In vivo action of IL-27: reciprocal regulation of Th17 and Treg cells in collagen-induced arthritis

Abstract

Interleukin (IL)-27 is a novel cytokine of the IL-6/IL-12 family that has been reported to be involved in the pathogenesis of autoimmune diseases and has a pivotal role as both a pro- and anti-inflammatory cytokine. We investigated the in vivo effects of IL-27 on arthritis severity in a murine collagen-induced arthritis (CIA) model and its mechanism of action regarding control of regulatory T (Tregs) and IL-17-producing T helper 17 (Th17) cells. IL-27-Fc-treated CIA mice showed a lower severity of arthritis. IL-17 expression in the spleens was significantly decreased in IL-27-Fc-treated CIA mice compared with that in the CIA model. The Th17 population was decreased in the spleens of IL-27-Fc-treated CIA mice, whereas the CD4(+)CD25(+)Foxp3(+) Treg population increased. In vitro studies revealed that IL-27 inhibited IL-17 production in murine CD4(+) T cells, and the effect was associated with retinoic acid-related orphan receptor γT and signal transducer and activator of transcription 3 inhibition. In contrast, fluorescein isothiocyanate-labeled forkhead box P3 (Foxp3) and IL-10 were profoundly augmented by IL-27 treatment. Regarding the suppressive capacity of Treg cells, the proportions of CTLA-4(+) (cytotoxic T-lymphocyte antigen 4), PD-1(+) (programmed cell death protein 1) and GITR(+) (glucocorticoid-induced tumor necrosis factor receptor) Tregs increased in the spleens of IL-27-Fc-treated CIA mice. Furthermore, in vitro differentiated Treg cells with IL-27 exerted a more suppressive capacity on T-cell proliferation. We found that IL-27 acts as a reciprocal regulator of the Th17 and Treg populations in CD4(+) cells isolated from healthy human peripheral blood mononuclear cells (PBMCs), as well as from humans with rheumatoid arthritis (RA) PBMCs. Our study suggests that IL-27 has the potential to ameliorate overwhelming inflammation in patients with RA through a reciprocal regulation of Th17 and Treg cells.

Figure 1

In vivo therapeutic effects of interelukin (IL)-27 via reciprocal regulation of T helper 17 (Th17) and Treg cells on collagen-induced arthritis (CIA) development. Eight days after CIA induction, IL-27-Fc was administered by hydrodynamic intravenous injection in IL-27-Fc-treated CIA animals. Eight and 10 days thereafter, the mice received two additional injections of IL-27-Fc in the muscles of both thighs by electroporation. (a) The clinical score and arthritis incidence rate of disease are shown for both CIA and IL-27-Fc-treated CIA mice over time. Data are representative of two independent experiments with six vanished animals per group (error bar, s.d.). (b) Mice were killed on day 28 after CIA induction, and the expression of IL-17, interferon (IFN)-γ and IL-10 was analyzed on the splenocytes from each group using flow cytometry. The representative flow cytometric dot plots for each group are shown. (c) The populations of CD4⁺CD25⁺Foxp3⁺ cells were analyzed by flow cytometry of the isolated splenocytes of each group. (d) Expression of CD4⁺IL-17⁺ or CD4⁺CD25⁺Foxp3⁺ cells in the spleens of CIA and IL-27-Fc-treated CIA mice was analyzed by immunostaining and confocal microscopy. The data are expressed as the mean±s.d. (error bar) for six animals per group. Original magnification, × 400. *P<0.05, **P<0.01, ***P<0.001 compared with CIA. Fosxp3, fluorescein isothiocyanate-labeled forkhead box P3.

Figure 2

In vitro regulatory effects of interleukin (IL)-27. (a) Splenic CD4⁺ T cells were isolated from normal C57BL/6 mice and then stimulated with anti-CD3 (0.5 μgml⁻¹) in the presence or absence of IL-23 (5 ngml⁻¹) or IL-27 (10 ngml⁻¹) for 3 days. Expression of mRNA levels of IL-17, retinoic acid-related orphan receptor γT (RORγT), signal transducer and activator of transcription 3 (STAT3) and interferon (IFN)-γ was analyzed by real-time polymerase chain reaction (PCR), and the IL-17 concentration of the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). *P<0.05, **P<0.01, ***P<0.001 compared with that in the absence of IL-27. (b) Splenic CD4⁺ T cells isolated from normal C57BL/6 mice were cultured in the presence or absence of IL-27 for 3 days. The mRNA expression of fluorescein isothiocyanate-labeled forkhead box P3 (Foxp3) and IL-10 was determined by real-time PCR, and the IL-10 concentration in the culture supernatant was measured by ELISA. The data are expressed as the mean±s.d. (error bar) for three independent experiments. *P<0.05, **P<0.01 compared with those without.

