The adaptor TRAF5 limits the differentiation of inflammatory CD4+ T cells by antagonizing signaling via the receptor for IL-6

Abstract

The physiological functions of members of the tumor-necrosis factor (TNF) receptor (TNFR)–associated factor (TRAF) family in T cell immunity are not well understood. We found that in the presence of interleukin 6 (IL-6), naive TRAF5-deficient CD4+ T cells showed an enhanced ability to differentiate into the TH17 subset of helper T cells. Accordingly, TH17 cell–associated experimental autoimmune encephalomyelitis (EAE) was greatly exaggerated in Traf5−/− mice. Although it is normally linked with TNFR signaling pathways, TRAF5 constitutively associated with a cytoplasmic region in the signal-transducing receptor gp130 that overlaps with the binding site for the transcription activator STAT3 and suppressed the recruitment and activation of STAT3 in response to IL-6. Our results identify TRAF5 as a negative regulator of the IL-6 receptor signaling pathway that limits the induction of proinflammatory CD4+ T cells that require IL-6 for their development.

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Figure 1: Naive polyclonal Traf5−/− CD4+ T cells display an enhanced TH17 phenotype in vitro.
Figure 2: Traf5−/− OT-II CD4+ T cells exhibit enhanced TH17 development in vitro and in vivo.
Figure 3: IL-6-mediated phosphorylation of STAT3 is enhanced in Traf5−/− CD4+ T cells.
Figure 4: Constitutive binding of TRAF5 to gp130 inhibits the IL-6-mediated recruitment of STAT3 to gp130.
Figure 5: The TRAF5-gp130 interaction limits the development of TH17 cells.
Figure 6: TRAF5 serves an inhibitory role in EAE.

Change history

  • 04 April 2014

    In the version of this article initially published online, the labels "WT" and "KO" in Figure 4b were transposed; the legend for Figure 5a erroneously stated that TH17 cells were differentiated with IL-6-IL-6R and TGF-β for 5 d instead of 3 d; and on page 2 of the article, the word "soluble" was erroneously used to describe the coreceptor CD28. These errors have been corrected for the print, PDF and HTML versions of the article.

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Acknowledgements

We thank W. Heath (University of Melbourne) for OT-II mice; S. Nagata (Kyoto University) and S. Akira (Osaka University) for the Flag-pEF-STAT3 vector. Supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (C) (24590571 to T.S.), the Ichiro Kanehara Foundation (T.S.), the Takeda Science Foundation (T.S.), the Suzuken Memorial Foundation (T.S.) and the US National Institutes of Health (AI049453 to M.C.).

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Contributions

H.Nag., M.C., N.I. and T.S. designed the experiments; H.Nag., Y.O., A.A., T.K., S.Y. and T.S. did the experiments; H.Nag., Y.O., A.A., T.K., S.Y., M.C., N.I. and T.S. analyzed data; H.Nak. contributed reagents and analytical tools; M.C., N.I. and T.S. supervised the project; M.C., N.I. and T.S. wrote the paper; and M.C., N.I. and T.S. provided funding for the project.

Corresponding author

Correspondence to Takanori So.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Expression of gp130 and IL-6R protein and Tbx21, Gata3, Foxp3 and Traf5 mRNA in wild-type and Traf5-/- CD4+ T cells.

(a) Expression of IL-6R–gp130 on purified wild-type and Traf5-/- naive CD4+ T cells. (b) Quantitative RT-PCR analysis of the expression of Tbx21, Gata3, and Foxp3 mRNAs in activated CD4+ T cells generated from naive wild-type or Traf5-/- B6 CD4+ T cells cultured for 48 h with anti-CD3 and anti-CD28 in various polarizing conditions (left margin) and presented relative to the expression of the gene encoding β-Actin (average and s.d. of triplicate wells). (c) Quantitative RT-PCR analysis of the expression of Traf5 mRNA in naive and activated wild-type CD4+ T cells cultured for 48 h with anti-CD3 and anti-CD28 in various cytokine conditions (below graph) and presented relative to the expression of the gene encoding β-Actin (average and s.d. of triplicate wells). NS, not significant; **P < 0.01 (Student t-test).

Supplementary Figure 2 Wild-type and Traf5-/- OT-II T cells proliferate and produce IL-6 in response to antigen similarly.

(a,b) Carboxylfluorescein diacetate, succinimidyl ester (CFSE)-dilution in antigen-responding CD4+ T cells generated from naive wild-type or Traf5-/- OT-II CD4+ T cells stimulated for 3 d with wild-type B6 splenic APCs (after depletion of T cells) and indicated concentrations (above lanes) of OVA peptide (amino acids 323-339) (a) or 0.1 μM OVA peptide in the presence of various concentration (above lanes) of IL-6 (b). (c) Primary IL-6 in supernatants of activated CD4+ T cells generated from naive wild-type or Traf5-/- OT-II CD4+ T cells stimulated for 3 d with indicated concentrations of OVA peptide (horizontal axis) and wild-type B6 APCs. (d) TGF-β-mediated proliferation arrest evaluated by CFSE-dilution in antigen-responding CD4+ T cells generated from naive wild-type or Traf5-/- OT-II CD4+ T cells stimulated for 3 d with wild-type B6 APCs and 0.1 μM OVA peptide with or without 10 ng/ml TGF-β.

