Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The signaling adaptor TRAF1 negatively regulates Toll-like receptor signaling and this underlies its role in rheumatic disease

Nature Immunology volume 18, pages 26–35 (2017)Cite this article

Subjects

Abstract

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis–associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex, SHARPIN, HOIP and HOIL-1, to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production, independently of tumor necrosis factor. Consistent with this, Traf1−/− mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling, providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Activated T cells from human subjects with an RA-associated SNP express less TRAF1 and produce less cytokines.
Figure 2: Monocytes from human PBMCs with an RA-associated SNP express lower TRAF1 protein and produce more inflammatory cytokines in response to LPS.
Figure 3: Effect of TRAF1 SNP on cytokine expression in PBMCs stimulated with LPS plus anti-CD3 and on expression of A versus G alleles in heterozygous subjects.
Figure 4: TRAF1 regulates LPS-induced inflammatory responses in the human monocytic cell line THP-1, independently of TNF signaling.
Figure 5: TRAF1 associates with the LUBAC complex by directly binding HOIP and HOIL-1 via its MATH domain.
Figure 6: TRAF1 limits linear ubiquitination of NEMO to control NF-κB activation.
Figure 7: LPS-stimulated monocytes with the RA-associated SNP exhibit enhanced linear ubiquitination.
Figure 8: Traf1−/− mice are more susceptible to endotoxin-induced septic shock.

Similar content being viewed by others

Accession codes

Accessions

NCBI Reference Sequence

References

  1. Rothe, M., Wong, S.C., Henzel, W.J. & Goeddel, D.V. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681–692 (1994).

    CAS  PubMed  Google Scholar 

  2. Wang, C.Y., Mayo, M.W., Korneluk, R.G., Goeddel, D.V. & Baldwin, A.S. Jr. NF-κB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281, 1680–1683 (1998).

    CAS  PubMed  Google Scholar 

  3. Carpentier, I. & Beyaert, R. TRAF1 is a TNF inducible regulator of NF-κB activation. FEBS Lett. 460, 246–250 (1999).

    CAS  PubMed  Google Scholar 

  4. Tsitsikov, E.N. et al. TRAF1 is a negative regulator of TNF signaling. enhanced TNF signaling in TRAF1-deficient mice. Immunity 15, 647–657 (2001).

    CAS  PubMed  Google Scholar 

  5. McPherson, A.J., Snell, L.M., Mak, T.W. & Watts, T.H. Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells. J. Biol. Chem. 287, 23010–23019 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Wicovsky, A. et al. Tumor necrosis factor receptor-associated factor-1 enhances proinflammatory TNF receptor-2 signaling and modifies TNFR1-TNFR2 cooperation. Oncogene 28, 1769–1781 (2009).

    CAS  PubMed  Google Scholar 

  7. Xie, P., Hostager, B.S., Munroe, M.E., Moore, C.R. & Bishop, G.A. Cooperation between TNF receptor-associated factors 1 and 2 in CD40 signaling. J. Immunol. 176, 5388–5400 (2006).

    CAS  PubMed  Google Scholar 

  8. Arron, J.R., Pewzner-Jung, Y., Walsh, M.C., Kobayashi, T. & Choi, Y. Regulation of the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF)2 by TRAF1 reveals mechanisms of TRAF2 signaling. J. Exp. Med. 196, 923–934 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Sabbagh, L., Pulle, G., Liu, Y., Tsitsikov, E.N. & Watts, T.H. ERK-dependent Bim modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8 T cell survival in vivo. J. Immunol. 180, 8093–8101 (2008).

    CAS  PubMed  Google Scholar 

  10. Sabbagh, L. et al. A critical role for TNF receptor-associated factor 1 and Bim down-regulation in CD8 memory T cell survival. Proc. Natl. Acad. Sci. USA 103, 18703–18708 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Speiser, D.E. et al. A regulatory role for TRAF1 in antigen-induced apoptosis of T cells. J. Exp. Med. 185, 1777–1783 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Lee, S.Y. & Choi, Y. TRAF1 and its biological functions. Adv. Exp. Med. Biol. 597, 25–31 (2007).

    PubMed  Google Scholar 

  13. Zheng, C., Kabaleeswaran, V., Wang, Y., Cheng, G. & Wu, H. Crystal structures of the TRAF2: cIAP2 and the TRAF1: TRAF2: cIAP2 complexes: affinity, specificity, and regulation. Mol. Cell 38, 101–113 (2010).

    PubMed  PubMed Central  Google Scholar 

  14. Vallabhapurapu, S. & Karin, M. Regulation and function of NF-κB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733 (2009).

