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Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO

Nature Immunology volume 15, pages 612–622 (2014)Cite this article

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Abstract

Excessive activation of dendritic cells (DCs) leads to the development of autoimmune and inflammatory diseases, which has prompted a search for regulators of DC activation. Here we report that Rhbdd3, a member of the rhomboid family of proteases, suppressed the activation of DCs and production of interleukin 6 (IL-6) triggered by Toll-like receptors (TLRs). Rhbdd3-deficient mice spontaneously developed autoimmune diseases characterized by an increased abundance of the TH17 subset of helper T cells and decreased number of regulatory T cells due to the increase in IL-6 from DCs. Rhbdd3 directly bound to Lys27 (K27)-linked polyubiquitin chains on Lys302 of the modulator NEMO (IKKγ) via the ubiquitin-binding–association (UBA) domain in endosomes. Rhbdd3 further recruited the deubiquitinase A20 via K27-linked polyubiquitin chains on Lys268 to inhibit K63-linked polyubiquitination of NEMO and thus suppressed activation of the transcription factor NF-κB in DCs. Our data identify Rhbdd3 as a critical regulator of DC activation and indicate K27-linked polyubiquitination is a potent ubiquitin-linked pattern involved in the control of autoimmunity.

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Figure 1: Rhbdd3−/− mice spontaneously develop systemic autoimmune disease.
Figure 2: Enhanced maturation and activation of Rhbdd3−/− DCs.
Figure 3: Rhbdd3 suppresses the development of TH17 cell–mediated colitis.
Figure 4: Rhbdd3 maintains T cell homeostasis by inhibiting the production of IL-6 by DCs.
Figure 5: Rhbdd3 inhibits TLR-induced activation of NF-κB in DCs via the UBA domain.
Figure 6: Rhbdd3 interacts with K27-linked polyubiquitin chains on NEMO.
Figure 7: Rhbdd3 recruits A20 and facilitates A20-mediated deubiquitination of NEMO.
Figure 8: Rhbdd3 inhibits TLR signaling dependent on A20 and Lys302 of NEMO.

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Acknowledgements

We thank J. Jiang for technical assistance; C. Dong (MD Anderson Cancer Center) for Il17−/− mice; S. Jung (Weizmann Institute of Science) for CD11c-DTR mice; and J. Jung (Harvard Medical School) for the pEBG plasmid. Supported by the National Key Basic Research Program of China (2013CB530503 and 2012CB910202), the National Natural Science Foundation of China (81230074, 81123006 and 81172787) and the National 125 Key Project (2012AA020901).

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Author notes

  1. Juan Liu, Chaofeng Han, Bin Xie and Yue Wu: These authors contributed equally to this work.

Authors and Affiliations

  1. National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China

    Juan Liu, Chaofeng Han, Shuxun Liu, Lijun Song, Yizhi Yu & Xuetao Cao

  2. National Key Laboratory of Medical Molecular Biology & Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China

    Juan Liu, Yuan Zhang & Xuetao Cao

  3. Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, China

    Bin Xie, Kun Chen, Meng Xia, Zhiqing Li, Ting Zhang, Feng Ma, Qingqing Wang, Jianli Wang & Xuetao Cao

  4. Institute of Developmental Biology and Molecular Medicine, School of Life Science, Fudan University, Shanghai, China

    Yue Wu, Kejing Deng, Yuan Zhuang, Xiaohui Wu & Tian Xu

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Contributions

X.C. designed and supervised the research; J.L., C.H., B.X., Y.W., S.L., K.C., M.X., Y. Zhang, L.S., Z.L., T.Z. and F.M. did experiments; Q.W., J.W., K.D., Y. Zhuang, X.W., Y.Y. and T.X. contributed reagents and analytical tools; J.L., C.H., B.X., Y.W., S.L., M.X. and X.C. analyzed data; and J.L., C.H. and X.C. wrote the paper.

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Correspondence to Tian Xu or Xuetao Cao.

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Integrated supplementary information

Supplementary Figure 1 Enhanced proliferation and Th1/Th17 differentiation of Rhbdd3–/– CD4+ T cells.

