Human TLR10 is an anti-inflammatory pattern-recognition receptor

Abstract

Toll-like receptor (TLR)10 is the only pattern-recognition receptor without known ligand specificity and biological function. We demonstrate that TLR10 is a modulatory receptor with mainly inhibitory effects. Blocking TLR10 by antagonistic antibodies enhanced proinflammatory cytokine production, including IL-1β, specifically after exposure to TLR2 ligands. Blocking TLR10 after stimulation of peripheral blood mononuclear cells with pam3CSK4 (Pam3Cys) led to production of 2,065 ± 106 pg/mL IL-1β (mean ± SEM) in comparison with 1,043 ± 51 pg/mL IL-1β after addition of nonspecific IgG antibodies. Several mechanisms mediate the modulatory effects of TLR10: on the one hand, cotransfection in human cell lines showed that TLR10 acts as an inhibitory receptor when forming heterodimers with TLR2; on the other hand, cross-linking experiments showed specific induction of the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra, 16 ± 1.7 ng/mL, mean ± SEM). After cross-linking anti-TLR10 antibody, no production of IL-1β and other proinflammatory cytokines could be found. Furthermore, individuals bearing TLR10 polymorphisms displayed an increased capacity to produce IL-1β, TNF-α, and IL-6 upon ligation of TLR2, in a gene-dose-dependent manner. The modulatory effects of TLR10 are complex, involving at least several mechanisms: there is competition for ligands or for the formation of heterodimer receptors with TLR2, as well as PI3K/Akt-mediated induction of the anti-inflammatory cytokine IL-1Ra. Finally, transgenic mice expressing human TLR10 produced fewer cytokines when challenged with a TLR2 agonist. In conclusion, to our knowledge we demonstrate for the first time that TLR10 is a modulatory pattern-recognition receptor with mainly inhibitory properties.

Keywords: SNPs; TLR10; cytokines; immunology.

Fig. 1.

Blocking TLR10 results in higher cytokine production. The 5 × 10⁵ PBMCs from 11 to 17 individuals were preincubated for 1 h at 37 °C with either 10 µg/mL aTLR10 antibody (aTLR10, clone 3C10C5) or 10 µg/mL IgG isotype control (IgG). After preincubation, cells were stimulated for 24 h with TLR2 ligand Pam3Cys (10 µg/mL), or live B. burgdorferi (B.b). After stimulation, cell supernatants were collected and proinflammatory cytokines were measured using ELISA (A–D). Bars represent the mean and the SEM. Paired t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

TLR10 transfected HEK cells inhibit cytokine production. HEK293 cells were transiently transfected with TLR2, TLR1/2, TLR2/6, or TLR2/10. (A) Twenty-four hours after transfection, cells were stimulated for 24 h with different concentrations of live B. burgdorferi (B.b.). (B) HEK293 cells transiently transfected using different ratios of plasmid concentrations of TLR10 plasmid. Cells were stimulated 24 h after transfection for 22 h with specific TLR ligands Pam3Cys or FSL-1. IL-8 production (in pg/mL) was measured in the cell-free supernatant using ELISA. (C) HEK293 cells transiently transfected with TLR5 or TLR5/10 plasmids. After 24 h of recovery, cells were stimulated for 22 h with a dose–response of recombinant flagellin. IL-8 production was measured by ELISA. Bars represent the mean of six separate experiments ± SEM. Mann–Whtiney U test, two-sided, **P < 0,01. (D) Monocyte-derived macrophages of eight individuals were transfected with scrambled siRNA as a negative control (NC siRNA) or siRNA directed against TLR10 (TLR10 siRNA). (E) After transfection, monocyte-derived macrophages were stimulated with 1.10⁶ live B. burgdorferi per mL. (F) Twenty-four hours after stimulation, IL-6 or TNF-α protein levels were measured in picrograms per milliliter (pg/mL) in the supernatants using specific sandwich-ELISAs.

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, B. burgdorferi, or LPS. IL-1β (A), IL-6 (B), IL-8 (C), and TNF-α (D) were measured in the supernatant using ELISA. Results are shown in nanograms per milliliter (ng/mL) (A–C) or picrograms per milliliter (pg/mL) (E–H). *P < 0.05, **P < 0.01, ***P < 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 μg/mL Poly IC, 5 μg/mL CpG, or 100 ng/mL flagellin. IL-1β (E), IL-6 (F), TNF-α (G), and IL-8 (H) were measured in the supernatant using ELISA. Results are shown in picrograms per milliliter (pg/mL). Bars represent the means ± SEM.