    CAS  PubMed  Google Scholar 

  15. Gerlach, B. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011).

    CAS  PubMed  Google Scholar 

  16. Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Tokunaga, F. et al. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex. Nature 471, 633–636 (2011).

    CAS  PubMed  Google Scholar 

  18. Damgaard, R.B. et al. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol. Cell 46, 746–758 (2012).

    CAS  PubMed  Google Scholar 

  19. Warner, N. et al. A genome-wide siRNA screen reveals positive and negative regulators of the NOD2 and NF-κB signaling pathways. Sci. Signal. 6, rs3 (2013).

    PubMed  Google Scholar 

  20. Kirisako, T. et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 25, 4877–4887 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Chang, M. et al. A large-scale rheumatoid arthritis genetic study identifies association at chromosome 9q33.2. PLoS Genet. 4, e1000107 (2008).

    PubMed  PubMed Central  Google Scholar 

  22. Plenge, R.M. et al. TRAF1-C5 as a risk locus for rheumatoid arthritis—a genomewide study. N. Engl. J. Med. 357, 1199–1209 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Kurreeman, F.A. et al. A candidate gene approach identifies the TRAF1/C5 region as a risk factor for rheumatoid arthritis. PLoS Med. 4, e278 (2007).

    PubMed  PubMed Central  Google Scholar 

  24. Panoulas, V.F., Smith, J.P., Nightingale, P. & Kitas, G.D. Association of the TRAF1/C5 locus with increased mortality, particularly from malignancy or sepsis, in patients with rheumatoid arthritis. Arthritis Rheum. 60, 39–46 (2009).

    CAS  PubMed  Google Scholar 

  25. Nishimoto, K. et al. Association study of TRAF1-C5 polymorphisms with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in Japanese. Ann. Rheum. Dis. 69, 368–373 (2010).

    CAS  PubMed  Google Scholar 

  26. Song, G.G., Bae, S.C., Kim, J.H. & Lee, Y.H. Associations between TRAF1-C5 gene polymorphisms and rheumatoid arthritis: a meta-analysis. Immunol. Invest. 43, 97–112 (2014).

    CAS  PubMed  Google Scholar 

  27. McInnes, I.B. & O'Dell, J.R. State-of-the-art: rheumatoid arthritis. Ann. Rheum. Dis. 69, 1898–1906 (2010).

    CAS  PubMed  Google Scholar 

  28. McInnes, I.B. & Schett, G. The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 365, 2205–2219 (2011).

    CAS  PubMed  Google Scholar 

  29. Davignon, J.L. et al. Targeting monocytes/macrophages in the treatment of rheumatoid arthritis. Rheumatology (Oxford) 52, 590–598 (2013).

    CAS  Google Scholar 

  30. Li, S., Wang, L., Berman, M., Kong, Y.Y. & Dorf, M.E. Mapping a dynamic innate immunity protein interaction network regulating type I interferon production. Immunity 35, 426–440 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Fujita, H. et al. Mechanism underlying IκB kinase activation mediated by the linear ubiquitin chain assembly complex. Mol. Cell. Biol. 34, 1322–1335 (2014).

    PubMed  PubMed Central  Google Scholar 

  32. Rieser, E., Cordier, S.M. & Walczak, H. Linear ubiquitination: a newly discovered regulator of cell signalling. Trends Biochem. Sci. 38, 94–102 (2013).

    CAS  PubMed  Google Scholar 

  33. Keusekotten, K. et al. OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell 153, 1312–1326 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Rothe, M., Sarma, V., Dixit, V.M. & Goeddel, D.V. TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40. Science 269, 1424–1427 (1995).

    CAS  PubMed  Google Scholar 

  35. Sakamoto, H. et al. Gliotoxin suppresses NF-κB activation by selectively inhibiting linear ubiquitin chain assembly complex (LUBAC). ACS Chem. Biol. 10, 675–681 (2015).

    CAS  PubMed  Google Scholar 

  36. Aderem, A. & Ulevitch, R.J. Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787 (2000).

    CAS  PubMed  Google Scholar 

  37. Masters, S.L., Simon, A., Aksentijevich, I. & Kastner, D.L. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease (*). Annu. Rev. Immunol. 27, 621–668 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Kondo, T., Kawai, T. & Akira, S. Dissecting negative regulation of Toll-like receptor signaling. Trends Immunol. 33, 449–458 (2012).

