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. 2016 May 1;196(9):3834-41.
doi: 10.4049/jimmunol.1502599. Epub 2016 Mar 28.

TLR10 Is a Negative Regulator of Both MyD88-Dependent and -Independent TLR Signaling

Song Jiang  1 , Xinyan Li  2 , Nicholas J Hess  2 , Yue Guan  2 , Richard I Tapping  3
Affiliations

TLR10 Is a Negative Regulator of Both MyD88-Dependent and -Independent TLR Signaling

Song Jiang et al. J Immunol. .

Abstract

TLRs are central components of the innate immune system which, upon recognition of bacterial, fungal or viral components, activate intracellular signals that lead to protective inflammatory responses. Among the 10-member human TLR family, TLR10 is the only remaining orphan receptor without a known ligand or signaling function. Murine TLR10 is a disrupted pseudogene, which precludes investigation using classic gene knockout approaches. We report here that TLR10 suppressed the production of an array of cytokines in stably transfected human myelomonocytic U937 cells in response to other TLR agonists. This broad TLR suppressive activity affects both MyD88- and TRIF-inducing IFN-β-mediated signaling pathways upstream of IκB and MAPK activation. Compared with nontransgenic littermate controls, monocytes of TLR10 transgenic mice exhibited blunted IL-6 production following ex vivo blood stimulation with other TLR agonists. After i.p. injection of LPS, lower levels of TNFα, IL-6, and type 1 IFN were measured in the serum of TLR10 transgenic mice compared to nontransgenic mice, but did not affect mouse survival in an LPS-induced septic shock model. Finally, treatment of human mononuclear cells with a monoclonal anti-TLR10 Ab suppressed proinflammatory cytokines released by LPS stimulation. These results demonstrate that TLR10 functions as a broad negative regulator of TLR signaling and suggests that TLR10 has a role in controlling immune responses in vivo.

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Figures

Fig. 1
Fig. 1. Stable TLR10 expression in U937 cells suppresses TLR-induced cytokine production
Parental U937 cells were transduced either with MMLV encoding FLAG-tagged TLR10 or an empty control MMLV. Stably transduced cells were isolated after two weeks of selection under 2 μg/ml Puromycin. (A) RT-PCR analysis of TLR10 expression in MMLV-U937 versus TLR10-U937 cell lines (B) Surface staining of TLR10 transfected and control cells using an anti-FLAG antibody. Cells were gated on forward and side scatter characteristics. (C) TLR10-U937 and MMLV-U937 control cells were differentiated for 48 hours in PMA. Cells were stimulated with the indicated agonist overnight, after which IL-6 was measured in cell-free supernatants using a paired-antibody ELISA and (D) Type 1 IFN was measured using an ISRE-L929 luciferase reporter assay. Luciferase activity was normalized to that of unstimulated MMLV control cells. Error bars represent the standard deviation of three independent samples and statistical analysis was performed using two-tailed paired Student’s t test. * p<0.01
Fig. 2
Fig. 2. TLR10 suppresses phosphorylation of MAPKs and degradation of IκB
TLR10-U937 and MMLV-U937 control cells were stimulated with either (A) 50 ng/mL PAM3CSK4, (B) 50 μg/mL polyI:C or (C) 50 ng/mL LPS. Cell lysates were analyzed by immunoblotting for the indicated signaling targets. Data are representative of at least three independent experiments.
Fig. 3
Fig. 3. TLR10 suppresses both MyD88 and TRIF signaling
HEK293T cells were co-transfected with the indicated TLR construct and either (A and C) MyD88 and an IL-8 promoter-driven luciferase construct or (B and D) TRIF and an IFN-β-driven luciferase reporter construct. Results indicate fold induction of luciferase over empty vector after normalizing each sample for transfection efficiency using Renilla luciferase. Error bars represent the standard deviation of three independent samples and statistical analysis was performed using two-tailed paired Student’s t test. * p < 0.05, ** p < 0.005
Fig. 4
Fig. 4. Generation of a TLR10 transgenic mouse under a constitutive CMV promoter
(A) Southern blot analysis of genomic DNA from 14 TLR10 positive founder mice with varying copy number insertions. (B) Reverse transcriptase dependent detection of FLAG-TLR10 in various tissues of a transgenic mouse. Western blots of (C) various tissues or (D) peripheral blood leukocytes from transgenic (tg) and non-transgenic (nc) mice using the anti-FLAG antibody to detect TLR10. FLAG-TLR10 migrates as a ~150 kDa band while a non-specific band appears at ~100 kDa.
Fig. 5
Fig. 5. Blood monocytes of TLR10 transgenic mice exhibit suppressed cytokine production in response to TLR agonists
(A) Whole blood from either TLR10-transgenic (tg) or non-transgenic (nc) littermate controls was stimulated with the indicated agonists and IL-6 release was measured in cell-free supernatants by ELISA. (B) Whole blood was stimulated with LPS and IL-6 production was measured by intracellular staining in monocyte (CD11b+, Ly6G−) and neutrophil (CD11b+, Ly6G+) populations. Dot plot represents ungated cell populations. (C) Median fluorescent intensity (MFI) of IL-6 is shown relative to unstimulated cells. Error bars represent the standard deviation of three independent samples and statistical analyses were performed using two-tailed paired Student’s t test. * p < 0.05, ** p < 0.01
Fig. 6
Fig. 6. TLR10 transgenic mice exhibit suppressed responses to injected LPS but are not protected in a model of septic shock
Four TLR10-transgenic (tg) or four non-transgenic littermate controls (nc) were injected IP with 25 mg/kg of LPS. Blood was collected at 1 hr and 4 h post injection and serum TNFα and IL-6 levels were measured using a paired antibody ELISA. Serum Type 1 IFN levels were measured using the ISRE-L929 reporter line. Error bars represent the standard deviation of at least three independent mice and statistical analysis was performed using one-tailed student’s t-test. * p < 0.05. (B) Six TLR10-transgenic (tg) and seven non-transgenic (nc) mice were injected IP with 25 mg/kg LPS and monitored for survival with results plotted in Kaplan-Meier format.
Fig. 7
Fig. 7. Anti-TLR10 antibody inhibits TLR-induced activation of peripheral blood mononuclear cells
Human peripheral blood mononuclear cells from two separate donors were pre-incubated with either an anti-TLR10 antibody or isotype matched control antibody prior to stimulation with the indicated agonist for 24 h. Cell-free supernatants were collected and IL-6 and TNFα production was assayed using an antibody-paired ELISA. Error bars represent standard error of the mean of three intra-donor replicates and statistical analysis was performed using the Holm-Sidak T-test assuming equal population scatter. * p < 0.05; ** p < 0.01
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