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The receptor TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity

Abstract

The molecules and pathways that fine-tune innate inflammatory responses mediated by Toll-like receptor 7 (TLR7) remain to be fully elucidated. Using an unbiased genome-scale screen with short hairpin RNA (shRNA), we identified the receptor TREML4 as an essential positive regulator of TLR7 signaling. Macrophages from Treml4−/− mice were hyporesponsive to TLR7 agonists and failed to produce type I interferons due to impaired phosphorylation of the transcription factor STAT1 by the mitogen-activated protein kinase p38 and decreased recruitment of the adaptor MyD88 to TLR7. TREML4 deficiency reduced the production of inflammatory cytokines and autoantibodies in MRL/lpr mice, which are prone to systemic lupus erythematosus (SLE), and inhibited the antiviral immune response to influenza virus. Our data identify TREML4 as a positive regulator of TLR7 signaling and provide insight into the molecular mechanisms that control antiviral immunity and the development of autoimmunity.

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Figure 1: TREML4 positively regulates TLR7 signaling.
Figure 2: Regulation of TLR7 signaling by TREML4.
Figure 3: In vivo regulation of TLR7 signaling by TREML4.
Figure 4: TLR7-induced phosphorylation of MAPKs and STAT1 is regulated by TREML4.
Figure 5: TREML4 regulates the trafficking and localization of MyD88 and TLR7.
Figure 6: Ameliorated autoimmunity in TREML4-deficient MRL/lpr mice.

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Acknowledgements

We thank H. Hemmi (Osaka University) and R. Steinman (The Rockefeller University) for cDNA encoding mouse TREML4; D. Golenbock (University of Massachusetts) plasmids encoding mouse TLR proteins; and B. Luo, M. Okamoto and members of the Genetic Perturbation Platform (Broad Institute) and The RNAi Consortium for shRNA lentiviral libraries and technical assistance. The vectors, embryonic stem cells and TREML4-deficient mouse strain were generated by the trans-NIH Knockout Mouse Project (KOMP) and were obtained from the KOMP Repository. The US National Institutes of Health funded the generation of 8,500 gene-targeted embryonic stem cells (by KOMP; U01HG004085 to Velocigene at Regeneron, and U01HG004080 to the CSD Consortium) and their archiving and distribution by the KOMP Repository at the University of California at Davis and Children's Hospital Oakland Research Institute (U42RR024244). Supported by the National Institutes of Allergy and Infectious Diseases (R01-AI084884 to T.K.M.; U24 AI082660 to J.E.K.; and T32-AI007061 to Z.G.R.-O.), the National Institutes of Arthritis, Musculoskeletal and Skin Diseases (K01-AR051367 to T.K.M.; and K01-AR066716 to Z.G.R.-O.), the National Institute of Diabetes and Digestive and Kidney Diseases (F32-DK097891 to W.F.P.), the Lupus Research Institute (T.K.M. and N.H.), the Alliance for Lupus Research (T.K.M. and N.H.) and the American Society of Nephrology (W.F.P.).

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Authors and Affiliations

Authors

Contributions

Z.G.R.-O., A.P., W.F.P., N.H. and T.K.M. planned the research, analyzed and interpreted data and wrote the manuscript; Z.G.R.-O., A.P. and T.K.M. did most of the experiments; A.P., J.W.G. and W.F.P. performed and analyzed ELISA, PCR and mouse pathology studies; G.S.C., D.E.R., A.D.L. and J.E.K. analyzed and interpreted data; M.T. helped with mouse breeding, genotyping and the production of lentiviruses encoding shRNA; M.T., J.E.K. and T.K.M. contributed to the generation of TREML4-deficient mice; and all authors participated in editing the manuscript.

Corresponding author

Correspondence to Terry K Means.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 TREML4 amplifies signaling via TLR7, TLR9 and TLR13.