Figure 3

Changes in CTLA-4⁺ (cytotoxic T-lymphocyte antigen 4), PD-1⁺ (programmed cell death protein 1) or GITR⁺ (glucocorticoid-induced tumor necrosis factor receptor) regulatory T (Treg) populations in the spleen of interleukin (IL)-27-Fc-treated collagen-induced arthritis (CIA). (a) The proportion of CTLA-4⁺, PD-1⁺ or GITR⁺ Treg cells was assessed ex vivo by intracellular flow cytometry. CD4⁺CD25⁺Foxp3⁺ cells were gated from splenocytes of each group of mice. Then, the expression of CTLA-4, PD-1 or GITR among these cells was identified by flow cytometric analysis. Data shown in left panel are representative of three results. (b) CD4⁺CD25⁺ regulatory T cells induced under Treg-polarizing condition with or without IL-27 were cocultured with murine CD4⁺ T cells and irradiated antigen-presenting cells (APCs) (7500 cGy) for 3 days. T-cell proliferation was determined using the [³H]thymidine incorporation assay. Treg cells with IL-27 stimulation exerted more suppressive properties. (c) Spleens from mice of each group were examined by confocal microscopy for staining with mouse antibodies against CD4 (white), CD25 (blue), fluorescein isothiocyanate-labeled forkhead box P3 (Foxp3) (red), PD-1 (green), GITR (green) and CTLA-4 (green) (left panel) (original magnification at × 400). The graph (right panel) represents the number of PD-1⁺, GITR⁺ or CTLA4⁺ Treg (represented by CD4⁺CD25⁺Foxp3⁺) cells that were found in the spleens of CIA- or IL-27-Fc-treated CIA. The number of PD-1⁺, GITR⁺ and CTLA4⁺ Treg cells increased in the spleens of IL-27-Fc-treated CIA mice compared with that of CIA. The data are expressed as the mean±s.d. (error bar) for six mice in each group. (d) CTLA-4-, PD-1- and the GITR-expressing cell populations among effector T cells (CD4⁺CD62L^lowCD44^high) were analyzed by flow cytometric analysis. In contrast to Treg cells, CTLA-4, PD-1 and GITR expression was similar between effector T cells isolated from each group. SSC, side scatter channel.

Figure 4

Reciprocal effects of interleukin (IL)-27 on the regulation of the T helper 17 (Th17) and regulatory T (Treg) lineage. CD4⁺ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of healthy subjects and stimulated with anti-CD3 plus anti-CD28 antibody stimulation with no cytokine added or in Th17-polarizing conditions in the presence or absence of IL-27. (a) After 3 days, the cells were stained intracellularly with antibodies against IL-17 and interferon (IFN)-γ and analyzed by flow cytometry. A representative plot shows the frequencies of CD4⁺IL-17⁺ cells and CD4+IFN-γ+ cells. (b) In vitro differentiated CD4⁺CD25⁺Foxp3⁺ Treg cells under Th17 conditions in the presence or absence of IL-27 were identified by flow cytometric analysis. (c) The concentrations of IL-17, IFN-γ and IL-10 in cultured supernatant from (a) were analyzed by enzyme-linked immunosorbent assay. Foxp3, fluorescein isothiocyanate-labeled forkhead box P3; SSC, side scatter channel.

Figure 5

Effects of interleukin (IL)-27 in rheumatoid arthritis (RA) patients. (a) CD4⁺ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) isolated from RA patients and were cultured in the presence of anti-CD3 plus anti-CD28 stimulation with or without IL-27 for 3 days. The proportion of CD4⁺IL-17⁺ cells and CD4⁺IFN-γ⁺ cells was measured by flow cytometry using intracellular staining for IL-17 and interferon (IFN)-γ. (b) The proportion of CD4⁺CD25⁺Foxp3⁺ cells under T helper 17 (Th17)-polarizing conditions in the presence or absence of IL-27 was determined by flow cytometric analysis. (c) Immunohistochemical staining was used to identify the expressions of IL-27 in RA and osteoarthritis (OA) synovium (original magnification at × 400). Foxp3, fluorescein isothiocyanate-labeled forkhead box P3; SSC, side scatter channel.