Daurinol Attenuates Autoimmune Arthritis via Stabilization of Nrp1-PTEN-Foxp3 Signaling in Regulatory T Cells

Abstract

Optimizing Treg function and improving Treg stability are attractive treatment strategies for treating autoimmune rheumatoid arthritis (RA). However, the limited number of circulating Tregs and questions about the functional stability of in vitro-expanded Tregs are potential limitations of Treg-based cell therapy. The aim of this study was to analyze the regulatory effect of daurinol, a catalytic inhibitor of topoisomerase IIα, on Th cell differentiation and to evaluate their therapeutic potential in a preclinical experimental model of RA. We investigated the effect of daurinol on T cell differentiation by flow cytometry. Foxp3 stability and methylation were analyzed by suppression assays and bisulfite pyrosequencing. Daurinol was treated in the collagen-induced arthritis (CIA) model, and the effects in vivo were determined. We found that daurinol can promote Treg differentiation and reciprocally inhibit Th17 differentiation. This Treg-inducing property of daurinol was associated with decreased activity of Akt-mTOR and reciprocally increased activity of neuropilin-1 (Nrp1)-PTEN. Daurinol treatment inhibited aerobic glycolysis in Th17 conditions, indicating the metabolic changes by daurinol. We found that the daurinol increase the Treg stability was achieved by Foxp3 hypomethylation. In vivo daurinol treatment in CIA mice reduced the clinical arthritis severity and histological inflammation. The Treg population frequency increased and the Th17 cells decreased in the spleens of arthritis mice treated with daurinol. These results showed the anti-arthritic and immunoregulating properties of daurinol is achieved by increased differentiation and stabilization of Tregs. Our study provides first evidence for daurinol as a treatment for RA.

Keywords: FOXP3 hypomethylation; daurinol; neuropilin 1; regulatory T cells; rheumatoid arthritis; stability.

Figure 1

Reciprocal effects of daurinol on Th17 and Treg cell differentiation in murine CD4+ T cells. Splenic CD4+ T cells from DBA/1J mice were cultured under the Th17-inducing condition (cultured with plate-bound anti-CD3 (0.5 μg/ml), soluble anti-CD28 (0.5 μg/ml), anti-interferon-γ anti-IFNγ; [5 μg/ml (A) or 2 μg/ml (D)], anti–IL-4 [5 μg/ml (A) or 2 μg/ml (D)], IL-6 (20 ng/ml), and TGFβ (2 ng/ml) for 72 h in the presence of vehicle or daurinol. Three days later, the cells were stained with antibodies to CD4, IL-17, and FoxP3. (A) A plot from one representative experiment shows the frequencies of IL-17+, CD25+, and FoxP3+ cells among CD+ T cells. (B) Mean ± SD values are presented in the form of a histogram. Data are representative of four independent experiments with similar results. (C) IL-17 levels in culture supernatants shown in (A,B) were measured by ELISA. (D) CD25+, Foxp3+, or IL-17+ cells among CFSE-labeled proliferating CD4+ T cells cultured under the Th17-skewing condition were analyzed by flow cytometry. (E) Expression of Treg-related genes (Foxp3, TGF-β, SOCS3; upper panel) and Th17-related genes (IL-17, RORγt, HIF-1α; lower panel) in the experiments shown in (D) were determined by real-time PCR. Data were obtained from three independent experiments, and values are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. –, untreated; Veh, vehicle-treated (DMSO); Dau, daurinol.

Figure 2

Daurinol induce the Neuropilin-1+Foxp3 Treg cell expansion. (A) Mouse splenic CD4+ T cells were cultured under the Th17-skewing condition for 72 h in the presence or absence of daurinol (2 μM). Cell lysates were analyzed by Western blotting to detect total and phosphorylated forms of STAT3 (pSTAT3), STAT5 (pSTAT5), PTEN (pPTEN), Akt (pAkt), and pmTOR, Nrp1, and Smad3. (B) The cell populations with the Treg phenotype (GITR+, ICOS+, CTLA-4+, CD103+, PD-1+, and Nrp1+ cells) after daurinol (2 μM) treatment were analyzed by flow cytometry. (C) Nrp1 was deleted in CD4+ T cells using the CRISPR–Cas9 system. Naïve CD4+ T cells and Nrp1-deleted cells were cultured under the Th17-polarizing condition in the presence or absence of daurinol. The populations of Foxp3+ T cells were analyzed by flow cytometry. Data were obtained from three independent experiments, and values are represented as the mean ± SD. *p < 0.05, **p < 0.01.