    CAS  PubMed  Google Scholar 

  39. Ma, A. & Malynn, B.A. A20: linking a complex regulator of ubiquitylation to immunity and human disease. Nat. Rev. Immunol. 12, 774–785 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Feldmann, M. & Maini, R.N. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned? Annu. Rev. Immunol. 19, 163–196 (2001).

    CAS  PubMed  Google Scholar 

  41. Abdul-Sater, A.A. et al. Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome. EMBO Rep. 14, 900–906 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Siegler, G., Kremmer, E., Gonnella, R. & Niedobitek, G. Epstein-Barr virus encoded latent membrane protein 1 (LMP1) and TNF receptor associated factors (TRAF): colocalisation of LMP1 and TRAF1 in primary EBV infection and in EBV associated Hodgkin lymphoma. Mol. Pathol. 56, 156–161 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Newton, K. et al. Using linkage-specific monoclonal antibodies to analyze cellular ubiquitylation. Methods Mol. Biol. 832, 185–196 (2012).

    CAS  PubMed  Google Scholar 

  44. Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).

    CAS  PubMed  Google Scholar 

  45. Matsumoto, M.L. et al. Engineering and structural characterization of a linear polyubiquitin-specific antibody. J. Mol. Biol. 418, 134–144 (2012).

    CAS  PubMed  Google Scholar 

  46. Wertz, I.E. et al. Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation. Nature 528, 370–375 (2015).

    CAS  PubMed  Google Scholar 

  47. Sanjabi, S. et al. A c-Rel subdomain responsible for enhanced DNA-binding affinity and selective gene activation. Genes Dev. 19, 2138–2151 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Fu, B., Li, S., Wang, L., Berman, M.A. & Dorf, M.E. The ubiquitin conjugating enzyme UBE2L3 regulates TNFα-induced linear ubiquitination. Cell Res. 24, 376–379 (2014).

    CAS  PubMed  Google Scholar 

  49. Smit, J.J. et al. The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension. EMBO J. 31, 3833–3844 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Genentech for providing linear ubiquitin–specific antibody, B. Ghumman and J. Warzyszynska for technical assistance, L.E. Wagar for collecting and genotyping some of the PBMC samples, G. Ehrhardt for providing the control-GST construct and D. Philpott, S. Girardin and M. Hayden for suggestions and comments. Supported by grants MOP-74492 and FDN-143250 from the Canadian Institutes of Health Research (CIHR) to T.H.W. T.H.W. is supported by the Sanofi Pasteur Chair in Human Immunology at the University of Toronto. D.L.C. was supported by a doctoral award from CIHR and the Canadian Association for HIV research.

Author information

Author notes

  1. Ali A Abdul-Sater

    Present address: Present address: School of Kinesiology and Health Science, Faculty of Health, York University, Toronto, Ontario, Canada.,

Authors and Affiliations

  1. Department of Immunology, University of Toronto, Toronto, Ontario, Canada

    Ali A Abdul-Sater, Maria I Edilova, Derek L Clouthier, Achire Mbanwi & Tania H Watts

  2. Institut für Molekulare Immunologie, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), München, Germany

    Elisabeth Kremmer

Authors
  1. Ali A Abdul-Sater
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria I Edilova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Derek L Clouthier
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Achire Mbanwi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Elisabeth Kremmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tania H Watts
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.A.A.-S. and T.H.W. conceived, designed and interpreted experiments and wrote the manuscript, with input from M.I.E. and D.L.C. A.A.A.-S. conducted experiments with the help of M.I.E., A.M. and D.L.C. E.K. provided anti-human TRAF1 monoclonal antibody.

Corresponding author

Correspondence to Tania H Watts.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gating strategy for flow cytometric analysis of T cells and frequency of T cell subsets

(a) Gating strategy for the flow cytometry analysis performed on T cells from healthy human subjects’ PBMCs for TRAF1 measurements in T cells, as in Fig. 1a. (b) Representative histograms showing TRAF1 expression in stimulated and unstimulated T cells from PBMCs of AA, AG and AG donors. (c) control knockdown (shCTRL) or TRAF1 knockdown (shTRAF1) RAJI cells were used to confirm specificity of TRAF1 stain. (d) Frequency of the various subsets of T cells from donor PBMCs with the AA, AG or GG genotype were measured by flow cytometry using the antibodies listed in the methods section. n = 26, 36, 15 human subjects of each genotype AA, AG, GG respectively. Results are representative from 2 similar experiments. Statistical analysis was performed using a non-parametric (Mann Whitney) unpaired t-test.

