Pathogenic conversion of Foxp3+ T cells into TH17 cells in autoimmune arthritis

Journal name:
Nature Medicine
Volume:
20,
Pages:
62–68
Year published:
DOI:
doi:10.1038/nm.3432
Received
Accepted
Published online

Abstract

Autoimmune diseases often result from an imbalance between regulatory T (Treg) cells and interleukin-17 (IL-17)-producing T helper (TH17) cells; the origin of the latter cells remains largely unknown. Foxp3 is indispensable for the suppressive function of Treg cells, but the stability of Foxp3 has been under debate. Here we show that TH17 cells originating from Foxp3+ T cells have a key role in the pathogenesis of autoimmune arthritis. Under arthritic conditions, CD25loFoxp3+CD4+ T cells lose Foxp3 expression (herein called exFoxp3 cells) and undergo transdifferentiation into TH17 cells. Fate mapping analysis showed that IL-17–expressing exFoxp3 T (exFoxp3 TH17) cells accumulated in inflamed joints. The conversion of Foxp3+CD4+ T cells to TH17 cells was mediated by synovial fibroblast-derived IL-6. These exFoxp3 TH17 cells were more potent osteoclastogenic T cells than were naive CD4+ T cell–derived TH17 cells. Notably, exFoxp3 TH17 cells were characterized by the expression of Sox4, chemokine (C-C motif) receptor 6 (CCR6), chemokine (C-C motif) ligand 20 (CCL20), IL-23 receptor (IL-23R) and receptor activator of NF-κB ligand (RANKL, also called TNFSF11). Adoptive transfer of autoreactive, antigen-experienced CD25loFoxp3+CD4+ T cells into mice followed by secondary immunization with collagen accelerated the onset and increased the severity of arthritis and was associated with the loss of Foxp3 expression in the majority of transferred T cells. We observed IL-17+Foxp3+ T cells in the synovium of subjects with active rheumatoid arthritis (RA), which suggests that plastic Foxp3+ T cells contribute to the pathogenesis of RA. These findings establish the pathological importance of Foxp3 instability in the generation of pathogenic TH17 cells in autoimmunity.

At a glance

Figures

  1. CD25loFoxp3+ T cells are unstable Foxp3+ T cells that convert to TH17 cells under arthritic conditions.
    Figure 1: CD25loFoxp3+ T cells are unstable Foxp3+ T cells that convert to TH17 cells under arthritic conditions.

    (a,b) Clinical score (a) and microcomputed tomography analysis of calcaneus in the ankle joints (b) of immunized DBA/1 mice adoptively transferred with 5 × 105 CD25hi (a, n = 3; b, n = 6) or CD25lo (a, n = 5; b, n = 10) Foxp3+CD4+ T cells purified from untreated DBA/1 Foxp3hCD2 mice. (c) Frequency of Foxp3+ cells in CFSE+ donor-derived T cells. Representative data of five mice are shown. (d,e) Results from the immunized C57BL/6 Ly5.2 mice that were adoptively transferred with total hCD2+ (n = 12), CD25hi (n = 6) or CD25lo (n = 7) Foxp3+CD4+ T cells from untreated B6.Ly5.1 Foxp3hCD2 mice. (d) Frequency of hCD2+ cells in donor-derived CD4+ T cells. (e) Top, representative plots of Foxp3 and IL-17 expression in CD25loFoxp3+ donor- or host-derived CD4+ T cells. Bottom, quantitative analysis of the frequency of IL-17+ cells in Foxp3CD4+ T cells derived from CD25loFoxp3+ donor or host cells (n = 5). (f) Frequency of IL-17+ (left) or CCR6+ (right) cells in Foxp3CD4+ T cells derived from CD25loFoxp3+ (Ly5.1+Ly5.2) or naive CD4+ T (Ly5.1+Ly5.2+) donor cells or host cells (Ly5.1Ly5.2+) (n = 10). All data are shown as the mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.005; NS, not significant). Each dot indicates a single mouse.

  2. Localization, marker gene expression and DNA methylation status of exFoxp3 T cells in arthritic mice.
    Figure 2: Localization, marker gene expression and DNA methylation status of exFoxp3 T cells in arthritic mice.

    Results are shown from fate mapping analyses of arthritic Foxp3-GFP-Cre × ROSA26-YFP mice that were performed 2 weeks after secondary immunization. (a) Frequency of exFoxp3 (GFPYFP+) cells in CD4+ T cells isolated from the indicated location (n = 3 for deep cervical LNs, n = 8 for all other groups). (b) Expression of CCR6 and RANKL in the indicated T cell populations in popliteal lymph nodes. Representative data of six independent experiments are shown. (c) Frequency of IL-17+ cells in the GFPYFP+ cell population (n = 6). (d) Expression of multiple Treg cell phenotypic markers in exFoxp3 T cells. Representative data of six independent experiments are shown. The percentages in each plot show the frequency of positive cells (indicated by the horizontal lines). (e) CpG methylation of the Foxp3, Il2ra and Ctla4 loci in exFoxp3 (GFPYFP+), GFP+YFP+ and GFPYFPCD4+ T cells. A horizontal row within each box corresponds to one sequenced clone in which specific CpGs were methylated (closed) or demethylated (open). All data are shown as the mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*P < 0.05, ***P < 0.005).

  3. Arthritic synovial fibroblasts promote the conversion of Foxp3+ T cells to TH17 cells in an IL-6-dependent manner.
    Figure 3: Arthritic synovial fibroblasts promote the conversion of Foxp3+ T cells to TH17 cells in an IL-6–dependent manner.

    (a) Coculture of Foxp3+CD4+ T cells and Thy1+CD11b synovial fibroblasts or Thy1CD11b+ synovial macrophages isolated from arthritic mice. The frequencies of hCD2+ (Foxp3) and IL-17+ cells were examined after 3 d of coculture. Representative data (left) and quantification (n = 3; right) are shown. (b) IFN-γ, IL-4 and IL-17 expression (n = 3 per analysis) in cocultures of total Foxp3+CD4+ T cells (left) or naive CD4+ T cells (right) with Thy1+CD11b synovial fibroblasts. (c,d) Frequency of IL-17+Foxp3 cells after 3 d of culture of total Foxp3+CD4+ T cells with the supernatant of Thy1+CD11b synovial fibroblasts (c; n = 3) or with Thy1+CD11b synovial fibroblasts using a transwell chamber (d; representative data). (e) Cytokine production by Thy1+CD11b synovial fibroblasts cultured in the presence or absence of IL-17 (n = 3). (f) Left, expression of Foxp3 and IL-17 in T cells after culture of Foxp3+CD4+ T cells with Thy1+CD11b synovial fibroblasts in the presence of neutralizing antibodies to IL-6, TNF-α or IL-1β (representative data). Right, quantitative analysis of the frequency of IL-17+Foxp3 cells in T cells (n = 3). All data are shown as the mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (***P < 0.005; NS, not significant; ND, not detected). Data are representative of three independent experiments with triplicate culture wells.

  4. exFoxp3 TH17 cells are osteoclastogenic T cells with distinct gene profiles.
    Figure 4: exFoxp3 TH17 cells are osteoclastogenic T cells with distinct gene profiles.

    (a,b) Osteoclast differentiation in a coculture of BMMs, Thy1+CD11b CIA synovial fibroblasts and the T cell subsets indicated. exFoxp3 TH17 indicates Foxp3 T cells developed from Foxp3+ T cells under TH17-polarizing conditions in this experiment. (a) Representative tartrate-resistant acid phosphatase (TRAP) staining. (b) Number of osteoclasts (TRAP+ MNCs) (n = 3 for exFoxp3 TH17 and Foxp3+ T cells, n = 4 for Il17a−/− TH17 cells, n = 6 for all other groups). WT, wild type. (c) Representative FACS profiles of RANKL and IL-17 expression in naive CD4+ T cell–derived TH17 cells (left) and exFoxp3 TH17 cells (middle and right) that differentiated under the conditions indicated. (d) Number of osteoclasts (TRAP+ MNCs) in a coculture of BMMs and the T cell subsets indicated (n = 3). (e,f) Number of osteoclasts (TRAP+ MNCs) in a coculture of BMMs, synovial fibroblasts and the T cell subsets indicated. (e) Analysis using exFoxp3 TH17 cells derived from Tnfsf11+/+ Foxp3hCD2 or Lck-Cre Tnfsf11flox/Δ Foxp3hCD2 mice. (f) Analysis using Tnfsf11+/+ or Tnfsf11Δ/Δ arthritic synovial fibroblasts. (g) Microarray analysis of selected TH17-related genes in exFoxp3 TH17 cells and TH17 cells. IL-17GFP+ Foxp3hCD2− cells developed from naive CD4+ T cells or Foxp3+CD4+ T cells under TH17-polarizing conditions were used as the TH17 cells and exFoxp3 TH17 cells, respectively. The mean fold change of three independent experiments is shown. (h) Quantitative RT-PCR analysis of differentially expressed genes in TH17 cells and exFoxp3 TH17 cells (n = 3). All data are representative of three independent experiments with triplicate culture wells and are shown as the mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*P < 0.05, ***P < 0.005).

  5. Pathogenic role of exFoxp3 T cells to arthritis in vivo.
    Figure 5: Pathogenic role of exFoxp3 T cells to arthritis in vivo.

    (ac) Clinical score (a), histological analysis (b) and bone morphometric analysis (c) of the ankle joints of DBA/1 Foxp3hCD2 mice adoptively transferred with the indicated T cell subsets that were purified from spleens and dLNs of collagen-immunized mice (n = 3 for total Foxp3 cells, n = 5 for CD25loFoxp3+ and CD25hiFoxp3+ cells, n = 6 for CD44hiFoxp3 cells) or OVA-immunized mice (n = 4). In b, toluidine blue (top and middle) and TRAP (bottom) staining of ankle joints were performed. The middle row is a magnification of the boxed areas in the top row. Scale bars, 100 μm. In c, the quantitative bone morphometric analysis was performed using histological sections (n = 3). Osteoclast number per bone surface (top left), osteoclast surface per bone surface (top right), articular cartilage dysfunction score (bottom left) and eroded surface per bone surface (bottom right) are shown. (d) Frequency of Foxp3+ cells in donor-derived CFSE+CD4+ T cells 1 week after secondary immunization (n = 5). (e) In vitro proliferative responses to type II collagen of CD25hiFoxp3+ or CD25loFoxp3+ T cells purified from the spleens and dLNs of collagen-immunized mice. The numbers indicate the frequency of proliferating cells as determined by CFSE dilution. Representative data of more than three independent experiments are shown. All data are shown as the mean ± s.e.m. Statistical analyses were performed using one-way analysis of variance with Newman-Keuls multiple comparison test (a) or unpaired two-tailed Student's t test (c,d) (*P < 0.05, **P < 0.01, ***P < 0.005).

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Author information

Affiliations

  1. Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

    • Noriko Komatsu,
    • Kazuo Okamoto,
    • Shinichiro Sawa &
    • Hiroshi Takayanagi
  2. Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology (ERATO) Program, Takayanagi Osteonetwork Project, Bunkyo-ku, Tokyo, Japan.

    • Noriko Komatsu,
    • Kazuo Okamoto,
    • Shinichiro Sawa,
    • Tomoki Nakashima &
    • Hiroshi Takayanagi
  3. Department of Cell Signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.

    • Tomoki Nakashima &
    • Masatsugu Oh-hora
  4. JST, Precursory Research for Embryonic Science and Technology Program, Bunkyo-ku, Tokyo, Japan.

    • Tomoki Nakashima &
    • Masatsugu Oh-hora
  5. Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Bunkyo-ku, Tokyo, Japan.

    • Masatsugu Oh-hora
  6. Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo, Japan.

    • Tatsuhiko Kodama
  7. Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

    • Sakae Tanaka
  8. Diabetes Center, University of California, San Francisco, San Francisco, California, USA.

    • Jeffrey A Bluestone
  9. Centre for Orthopaedic Research, School of Surgery, The University of Western Australia, Nedlands, Western Australia, Australia.

    • Hiroshi Takayanagi

Contributions

N.K. designed and performed experiments, interpreted the results and prepared the manuscript. K.O., S.S., T.N. and M.O. contributed to study design and manuscript preparation. T.K. contributed to microarray analysis. S.T. contributed to the analysis of human RA and osteoarthritis samples. J.A.B. generated Foxp3-GFP-Cre mice and contributed to study design and data interpretation. H.T. directed the project and wrote the manuscript.

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The authors declare no competing financial interests.

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