(a) Quantitative PCR analysis of human IL8 mRNA in HEK cells transfected with the specified mouse TLR in the absence (open columns) or presence (solid columns) of mouse TREML4 and treated with Pam3CSK4 (1 μg/mL), pIC (10 μg/mL), LPS (100 ng/mL), GRD (10 μg/mL), CpG-DNA (10 μg/mL) or rRNA (10 μg/mL) for 3 hours, data presented as fold over untreated. (b) Quantitative PCR analysis of human IL8 mRNA in HEK reporter cells mock transfected (Mock) or transfected to express mouse DECTIN1-TNF-R1, SCARF1-TNF-R1, or TREML4-TNF-R1 and left untreated (UT) or cultured for 3 h with ultraviolet-irradiated late apoptotic mouse embryonic fibroblasts (apoptotic UV-MEF, 5:1 ratio), heat-killed Candida albicans (HK-C. albicans, 5:1 ratio), GRD (10 μg/mL), CpG-DNA (10 μg/mL), or rRNA (10 μg/mL); results are presented relative to those of mock-transfected cells left untreated. * P<0.01 and **P<0.001 (Mann-Whitney test). Data are from one experiment representative of at least three independent experiments (error bars, median and s.d. of triplicates), all data presented as fold over untreated.

Supplementary Figure 2 TREML4 expression profile in tissues and cells.

(a) Quantitative PCR analysis of Treml4 mRNA in tissues from Treml4+/+ and Treml4−/− mice. (b) Quantitative PCR analysis of Treml4 mRNA abundance in different cell populations isolated from the spleen and bone marrow (BM) of Treml4+/+ and Treml4−/− mice. (a,b) Copies of Treml4 mRNA normalized to copies of Gapdh mRNA. Bars denote means and s.d. of three samples per group with duplicate measurements. (c) The targeting vector used to generate Treml4−/− mice contained 6239-bp of 5’ sequence upstream of the transcriptional start site, a neomycin resistance cassette replacing exons 2-4 (which encode the entire extracellular domain), and 4366-bp of 3’ sequence spanning exons 5-6. The linearized vector was electroporated into C57BL/6 ES cells, and targeted clones were selected in the presence of the antibiotic neomycin were identified by PCR and Southern blot analysis. Targeted ES clones were injected into B6 mice blastocysts, yielding several lines of chimeric mice that transmitted the disrupted allele through the germline DNA. (d) Quantitative PCR of Cxcl2 mRNA in BM derived neutrophils isolated from Treml4+/+ and Treml4−/− mice treated with the specified TLR ligands for 4 h. Data presented as fold over untreated. (e) Cell migration of neutrophils to fMLP in a transwell chemotaxis chamber for 30 min. (f) Opsonized latex beads were incubated with neutrophils at a ratio of 25:1 for 30 min. Phagocytosis was measured by fluorescent microscopy. Data are presented as the number of internalized particles per 100 cell X 100. *P<0.01 (Mann-Whitney test). Data are from one experiment representative of at least three independent experiments performed (d-f; error bars, median and s.d. of triplicates).

Supplementary Figure 3 Expression of TLR7 and TLR signaling molecules in TREML4-deficient cells.

(a) Quantitative PCR analysis of Tnf mRNA in F4/80+ splenic macrophages isolated from Treml4+/+ and Treml4−/− infected with live VSV and CMV for 8 h. Data presented as fold over untreated. (b) Flow cytometry analysis of TLR7 expression in splenic macrophages and splenic DCs from Treml4+/+ and Treml4−/− mice. Histograms are representative of one experiment of three independent experiments performed. (c) Quantitative PCR analysis of TLR7 signaling intermediates in splenic macrophages isolated from Treml4+/+ and Treml4−/− mice. Data are from one experiment representative of three independent experiments performed. Bars denote means and s.d. of three samples per group with duplicate measurements. *P<0.01 (a, Mann-Whitney test).

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Ramirez-Ortiz, Z., Prasad, A., Griffith, J. et al. The receptor TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity. Nat Immunol 16, 495–504 (2015). https://doi.org/10.1038/ni.3143

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