Supplementary Figure 2 TRAF1 protein levels in unstimulated T cells and gating strategies for T cell cytokines and monocytes

(a) TRAF1 levels were measured by flow cytometry using a PE labeled anti-hTRAF1 antibody in unstimulated T cells from donor PBMCs with the AA, AG or GG genotypes. Each symbol represents one donor; n = 26, 36, 15 human donors of each genotype AA, AG, GG, respectively. Results are representative of 2 similar experiments. Statistical analysis was performed using a non-parametric (Mann Whitney) unpaired t-test. * p <0.05 (b) Gating strategy for intracellular flow cytometry analysis performed on T cells from healthy human donor PBMCs for cytokine measurements, as in Fig. 1b. (c) Gating strategy for the flow cytometry analysis performed on purified monocytes from healthy human donor PBMCs, as in Fig. 2.

Supplementary Figure 3 Flow cytometric measurements of TRAF1 and cytokines from human PBMC monocytes

(a) Purified monocytes from healthy human subjects’ PBMCs were treated or not with LPS (100 ng/ml for 2, 4 or 24 hrs), and TRAF1 levels were measured by flow cytometry using a PE-labeled anti-hTRAF1 antibody. (a) Graphical representation of the mean fluorescence intensities (dMFI = MFI of sample – MFI of FMO) of TRAF1 in purified monocytes of donors with the CC, CT and TT genotypes of the TRAF1 SNP rs2900180. Each symbol represents one donor; n = 29, 19, 10 human donors of each genotype CC, CT, TT, respectively. (b-d) Purified monocytes from healthy human donor PBMCs were treated or not with LPS (100 ng/ml for 4, 6 or 8 hrs), and percentage of cytokine producing cells was assessed by flow cytometry using an (b) IL-1β antibody, IL-6 antibody (c) or TNF antibody (d). (b-d) Each symbol represents one donor; n = 22, 22, 14 human donors of each genotype AA, AG, GG, respectively. Results are representative of 2 similar experiments. (a-d) Statistical analysis was performed using a non-parametric (Mann Whitney) unpaired t-test. * p < 0.05 and ** p < 0.01

Supplementary Figure 4 shTRAF1 THP-1 cells have enhanced NF-κB activation and express higher levels of inflammatory cytokines than shCTRL cells after TLR or NOD ligand treatment

(a) shCTRL (shC) and shTRAF1 (shT) THP-1 cells were stimulated with Pam3CSK4 (P3C; 10 ng/ml), Flagellin (Fla; 0.5 μg/ml), Resiquimod (R848; 10 ng/ml), Poly(I:C) (I:C; 10 μg/ml), L18-muramyl dipeptide (MDP; 10 ng/ml) or LPS (100 ng/ml) for the indicated times and whole cells extracts (WCE) were immunoblotted for phospho-IκB-α (p-IκB-α). Blots are representative of two independent experiments (b) Gene expression of TNF, CCL5, IL6 and p40 (IL12) was evaluated by real-time PCR (Q-PCR) in 100 ng/ml LPS stimulated shCTRL or shTRAF1 THP-1 cells for the indicated times as in Fig. 4e. Results were normalized to GAPDH and reported as relative fold change w.r.t. untreated control. Graphs show the mean ± standard deviation of three independent experiments, which were compared using a 2 way ANOVA with multiple comparisons. * p < 0.05, ** p < 0.01 and *** p < 0.001 (c) TNF levels were measured by Enzyme-linked immunosorbent assay (ELISA) in supernatants of anti-CD3/anti-CD28 stimulated T cells from 23 human donors PBMCs with or without co-treatment with 1 μg/ml Adalimumab (Ada) for 48 hrs. Graphs show the mean ± SEM. Statistical analysis was performed using a paired t-test. *** p < 0.001

Supplementary Figure 5 Status of NEMO ubiquitination following LPS and TNF stimulation