Supplementary Figure 3 Expression of gp130, STAT3 and phosphorylated STAT3 protein and Il6st and Traf5 mRNA in various cells from wild-type and Traf5-/- B6 mice.

(a) Expression of gp130 (left panel) and immunoblot analysis of phosphorylated STAT3 and total STAT3 after stimulation for various times (above lanes) with 200 ng/ml of IL-6–IL-6R (middle panel) and ratio of phosphorylated STAT3 to total STAT3 after stimulation for 10 min with 200 ng/ml IL-6–IL-6R (average and s.d. of triplicate wells, right panel) in purified splenic wild-type and Traf5-/- polyclonal CD8+ T cells. Isotype, isotype-matched control antibody. (b) Expression of gp130 on T cells, NKT cells, NK cells, B cells, and macrophages from wild-type and Traf5-/- B6 mice. (c) Quantitative RT-PCR analysis of the expression of Traf5 and Il6st mRNAs in different cell populations described above and presented relative to the expression of the gene encoding β-Actin (average and s.d. of triplicate wells). (d) Immunoblot analysis of phosphorylated STAT3 and total STAT3 in wild-type and Traf5-/- macrophages stimulated for 10 min with various concentrations (above lanes) of IL-6–IL-6R (left panel). Ratio of phosphorylated STAT3 to total STAT3 in wild-type and Traf5-/- macrophages stimulated for 10 min with 200 ng/ml of IL-6–IL-6R (average and s.d. of triplicate wells, right panel). (e) Expression of IL-10R (left panel) and immunoblot analysis of phosphorylated STAT3 and total STAT3 (middle panel) and ratio of phosphorylated STAT3 to total STAT3 (average and s.d. of triplicate wells, right panel) after stimulation for 10 min with 50 ng/ml of IL-10 in purified splenic wild-type and Traf5-/- polyclonal CD4+ T cells. (f) Expression of IL-21R (left panel) and immunoblot analysis of phosphorylated STAT3 and total STAT3 (middle panel) and ratio of phosphorylated STAT3 to total STAT3 (average and s.d. of triplicate wells, right panel) after stimulation for 10 min with 10 ng/ml of IL-21 in purified splenic wild-type and Traf5-/- polyclonal CD4+ T cells. NS, not significant; *P < 0.05 and **P < 0.01 (Student t-test).

Supplementary Figure 4 The cytoplasmic amino acid residues in gp130 responsible for TRAF5 binding.

(a) Immunoassay of HEK cells transduced with plasmids encoding gp130 mutants (above lanes) with various deletions in the cytoplasmic region in positions 641–917 (above blot) and cotransfected to express V5-TRAF5 (242–558), followed by immunoprecipitation of proteins from lysates with control IgG or anti-c-Myc and immunoblot analysis with anti-V5 or anti-c-Myc. (b) Amino acid sequence alignment of the TRAF5 binding sites in gp130 from various species. (c) Immunoprecipitation of V5-TRAF5 (242–558) from lysates of HEK cells transiently transfected with plasmid vectors encoding V5-TRAF5 (242–558) and GFP-tagged gp130 (769–800) with wild-type sequence (WT) or alanine substitutions (Ala-mut) (above blot), followed by immunoblot analysis with anti-V5 or anti-GFP. Input (bottom), immunoblot analysis of lysates without immunoprecipitation. (d) Immunoprecipitation of c-Myc-gp130 (1–917) from lysates of HEK cells transiently transfected with plasmid vectors encoding c-Myc-gp130 (1–917), V5-TRAF5 (242–558), and GFP-tagged gp130 (769-800) with wild-type sequence (WT) or alanine substitutions (Ala-mut) (above blot), followed by immunoblot analysis with anti-V5, anti-c-Myc, or anti-GFP.

Supplementary Figure 5 The role of TRAF5 in active and passive EAE.

(a) Body weight changes in wild-type and Traf5-/- mice after induction of active EAE as in Fig. 6a–c, monitored over 22 d (average and s.e.m. of n = 10 mice per genotype). (b) The percentages of donor CD4+CD45.2+ cells in the peripheral blood CD4+ T cells from recipient CD45.1+ B6.SJL mice 7 days after induction of passive EAE as in Fig. 6d (average and s.e.m. of n = 3 mice (no cell transfer) or 6 mice (adoptive transfer)). NS, not significant; *P < 0.05 (Student t-test).

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Nagashima, H., Okuyama, Y., Asao, A. et al. The adaptor TRAF5 limits the differentiation of inflammatory CD4+ T cells by antagonizing signaling via the receptor for IL-6. Nat Immunol 15, 449–456 (2014). https://doi.org/10.1038/ni.2863

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