(a) Appearances of spleens and inguinal lymph nodes (LNs) from 6-month-old Rhbdd3–/– and Rhbdd3+/+ mice (n=3 mice per group). (b) Rhbdd3 mRNA expression in each of the indicated organs and immune cell types were measured by RT-PCR. (c, d) The percentages of CD4+CD25+Foxp3+ Treg in the spleen of Rhbdd3–/– and Rhbdd3+/+ mice were identified by FACS (c) and quantified (d, n=3 mice per group). The dot plots were gated on CD4+ T cells. (e) Proliferation of splenic CD4+ T cells stimulated with α-CD3/28. (f) Production of IFN-γ and IL-17 by MLN (mesenteric LNs) cells, splenocytes or CD4+ T cells stimulated with α-CD3/28. (g, h) Percentages of Th1 and Th17 cells in splenic T cells after α-CD3/CD28 stimulation were analyzed by FACS (g) and quantified (h). Med, Medium. *P<0.05, **P<0.01, ns, not significant (two tailed Student's t-test). Data are from three independent experiments (mean±s.d. of three mice (d) or of technical triplicates (f, h)) or are representative of three independent experiments (a-c, e, g).

Supplementary Figure 2 More severe colitis and overactivation of immune cells in Rhbdd3–/– mice upon TNBS induction.

(a-d) TNBS-colitis was induced in 8-week-old Il17+/+ and Il17–/– mice (n=5 mice per group). Production of IL-17 in colon tissue was measured by CBA test (a). Percentages of weight changes were monitored daily (b). Appearances and histological sections of colons were examined at day3 after TNBS-induction (c, d). (e, f) TNBS-colitis was induced in 8-week-old Rhbdd3+/+ and Rhbdd3–/– mice (n=5 per group). Reduction of colon length was examined at day 3 after TNBS induction (e). Percentages of CD40, CD80, CD86 and I-Ab positive CD11c+ cells and CD44 positive CD4+ T cells in splenocytes were determined at day 3 after TNBS induction (f). *P<0.05, **P<0.01 (two tailed Student's t-test). Data are from three independent experiments (mean±s.d. of five mice (a, b, c (right), e (right), f) or are representative of three independent experiments with similar results (c (left), d, e (left)).

Supplementary Figure 3 Rhbdd3 promotes the generation of Treg and inhibits the differentiation of Th1 and Th17 by inhibiting IL-6 production from DC.

(a-c) CFSE-labeled OT-II CD4+ T cells were co-cultured with TLR-activated BMDCs (a) or splenic DCs (b, c) which were pretreated with OVA323-339 peptide at a ratio of 10:1. After 4 days of culture, proliferation rate of T cells were determined by CFSE dilution (a, b) and secretion of IFN-γ and IL-17 in co-culture supernatants were measured by CBA kit (c). (d) Percentages of CD4+CD25+ and CD4+Foxp3+ Treg in the spleens of Rhbdd3+/+, Rhbdd3–/–, Rhbdd3–/–Il6–/– or Il6–/– mice were measured by FACS (n=3 mice per group). (e) Production of IFN-γ and IL-17 by CD4+ T cells from Rhbdd3+/+, Rhbdd3–/–, Rhbdd3–/–Il6–/– or Il6–/– mice stimulated with α-CD3/CD28 were determined by CBA test. (f, g) CD11c-DTR mice depleted of DC were administrated with Rhbdd3+/+ or Rhbdd3–/– BMDCs. After 3 days, TNBS-induced colitis was performed in recipient mice. Weight loss were monitored daily (f, n=3 mice per group) and histology of colon sections were examined by H&E staining at day3 after TNBS induction (g). *P<0.05, **P<0.01 (two tailed Student's t-test). Data are from three independent experiments (mean±s.d. of three mice (d, f) or of technical triplicates (a, c, e) or are representative of three independent experiments with similar results (b, g).

Supplementary Figure 4 Rhbdd3 controls T cell homeostasis through inhibiting IL-6 production while does not affect T cell-intrinsic signaling activation.

(a) CD4+ T cells from Rhbdd3+/+ and Rhbdd3–/– mice were cultured under distinct T cell subset polarization conditions and analyzed for Th1, Th2, Th17, iTreg cell differentiation. (b, c) CD4+ T cells (b) and CD4+CD44lowCD62Lhigh naïve T cells (c) were stimulated with α-CD3/28 and assessed for MAPK and NF-κB signaling activation. Data are from one representative of three independent experiments with similar results.

Supplementary Figure 5 Rhbdd3 negatively regulates TLR and TNF-α-induced NF-κB pathways in BMDCs but not in BMDMs and MEF.

(a) Immunoblot of NF-κB and MAPK signaling proteins in Rhbdd3+/+ and Rhbdd3–/– BMDCs stimulated with CpG, Poly(I:C) and TNF. (b) EMSA analysis of NF-κB DNA binding ability in nuclear extracts of Rhbdd3+/+ and Rhbdd3–/– BMDCs stimulated with LPS. (c, d) Immunoblot of NF-κB and/or MAPK signaling proteins in Rhbdd3+/+ and Rhbdd3–/– BMDCs and BMDMs (c) and MEFs (d) stimulated with LPS. Data are from one representative of three independent experiments with similar results.

