Synthetic Pam3Cys was purchased from EMC Microcollections. for the first time that TLR10 is a modulatory pattern-recognition receptor with mainly inhibitory properties. Highly conserved molecular structures of invading microorganisms are recognized by immune cells through pattern-recognition receptors, of which Toll-like receptors (TLRs) Rabbit Polyclonal to E-cadherin are the most documented family. In humans, 10 members of the TLR family have been described (1). In general, specific ligation of TLRs leads to induction of proinflammatory mediators, such as cytokines and chemokines. One member of the TLR family however, TLR10, is considered an orphan receptor because of its still-unknown ligands and function. Human is encoded on chromosome 4 within the gene cluster, together with 0.001) (Fig. 1 and 0.05), and to a somewhat lesser extent for TNF- (Fig. 1 and and (B.b). After stimulation, cell supernatants were collected and proinflammatory cytokines were measured using ELISA ABT-751 (E-7010) (test; * 0.05, ** 0.01, *** 0.001. TLR10 Overexpression Inhibits TLR2-Mediated Signaling. To explore the mechanisms through which TLR10 exerts the suppressive function, HEK293 cells were transfected with either TLR2 alone, or TLR2 together with TLR1, TLR6, or TLR10. HEK cells were cultured in the presence of either the TLR2 ligand Pam3Cys or for 24 h. IL-8 production was observed when HEK293 cells transfected with TLR2 alone were exposed to TLR2 ligands. In contrast, HEK293 cells cotransfected with both TLR2 and TLR10 showed a strong reduction of IL-8 production (Fig. 2 and (B.b.). (test, two-sided, ** 0,01. (per mL. (in monocyte-derived macrophages. To assess the efficiency of the siRNA approach, we determined the mRNA levels in the silenced macrophages (Fig. 2After incubation of the cells with the specific siRNA, cells were exposed to for 24 h. Up-regulation of IL-6 production by the Gene. A strong argument in favor of TLR10 being a functional receptor is that the gene is under selection pressure in European populations; nonsynonymous polymorphisms within the human gene are considered to influence TLR10 function (11). Two of these SNPs, N241H (rs11096957) and M326T (rs11466653), ABT-751 (E-7010) have been found to be associated with reduced risk of prostate cancer and were described to be possibly damaging to the gene (11, 12). We investigated whether the presence of these SNPs in affects the TLR2-induced cytokine response by PBMCs obtained from healthy individuals with different genotypes. Within the group of the 112 volunteers screened, 40 were identified as bearing the wild-type allele, 60 were heterozygous, and 12 were homozygous for the N241H SNP. PBMCs isolated from individuals bearing the three different genotypes were incubated with RPMI, Pam3Cys, live (Fig. 3 polymorphisms, again underscoring the selective interaction of TLR10 with TLR2 (Fig. 3 polymorphisms (Fig. S2). Open in a separate window Fig. 3. SNP Asn241His leads to increased cytokine production and is dependent on TLR2-mediated responses. 5 105 PBMCs from 40 individuals without the N241H polymorphism (wt; wild-type; white bars), 60 individuals with the SNP in one of both alleles (he; heterozygous; gray bars), and 12 homozygous individuals (ho; black bars) were stimulated for 24 h at 37 C with RPMI, Pam3Cys, 0.05, ** 0.01, *** 0.001. The 5 105 PBMCs from individuals without the Asn241His polymorphism (wt; wild-type; white bars), individuals with the SNP in one of both alleles (he; heterozygous; gray bars), and homozygous individuals (ho; black bars) were stimulated for 24 h at 37 C with either medium (RPMI), 50 g/mL Poly IC, 5 g/mL CpG, ABT-751 (E-7010) or 100 ABT-751 (E-7010) ng/mL flagellin. IL-1 (and Table S1). Among these genes, the gene encoding for IL-1 receptor antagonist (testing. Experiment represents six separate measurements. ** 0.01. (and and.