistant to lipopolysaccharide-induced sepsis. These results suggest that PRAT4A regulates the subcellular distribution and response of multiple TLRs and is required for both innate and adaptive immune responses.

Cathepsins are required for Toll-like receptor 9 responses

Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9.

A Protein Associated with Toll-Like Receptor 4 (PRAT4A) Regulates Cell Surface Expression of TLR4

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.

High-Density Lipoprotein Suppresses the Type I Interferon Response, a Family of Potent Antiviral Immunoregulators, in Macrophages Challenged With Lipopolysaccharide

Background— High-density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that HDL might also inhibit atherogenesis by combating inflammation. Methods and Results— To identify potential antiinflammatory mechanisms, we challenged macrophages with lipopolysaccharide, an inflammatory microbial ligand for Toll-like receptor 4. HDL inhibited the expression of 30% (277 of 911) of the genes normally induced by lipopolysaccharide, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by Toll-like receptor 4 and the TRAM/TRIF signaling pathway. Unexpectedly, the ability of HDL to inhibit gene expression was independent of macrophage cholesterol stores. Immunofluorescent studies suggested that HDL promoted TRAM translocation to intracellular compartments, which impaired subsequent signaling by Toll-like receptor 4 and TRIF. To examine the potential in vivo relevance of the pathway, we used mice deficient in apolipoprotein A-I, the major protein of HDL. After infection with Salmonella typhimurium, a Gram-negative bacterium that expresses lipopolysaccharide, apolipoprotein A-I–deficient mice had 6-fold higher plasma levels of interferon-&bgr;, a key regulator of the type I interferon response, than did wild-type mice. Conclusions— HDL inhibits a subset of lipopolysaccharide-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection.

Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation

Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3–coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase Cγ1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

Serum Amyloid A3 Binds MD-2 To Activate p38 and NF-κB Pathways in a MyD88-Dependent Manner

Serum amyloid A (SAA) 3 is a major component of the acute phase of inflammation. We previously reported that SAA3 served as an endogenous peptide ligand for TLR4 to facilitate lung metastasis. Because these experiments were performed with SAA3 recombinant proteins purified from Escherichia coli or mammalian cells, we could not rule out the possibility of LPS contamination. In this study, we used SAA3 synthetic peptides to eliminate the presence of LPS in SAA3. We found that the SAA3 synthetic peptide (aa 20–86) (20–86) stimulated cell migration and activated p38 in a manner dependent on TLR4, MD-2, and MyD88. SAA3 (20–86) also activated NF-κB and Rho small GTPase. Using surface plasmon resonance analysis, the binding constant KD values between SAA3 (20–86) or SAA3 (43–57) and TLR4/MD-2 protein highly purified by the baculovirus system were 2.2 and 30 μM, respectively. FLAG-tagged SAA3 tightly bound to protein A–tagged MD-2, but not to TLR4 in baculovirus coinfection experiments. Although SAA3 (20–86) caused a low, but appreciable level of endocytosis in TLR4, it induced the upregulation of both IL-6 and TNF-α, but not IFN-β1. An i.v. injection of SAA3 (43–57) induced the lung recruitment of CD11b+Gr-1+ cells at an estimated serum concentration around its KD value toward TLR4/MD-2. Taken together, these results suggest that SAA3 directly binds MD-2 and activates the MyD88-dependent TLR4/MD-2 pathway.

Chemical synthesis of Helicobacter pylori lipopolysaccharide partial structures and their selective proinflammatory responses.

Helicobacter pylori is a common cause of gastroduodenal inflammatory diseases such as chronic gastritis and peptic ulcers and also an important factor in gastric carcinogenesis. Recent reports have demonstrated that bacterial inflammatory processes, such as stimulation with H. pylori lipopolysaccharide (LPS), initiate atherosclerosis. To establish the structures responsible for the inflammatory response of H. pylori LPS, we synthesized various kinds of lipid A structures (i.e., triacylated lipid A and Kdo-lipid A compounds), with or without the ethanolamine group at the 1-phosphate moiety, by a new divergent synthetic route. Stereoselective α-glycosylation of Kdo N-phenyltrifluoroacetimidate was achieved by use of microfluidic methods. None of the lipid A and Kdo-lipid A compounds were a strong inducer of IL-1β, IL-6, or IL-8, suggesting that H. pylori LPS is unable to induce acute inflammation. In fact, the lipid A and Kdo-lipid A compounds showed antagonistic activity against cytokine induction by E. coli LPS, except for the lipid A compound with the ethanolamine group, which showed very weak agonistic activity. On the other hand, these H. pylori LPS partial structures showed potent IL-18- and IL-12-inducing activities. IL-18 has been shown to correlate with chronic inflammation, so H. pylori LPS might be implicated in the chronic inflammatory responses induced by H. pylori. These results also indicated that H. pylori LPS can modulate the immune response: NF-κB activation through hTLR4/MD-2 was suppressed, whereas production of IL-18 and IL-12 was promoted.

Palmitoylation-Dependent Plasma Membrane Transport but Lipid Raft-Independent Signaling by Linker for Activation of T Cells

Linker for activation of T cells (LAT) is a dually palmitoylated transmembrane adaptor protein essential for T cell development and activation. However, whether LAT palmitoylation and/or lipid raft localization are required for its function is controversial. To address this question, we used a combination of biochemical, imaging, and genetic approaches, including LAT retrovirus-transduced mouse T cells and bone marrow chimeric mice. A nonpalmitoylated, non-lipid raft-residing mutant of transmembrane LAT could not reconstitute T cell development in bone marrow chimeric mice. This mutant was absent from the plasma membrane (PM) and was restricted mainly to the Golgi apparatus. A chimeric, nonpalmitoylated LAT protein consisting of the PM-targeting N-terminal sequence of Src kinase and the LAT cytoplasmic domain (Src-LAT) localized as a peripheral membrane protein in the PM, but outside lipid rafts. Nevertheless, Src-LAT restored T cell development and activation. Lastly, monopalmitoylation of LAT on Cys26 (but not Cys29) was required and sufficient for its PM transport and function. Thus, the function of LAT in T cells requires its PM, but not raft, localization, even when expressed as a peripheral membrane protein. Furthermore, LAT palmitoylation functions primarily as a sorting signal required for its PM transport.