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.

The purpose of this study was to examine the regeneration of periodontal tissue after the application of recombinant human bone morphogenetic protein-2 (rhBMP-2) to horizontal circumferential defects created by experimental periodontitis. Twelve mandibular second premolars in 6 adult beagle dogs were subjected to experimental periodontal breakdown by placing silk ligatures around the teeth until the bone loss exceeded half of the root length. Flap surgery was then performed and the exposed cementum removed. The distance between the bone crest and cemento-enamel junction (CEJ) was about 5 mm. RhBMP-2 (40 micrograms/100 microliters) with a sponge-type carrier material made of gelatin and polylactic acid polyglycolic acid copolymer was placed in the furcation area (5 mm x 5 mm x 5 mm) and around the roots (10 mm x 5 mm x 2.5 mm x 2 pieces). In the control group, the same carrier material without rhBMP-2 was placed in the same manner. The flaps were replaced and sutured to cover these materials completely. Twelve weeks after surgery, the animals were sacrificed and serial sections were prepared in a bucco-lingual plane. Considerable new bone formation was observed in the rhBMP-2-treated sites. New cementum with Sharpey's fibers was observed on the instrumented root surface. On histometric analysis, the amount of new bone, new cementum, and connective tissue attachment was significantly greater in the rhBMP-2-treated group (paired t test; P < 0.01). These results indicate that suitable application of rhBMP-2 can produce considerable periodontal tissue regeneration, even in cases of horizontal circumferential defects.

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.

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.