Cutting edge: all-trans retinoic acid sustains the stability and function of natural regulatory T cells in an inflammatory milieu

Abstract

Recent studies have demonstrated that plasticity of naturally occurring CD4(+)Foxp3(+) regulatory T cells (nTregs) may account for their inability to control chronic inflammation in established autoimmune diseases. All-trans retinoic acid (atRA), the active derivative of vitamin A, has been demonstrated to promote Foxp3(+) Treg differentiation and suppress Th17 development. In this study, we report a vital role of atRA in sustaining the stability and functionality of nTregs in the presence of IL-6. We found that nTregs treated with atRA were resistant to Th17 and other Th cell conversion and maintained Foxp3 expression and suppressive activity in the presence of IL-6 in vitro. atRA decreased IL-6R expression and signaling by nTregs. Of interest, adoptive transfer of nTregs even from arthritic mice treated with atRA suppressed progression of established collagen-induced arthritis. We suggest that nTregs treated with atRA may represent a novel treatment strategy to control established chronic immune-mediated inflammatory diseases.

FIGURE 1

Addition of atRA confers nTregs resistant to Th17 conversion and sustains Foxp3 expression following stimulation with IL-6. A, Splenic nTregs were stimulated with immobilized anti-CD3 (1 μg/ml), soluble anti-CD28 (1 μg/ml), and IL-6 (10 ng/ml) ± atRA (0.05 μM). Three days later, these cells were collected for intracellular IL-17 and IFN-γ staining. Values indicate mean ± SEM of four separate experiments and representative of these experiments (B). C, nTregs were activated with anti-CD3/CD28–coated beads ± atRA solvent (DMSO) or atRA (0.05 μM) or IL-6 (10 ng/ml) for 3 d, and Foxp3 expression was determined by FACS staining. Data are representative of four independent experiments. D, The suppressive activities of nTregs against dep-CD25 T cells ± IL-6 and/or atRA were determined by the inhibition of tritiated thymidine ([³H]thymidine deoxyribose) uptake. Values indicate the mean ± SEM of four independent experiments (D). The p values were calculated by Student t test and indicate significant differences between cultures ± atRA (p < 0.05; below is same).

FIGURE 2

Expanded nTregs treated with atRA maintain Foxp3 expression and suppressive function while restraining to Th17 and Th1 conversion in the presence of IL-6. A, nTregs were stimulated as in Fig. 1B for 7 d, and Foxp3 expression was determined. B, The suppressive activity of these cells was similarly analyzed by inhibition of [³H]thymidine deoxyribose incorporation as in Fig. 1D. Values indicate the mean ± SEM of triplicate wells and data representative of three independent experiments. nTregs were activated as in A and restimulated with IL-6 as in Fig. 1A. Intracellular IL-17 and IFN-γ expression (C) and Foxp3 expression (D) were determined by FACS staining. Results are either representative or mean ± SEM of three independent experiments. E, The suppressive activities of atRA or DMSO treated nTregs ± IL-6 in vitro was determined by similar methods as Fig. 1D.

FIGURE 3

nTregs treated with atRA suppress the progression of established CIA. A, nTregs isolated from naive DBA/1 mice were expanded as in Fig. 1B for 4 d. Mice with established CIA were injected i.v. with 1 × 10⁶ atRA-treated nTregs, DMSO-treated nTregs, or PBS (control group) (n = 8/group). The mice were examined every 3 d postinjection, and the clinical scores are indicated. B, CII-specific IgG1 and IgG2a levels in sera on day 45 after CII/CFA immunization were measured by ELISA. Values indicate the mean ± SEM of two independent experiments (n = 8/group). C, nTregs isolated from CIA were treated ± atRA as described in Fig. 2A, and their suppressive activity was determined by similar methods as Fig. 2A. Values indicate the mean ± SEM of three independent experiments. D, nTregs from CIA mice were expanded with anti-CD3/CD28 beads (1:5) and IL-2 (100 U/ml) ± atRA (0.05 μM) for 4 d. Mice with established CIA were injected i.v. with 1 × 10⁶ atRA-treated nTregs, DMSO-treated nTregs, or PBS (control group) (n = 6/group). The mice were examined every 5 d postinjection, and the clinical scores are indicated.

FIGURE 4

nTregs treated with atRA maintain their phenotype and function via downregulation of IL-6R expression and phospho-STAT3 activation. A, nTregs were treated ± atRA as described in Fig. 2A, and CD126 (IL-6R α-chain) expression was determined by FACS. The figure shows data from one of four separate experiments. B indicates the mean ± SEM of surface CD126, CD130 (IL-6R β-chain), intracellular phosphorylated STAT3, and transcription factor RORγt expression as determined by FACS (n = 4).