Figure 3

Daurinol inhibits aerobic glycolysis in CD4+T cells. (A) The expression levels of aerobic glycolysis-associated genes were determined by real-time PCR in murine CD4+ T cells cultured under the Th17-polarizing condition in the presence of absence of daurinol (2 μM). (B) ECAR in CD4+ T cells cultured under the Th17-polarizing condition in the presence or absence of daurinol. The data are representative of three independent experiments and are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Stable immunoregulatory potential of daurinol-induced Tregs associated with Foxp3 hypomethylation. (A) Murine CD4+ T cells were cultured under the Treg-inducing condition and cultured for a further 72 h under the Th17-inducing condition in the presence or absence of daurinol (2 μM). The CD4+CD25+Foxp3+ Treg population was analyzed by flow cytometry. (B) IL-17 and IFN-γ levels in culture supernatants from cells described in (A) were measured by ELISA. –, untreated (C) T cell stimulatory capacity of daurinol-induced Tregs. Anti-CD3-stimulated CD4+ T cells were cultured with daurinol-induced Tregs at different Treg:effector T cell ratios for 3 days. T cell proliferation was determined on day 3 of culture by measuring the incorporation of ³H-thymidine. Data are presented as mean ± SD (bars) of triplicates. **p < 0.01, ***p < 0.001. (D) Representative pyrograms showing hypomethylation of Foxp3 in CD4+ T cells cultured under the Th17-skewing condition with daurinol treatment.

Figure 5

In vivo therapeutic effects of oral administration of daurinol on the development of autoimmune arthritis. Three weeks after immunization with type II collagen (CII), mice with CII-induced arthritis (CIA) were orally administered vehicle or daurinol (5 mg/kg or 25 mg/kg) once every 2 days for 3 weeks. (A) Clinical scores for arthritis (left) and incidence of arthritis (right) are shown for each treatment group over time (representative results from one of two independent experiments). (B) At 46 days after the first CII immunization, tissue sections were obtained from the ankle joints of mice with CIA and stained with hematoxylin and eosin (H&E; original magnification ×40) and Safranin O (original magnification ×200) to examine the severity of arthritis (left). Histological scores for inflammation, bone damage, and cartilage damage were determined (right). (C) Synovial tissue sections of ankle joints from each group of mice were stained with antibodies to IL-1β, IL-17, TNF-α, IL-6, RANK, and RANKL. (D) Concentrations of total and CII-specific IgG1 and IgG2a in the serum of mice from each group were measured by ELISA. Data show the mean ± SD (bars) for six mice per group. (E) RNA was extracted from splenic CD4+ T cells from vehicle- or daurinol (25 mg/kg)-treated CIA mice and analyzed by real-time PCR for the expression of Foxp3, Nrp1, IL-19, KLF2, IL-17, HIF-1α, RORγt, and TNF-α mRNA. Bars show the mean ± SD mRNA expression relative to that of GAPDH for six mice per group from two independent experiments. (F) Left, Spleens were examined by immunofluorescence staining with monoclonal antibodies against CD4 (red), IL-17 (green), CD25 (blue), Foxp3 (yellow), and Nrp1 (green). Original magnification ×400. Scale bar represents 20 μM. Right, CD4+IL-17+ Th17 cells and CD4+CD25+Fop3+Nrp1+ Tregs were enumerated visually at higher magnification as projected on a screen, with each confocal image representative of four fields of view. Values represent the mean ± SD for six mice per group from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

Reciprocal regulation of Nrp1 and PTEN signaling-dependent human Th17 and Treg differentiation by daurinol. Human CD4+ T cells from peripheral blood mononuclear cells were treated with daurinol (dose, 0.5–10 μM) or vehicle under the Th17 cell-polarizing condition for 3 days. (A) The populations of CD4+ Foxp3+ cells and CD4+Foxp3+ cells were analyzed by flow cytometry. Th17 cell differentiation was inhibited by daurinol treatment in a dose-dependent manner, whereas Treg cell differentiation was reciprocally increased. (B) IL-17 levels in culture supernatants were measured by ELISA. (C) Expression of Nrp1, Foxp3, TGF-β, and IL-17 mRNA was determined by real-time PCR. (D) The populations of Foxp3+CD4+ cells altered by Nrp1 and PTEN knockdown in the CRISPR–Cas9 system in human CD4+ T cells were analyzed by flow cytometry. Foxp3+CD4+ T cell differentiation was inhibited by attenuating the expression of Nrp1 and PTEN compared with untreated cells. (E) IL-17 and IFN-γ levels in culture supernatants in the experiments shown in (D) were measured by ELISA. Data were obtained from at least three independent experiments, and values are represented as the mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.