Supplementary Figure 6 Rhbdd3 interacts with K27-linked polyubiquitin chains on NEMO.

(a) Immunoblot in HEK293 cell lysates after single and sequential V5 IP. (b) Immunoblot in HEK293 cell lysates after sequential V5 IP as in (a). Numbers below HA-lanes indicate densitometry of HA relative to V5 expression in that same lane. (c) Immunoblot in HEK293 cell lysates after Flag IP. (d) Immunoblot in HEK293 cell lysates after V5 IP. (e) Silver staining of in vitro purified GST proteins. (f) Schematic of IP procedure in Fig. 6f. (g) Immunoblot in HEK293 cell lysates after V5 IP. Data are from one representative of three independent experiments with similar results.

Supplementary Figure 7 K27-linked polyubiquitination mediated the interaction among Rhbdd3, A20 and NEMO.

(a) Immunoblot of cell lysates immunoprecipitated by single or sequential V5 IP in HEK293 cells overexpressed with V5-tagged Rhbdd3 and HA-tagged one-K ubiquitin mutants. In single V5 IP, cell lysates were immunoprecipitated with anti-V5; in sequential V5 IP, proteins immunoprecipitated by anti-V5 were boiled in presence of 1% SDS and subjected to a second IP by anti-V5. (b) Immunoblot of cell lysates immunoprecipitated by Myc IP in HEK293 cells overexpressed with Flag-tagged Rhbdd3 and Myc-tagged A20. (c) Schematic of IP procedure in Fig. 7f. (d) Immunoblot of cell lysates immunoprecipitated by Myc IP followed by HA IP in HEK293 cells transfected as indicated. Data are from one representative of three independent experiments with similar results.

Supplementary Figure 8 Proposed working model for negative regulation of TLR-triggered NEMO/NF-κB activation and control of autoimmunity by Rhbdd3.

(a) (I) Activation of NEMO/NF-κB pathway upon TLR stimulation. Stimulation of TLR agonists activates IRAK1 and TRAF6. TRAF6 acts as an E3 ubiquitin ligase to activate the TAK1 complex, inducing IKK complex consisting of IKKα, IKKβ and NEMO. Activated IKK complex then phosphorylates IκB and leads to its dissociation from NF-κB. Freed NF-κB translocates into the nucleus and eventually initiates production of proinflammatory cytokines such as IL-6. (II) Inhibition of TLR-triggered NEMO/NF-κB activation by Rhbdd3. TLR signal promotes aggregation of Rhbdd3 in endosomes. Rhbdd3 directly binds to K27-linked polyubiquitin chains on K302 of NEMO via UBA domain. Rhbdd3 also recruits and interacts with A20 via K27-linked polyubiquitin chains on K268 and facilitates A20-mediated K63-linked deubiquitination on NEMO. Above signals lead to inhibition of IκBα degradation and NF-κB activation, and eventually inhibit the production of proinflammatory cytokines such as IL-6. (b) Activation of DC via TLRs leads to activation of NF-κB and production of IL-6, which then potently promotes Th17 generation while inhibits Treg generation, contributing to the development of autoimmune diseases. Rhbdd3 accumulates in endosome upon TLR stimulation and selectively inhibits TLR-triggered NF-κB activation and IL-6 production in DC by interacting with A20 and NEMO and facilitating K63-linked deubiquitination of NEMO. The inhibition of IL-6 production from DC by Rhbdd3 leads to the suppression of Th17 generation but promotion of Treg generation, and eventually contributing to the control of autoimmunity and prevention of autoimmune diseases. Abbreviations: IκBα, Inhibitor of NF-κB alpha; IKKα/β, IκB kinase α/β IRAK1, Interleukin-1 receptor-associated kinase 1; K27/K63, K27/K63-linked polyubiquitin chains; TAB1, TAK-binding protein 1; TAB2/3, TAK-binding protein 2/3 ; TAK1, Transforming growth factor β-activated kinase 1; TRAF6, TNF receptor-associated factor 6; NEMO, NF-κB essential modulator; NF-κB, Nuclear factor kappa B; UBA, Ubiquitin-binding associated domain.

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Supplementary Table 1

Primer sequence used for RT-PCR and quantitative PCR. (PDF 69 kb)

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Liu, J., Han, C., Xie, B. et al. Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO. Nat Immunol 15, 612–622 (2014). https://doi.org/10.1038/ni.2898

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