Fig. 4.

TLR10 induces IL-1Ra through MAPK-dependent pathways. Ten micrograms per milliliter anti-TLR10 antibody or 10 µg/mL IgG isotype control were coated to 24-well flat-bottom plates and incubated for 2 h at 37 °C. After washing and blocking, 5 × 10⁶ PBMCs from two donors were added and incubated overnight at 37 °C. (A) Heatmap of the genes found to be up- and down-regulated after adding anti-TLR10 antibody. A magnification of the upper part of the heatmap is shown in the table (Right). Numbers represent fragments per kilobase per million mapped reads values. (B) mRNA expression levels and (C) protein secretion of IL-1Ra were determined. Ten micrograms per milliliter anti-TLR10 antibody or 10 µg/mL IgG isotype control were coated to 96-well flat-bottom plates. After blocking, 5 × 10⁵ PBMCs were added to the coated plates and incubated for 2 h at 37 °C. Bars represent mean ± SEM for at least four donors. (D) The 5 × 10⁵ PBMCs from individuals without polymorphisms (wt; wild-type; white bars), individuals with the I369L SNP (D) or I775V SNP (E) in one of both alleles (he; heterozygous; gray bars), and individuals with the SNP in both alleles (ho; homozygous; black bars), were stimulated for 24 h at 37 °C with RPMI, or B. burgdorferi. IL-1Ra was measured in the supernatant using ELISA. Results are shown in picrograms per milliliter (pg/mL). Bars represent the means ± SEM. (F) Cytokine secretion by PBMC after cross-linking anti-TLR10 antibody. Detection limit IL-1β, IL-6, TNF-α, and IL-8 are, respectively, 39 pg/mL, 156 pg/mL, 78 pg/mL, and 1,560 pg/mL. (G) 5 × 10⁴ A549 cells in RPMI (10% FCS) were preincubated for 30 min at 37 °C with supernatant from cross-linking experiments. In these cross-linking experiments, either IgG or specific anti-TLR10 antibodies (10 µg/mL) were cross-linked to PBMCs for 24 h. Before addition to the A549 cells, this supernatant was preincubated for 1 h at 37 °C with either specific neutralizing antibodies against IL-1Ra (aIL-1Ra), or isotype control antibodies (IgG Goat), both 10 µg/mL. Thereafter, the A549 cells were either stimulated for 24 h with medium or 3 pg/mL active IL-1β. Bars represent means plus SEM, paired t testing. Experiment represents six separate measurements. **P < 0.01. (H) IL-1Ra mRNA expression levels or protein in picrograms per milliliter (pg/mL) (I) in PBMCs (in the absence/presence of anti-TLR10 coated antibodies) with or without PI3k inhibitors 3MA or Wortmannin (Wort). (J) Model of TLR10 function. TLR10 inhibits MAPK signaling, and also induces PI3k which finally results in IL-1Ra production.

Fig. 5.

Human TLR10 inhibits TLR2-mediated responses in vivo. (A) Generation of human TLR10 transgenic C57BL/6 mice. Using RMCE, a CAG promoter cassette, the human TLR10 ORF, the hGH polyadenylation signal, and an additional polyadenylation signal was cloned into the ROSA26 locus. (B) The humanTLR10 fragment was amplified using specific hTLR10 primers. The amplification of the positive control fragment (585 bp) refers to wild-type mice. Mice expressing the human TLR10 gene have a fragment of 368 bp. (C) Plasma IL-6 and KC levels in wild-type (n = 13) and hTLR10 transgenic (n = 13) mice, 4 h after intraperitoneal injection of 50 µg Pam3Cys. IL-6 and KC were measured using ELISA. Data are mean ± SEM in pg/mL; *P < 0.05, Mann–Whitney U test. (D) The 5 × 10⁶ spleen cells of five wild-type C57BL/6 and six hTLR10tg mice were incubated with medium alone (RPMI), or 10 µg/mL Pam3Cys for 24 h at 37 °C, 5% CO₂. Supernatants were collected for measurement of IL-6 by ELISA. Data are mean ± SEM in picrograms per milliliter (pg/mL); five WT mice, six hTLR10tg mice, *P < 0.05, **P < 0.01, two-tailed Mann–Whitney U test. (E) After isolation of peritoneal macrophages, IL-6 and mKC levels were measured using ELISA. Data are mean ± SEM in picrograms per milliliter (pg/mL); *P < 0.05, ***P < 0.001, Mann–Whitney U test, seven mice per group.