(a) IP of endogenous Lys48 ubiquitin chains (K48 Ub) or Lys63 ubiquitin chains (K63 Ub) followed by immunoblotting for NEMO as in Fig. 6 panel c, with whole cell lysates, as a loading control, shown to the right. (b) Analysis of TNFR1-associated NEMO ubiquitination status and TNFR1 immunoprecipitates in Flag-TNF-treated shCTRL and shTRAF1 THP-1 cells. Lysates were first immunoprecipitated with anti-Flag (IP1: Flag), dissociated and re-immunoprecipitated with anti-linear ubiquitin antibody (IP2: Linear Ub) or anti-K63 ubiquitin antibody (IP2: K63 Ub), and blotted for TNFR1. Whole cell lysates are shown in the bottom panel. (c) NF-κB firefly luciferase reporter was cotransfected into HEK293 FT cells with control Renilla luciferase and LUBAC (HOIP + HOIL-1). Some cells were additionally transfected with increasing amounts of TRAF1 or with 125 ng of TRAF2 expression vectors, as indicated. Luciferase activity was measured after 48 h. Graphs show the mean ± standard deviation of three independent experiments (d) Gene expression of TRAF1, TNF, CCL5 and p40 (IL12 β) was evaluated by real-time PCR (Q-PCR) in 100 ng/ml LPS stimulated shCTRL or shTRAF1 THP-1 cells (treated with non-targeting siRNA (siCTRL) or HOIP targeting siRNA (siHOIP) for 48 hrs) for the indicated times as in panel a. Results were normalized to GAPDH and reported as relative fold change w.r.t. untreated control. Graphs show the mean ± standard deviation of three independent experiments, which were compared using a 2 way ANOVA with multiple comparisons. (e) LUBAC inhibition by gliotoxin reverses the enhanced NF-κB activation in shTRAF1 THP-1 cells. WCE from control knockdown (shCTRL) and TRAF1 knockdown (shTRAF1) THP-1 cells treated with LPS (100 ng/ml) for the indicated times were immunoblotted for IκB-α (left panels) or as a loading control GAPDH (right panels). Some shCTRL THP-1 cells were pretreated (15’) with 1 μM Gliotoxin. Blots are representative of 3 independent experiments.

Supplementary Figure 6 Proposed mechanism for TRAF1 role in TLR and TNFR1 signaling

TRAF1 limits linear ubiquitination of NEMO in both the TLR and TNFR1 signaling pathways, but these effects in TNFR1 signaling are counterbalanced by the role of TRAF1 in recruitment of cIAP1 to enhance K63-linked polyubiquitination of NEMO.

Supplementary Figure 7 Macrophages from Traf1–/– mice are hyper-responsive to TLR stimulation

(a) d5 bone marrow macrophages (BMMs) from C57BL/6NCrl (WT) mice were stimulated with 10 ng/ml LPS for the indicated times, and gene expression was assessed by reverse-transcriptase PCR (RT-PCR). (b) BMMs from WT (left) or Traf1-/- (right) littermate mice were stimulated with 10 ng/ml LPS for the indicated times, and whole cells extracts (WCE) were immunoblotted for total IκB-α (IκB-α), phospho-ERK1/2 (p-ERK1/2) and as a loading control GAPDH. Blots are representative of at least three independent experiments. (c) Gene expression of Ifn-β, Tnf, Il1-β and Traf1 was evaluated by real-time PCR (Q-PCR) in LPS stimulated BMMs for the indicated times as in panel a (10ng/ml LPS). (d) d5 bone marrow macrophages (BMMs) from WT or Traf1-/- littermate mice were stimulated with 100 ng/ml or 1 μg/ml LPS for the indicated times, and gene expression of Ifn-β, Ip-10, Tnf, Il-1β and Kc was evaluated by real-time PCR (Q-PCR). (c-d) Results were normalized to GAPDH and reported as relative fold change w.r.t. untreated control. Graphs show the mean ± standard deviation of three independent experiments, which were compared using a 2 way ANOVA with multiple comparisons. * p < 0.05, ** p < 0.01 and *** p < 0.001

Supplementary Figure 8 Traf1–/– macrophages produce higher levels of cytokines than WT

(a) Tnf levels were measured by Enzyme-linked immunosorbent assay (ELISA) in supernatants from WT (Traf1+/+) or KO (Traf1-/-) BMMs treated with 10 ng/ml, 100 ng/ml or 1 μg/ml LPS for the indicated times. Graphs show the mean ± standard deviation of three independent experiments, which were compared using a 2 way ANOVA with multiple comparisons. *** p < 0.001 (b) The levels of Tnf, Il6 and Ccl5 secreted into the supernatants of LPS stimulated WT (Traf1+/+) heterozygous (Traf1+/-) or KO (Traf1-/-) BMMs were measured using Bio-plex cytokine assay. Graphs show the mean ± standard deviation of three independent experiments, which were compared using unpaired t-test; * p < 0.05 and *** p < 0.001

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1569 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Abdul-Sater, A., Edilova, M., Clouthier, D. et al. The signaling adaptor TRAF1 negatively regulates Toll-like receptor signaling and this underlies its role in rheumatic disease. Nat Immunol 18, 26–35 (2017). https://doi.org/10.1038/ni.3618

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3618

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing