A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17

Abstract

Interleukin 17 (IL-17) has been linked to autoimmune diseases, although its regulation and function have remained unclear. Here we have evaluated in vitro and in vivo the requirements for the differentiation of naive CD4 T cells into effector T helper cells that produce IL-17. This process required the costimulatory molecules CD28 and ICOS but was independent of the cytokines and transcription factors required for T helper type 1 or type 2 differentiation. Furthermore, both IL-4 and interferon-gamma negatively regulated T helper cell production of IL-17 in the effector phase. In vivo, antibody to IL-17 inhibited chemokine expression in the brain during experimental autoimmune encephalomyelitis, whereas overexpression of IL-17 in lung epithelium caused chemokine production and leukocyte infiltration. Thus, IL-17 expression characterizes a unique T helper lineage that regulates tissue inflammation.

Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Figures

Figure 1

The generation of IL-17-producing T cells requires CD28 and ICOS costimulation. (a,b) B6 mice were immunized with MOG(35–55) in CFA or CFA alone; 7 d later, lymph node and spleen cells from immunized mice (two mice in each group) were collected. (a) ELISA of cytokines in supernatants of lymph node and spleen cells from nonimmunized (Nonimm) and immunized mice stimulated in triplicate with MOG peptide for 72 h in vitro. (b) Intracellular staining for IL-17, IFN-γ or TNF after MOG restimulation. Data are of gated CD4+ cells; numbers beside outlined areas indicate percent positive cells in that area. (c) Intracellular detection of IL-17 and IFN-γ in conjunction with anti-CD4 or anti-CD8. B6 mice were immunized for 7 d with KLH in CFA and then spleen and lymph node cells were collected and restimulated with PMA and ionomycin. Numbers beside outlined areas indicate percent positive cells in that area. (d) ELISA of cytokine production by CD4 T cells isolated from Icos-sufficient (Icos+/+) or Icos-deficient (Icos−/−) OTII mice and activated for 4 d with splenic APCs from B6 mice (B7 WT) or mice doubly deficient in B7.1 and B7.2 (B7 KO) in the presence of OVA peptide; activated cells were then washed and restimulated for 24 h with anti-CD3. (e) ELISA of cytokine production by naive T cells isolated from Icos+/1 and Icos−/−mice with or without the Maf transgene and activated for 4 d with anti-CD3, APCs and IL-2. (f) ELISA of cytokine production by spleen cells isolated from B6, B7-deficient (B7 KO), Icos−/− or B7-deficient Icos−/− mice (three in each group, analyzed individually) immunized with KLH in CFA; spleen cells were restimulated ex vivo with various doses of KLH. Data are representative of at least two independent experiments with similar results.

Figure 2

Regulation of IL-17 expression by cytokines and transcriptional factors. (a) ELISA (top) and intracellular staining (bottom) to assess cytokine production by OTII cells. Naive T cells isolated from OTII mice were cultured for 5 d together with APCs from B6 mice along with 10 μg/ml of OVA peptide in presence or absence of anti-IL-4 (α-IL-4), anti-IFN-γ (α-IFN-γ) or IL-23. Cells were restimulated for 24 h with anti-CD3 for ELISA or for 5 h with PMA and ionomycin for intracellular staining. (b) Flow cytometry (dot plots) and ELISA (graph) of spleen and lymph node cells collected from MOG-immunized B6 and Ifng−/− mice (two per group), analyzed with a CD4+ gate for expression IL-17 and IFN-γ after MOG restimulation. (c) ELISA of cytokine expression by spleen and lymph node cells from BALB/c, STAT4-deficient (Stat4−/−) and STAT6-deficient (Stat6−/−) mice immunized with KLH in CFA, analyzed 7 d later after ex vivo restimulation of cells with KLH. (d) Intracellular staining (dot plots) or ELISA (graphs) of cytokine production by draining lymph node cells and splenocytes from T-bet-knockout mice (Tbx21−/−) and B6 mice immunized with MOG in CFA at the tail base and analyzed 7 later. Numbers above bracketed lines (a) or beside oval areas (b,d) indicate percent of cells in that area. Data are representative of at least two independent experiments with similar results.

Figure 3

Regulation of IL-17 production by effector and memory T cells. (a) ELISA of IL-17 production by enriched CD62L−CD4+ cells treated for 36 h with various stimuli (horizontal axis) in the presence or absence of plate-bound anti-CD3 and anti-CD28. (b) ELISA of IL-17 production by sorted CD62L−CD4+ cells preactivated with plate-bound anti-CD3, washed and then treated for 24 h with various stimuli (horizontal axis) plus IL-23. None, absence of anti-CD3 plus anti-CD28; –, without other stimuli. Data are representative of two experiments.

Figure 4

IL-17 regulates genes encoding inflammatory molecules. RT-PCR of the expression of chemokines and matrix metalloproteinases (genes, right margin) in MEFs stimulated for 6 h with human IgG (hIgG), IL-17–Ig, IL-1β or TNF.

Figure 5

IL-17 regulates chemokine expression in brain tissue during EAE. Anti-IL-17 or control rat IgG (100 μg/mouse) was administered intraperitoneally to mice with EAE on days 9, 11 and 13 after the first MOG immunization. (a) EAE incidence is reduced by treatment with anti-IL-17 (α-IL-17). Data are representative of two independent experiments; anti-IL-17, n = 10 mice, and control, n = 11 mice. Mean clinical scores of sick mice are for nine and six mice for the control and anti-IL-17 groups, respectively. (b) ELISA of proinflammatory cytokines in spleen and lymph node cells from control or anti-IL-17-treated mice (at least three in each group) restimulated for 72 h in triplicate in vitro with MOG and analyzed on day 18 after MOG immunization. Data combine more than three mice in each group. (c) Taqman PCR expression of chemokines CCL2, CCL7 and CXCL1 in brains from normal B6 mice and mice subjected to EAE and treated with rat IgG or anti-IL-17. Results are normalized to β-actin expression and were analyzed in triplicate. Expression in normal mice was considered to be 1.

Figure 6

Generation and analysis of Cc10-Il17–transgenic mice. (a) CC10–IL-17 construct. hGH, human growth hormone. (b) ELISA of IL-17 in bronchoalveolar lavage fluid collected from transgene-positive mice (Tg+) and transgene-negative littermates (Tg−). (c) RT-PCR of mRNA (genes, left margin) in lungs obtained from 5-month-old transgene-positive and transgene-negative mice. (d–f) Histological comparison of lung tissue. Lungs from 10-month-old transgene-positive and transgene-negative mice (d) or 10-month-old (e) or 5-month-old (f) transgene-positive mice are stained with hematoxylin and eosin (H&E); with periodic acid Schiff (PAS) to compare mucus production in airway epithelium; and with Masson’s trichrome staining to compare airway collagen deposition. Arrows, blue-staining collagen. (g) RT-PCR of expression of genes (left margin) in MLE12 cells treated for 6 h with various stimuli (above lanes). Csf2 encodes granulocyte colony-stimulating factor.

Supplementary Figure 1. Anti-IL-17 antibody specifically blocks IL-17 biological activity

MEF were treated with 500 ng/ml IL-17-Ig, IL-17F-Ig or human IgG in the presence of a rat Ig control or anti-IL-17 for 24 hrs. IL-6 production was measured by ELISA.

Supplementary Figure 2. Blockage of IL-17 after disease onset reduces inflammation during EAE

(a) Anti-IL-17 treatment attenuates EAE incidence and severity. C57BL/6 mice were immunized with MOG/CFA and administered with pertussis toxin (see “Experimental Procedures”). Three doses of anti-IL-17 or rat IgG was administered after the disease onset (clinic score >1) on day 13, 14 and 16 post first immunization shown as arrows (n=5 in each group). (b) Less infiltration of CD4 T cells and macrophages were observed in anti-IL17 treatment group than rat IgG group. On day 17, mononuclear cells isolated from brains and spinal cords were analyzed for surface markers CD11b and CD4. Student t-test is shown as P values. (c) Splenocytes were ex vivo restimulated with MOG for 48 hours and cytokines expression were measured by ELISA. (d) Splenocyte proliferation in anti-IL-17 treatment group was similar with control group.

Supplementary Figure 3. Summary of TH differentiation

THi is a novel arm of TH cells. Cytokine and transcription factor for TH progenitor to differentiate into THi cells are distinct from those required by conventional TH1 and TH2.