A guanidine-rich regulatory oligodeoxynucleotide improves type-2 diabetes in obese mice by blocking T-cell differentiation

Abstract

T lymphocytes exhibit pro-inflammatory or anti-inflammatory activities in obesity and diabetes, depending on their subtypes. Guanidine-rich immunosuppressive oligodeoxynucleotides (ODNs) effectively control Th1/Th2-cell counterbalance. This study reveals a non-toxic regulatory ODN (ODNR01) that inhibits Th1- and Th17-cell polarization by binding to STAT1/3/4 and blocking their phosphorylation without affecting Th2 and regulatory T cells. ODNR01 improves glucose tolerance and insulin sensitivity in both diet-induced obese (DIO) and genetically generated obese (ob/ob) mice. Mechanistic studies show that ODNR01 suppresses Th1- and Th17-cell differentiation in white adipose tissue, thereby reducing macrophage accumulation and M1 macrophage inflammatory molecule expression without affecting M2 macrophages. While ODNR01 shows no effect on diabetes in lymphocyte-free Rag1-deficient DIO mice, it enhances glucose tolerance and insulin sensitivity in CD4(+) T-cell-reconstituted Rag1-deficient DIO mice, suggesting its beneficial effect on insulin resistance is T-cell-dependent. Therefore, regulatory ODNR01 reduces obesity-associated insulin resistance through modulation of T-cell differentiation.

Figure 1. Immunosuppressive activity of ODNR01 on Th1- and Th17-cell differentiation

1. CD4⁺ T-cell culture media IFN-γ, IL-4 and IL-17 levels as determined by ELISA. CD4⁺ T cells were treated with anti-CD3 and anti-CD28 mAb with or without (−) 5 µM of the different regulatory ODNs under Th0, Th1 or Th17 conditions for 3 days. Immunosuppressive ODNA151 was used as a positive control, and random ODN1612 was used as a negative control. *p < 0.05 versus (−) and ODN1612; #p < 0.05 versus ODNA151.

2. ELISA determined CD4⁺ T-cell culture media IFN-γ, IL-4 and IL-17 levels after cells were cultured with or without ODN1612 (5 µM) and different doses of ODNR01 (1, 5 and 10 µM) under Th0, Th1 or Th17 conditions. *p < 0.05 versus (−) and ODN1612.

3. RT-PCR determined CD4⁺ T-cell IFN-γ, IL-4 and IL-17 mRNA levels after cells were treated as in B. *p < 0.05 versus (−) and ODN1612.

4. Representative FACS histograms of Th1 and Th17 cells for purified CD4⁺ T cells treated under Th0, Th1 or Th17 conditions with or without ODN (5 µM) for 4 days. Lymphocytes were first gated on the SSC/FSC plots and then the expression of T-bet and RORγt on the purified CD4⁺ T cells was analyzed.

5. Th1- or Th17-cell frequencies in CD4⁺ T cells after cells were treated with ODNs as in B for 4 days, *p < 0.05 versus (−) and ODN1612. Data are representative of three independent experiments.

Figure 2. Immunosuppression of STAT1/3/4 phosphorylation by ODNR01 selective binding

1. A,B. STAT and phospho-STAT Western blot analysis of anti-CD3/28 mAb-stimulated CD4⁺ T cells treated with indicated cytokines, with and without 5 µM of ODNR01, ODNA151 or ODN1612.

2. C. Confocal microscopy to detect co-localization of FITC-conjugated ODNs (2.5 µM; green) with different Alex Fluor 555-conjugated anti-STAT rabbit polyclonal antibodies (red) in CD4⁺ T cells after 24 h of incubation.

3. D. Left panels: Immunoblot analysis of STATs from CD4⁺ T cells pre-incubated with 5 µM biotinylated-ODNs, lysed, and precipitated with avidin agarose beads for STAT detection. Right panels: Immunoblot analysis of STATs from CD4⁺ T-cell lysates that were pre-incubated with 1 µM biotinylated ODN and 5 µM unlabelled ODN for 1 h and immunoprecipitated with avidin beads for detection of different STAT. The experiments were repeated three times with similar results.

Figure 3. Effects of ODNs on obesity and diabetes in mice

1. A. Body weight gain in DIO mice (on a high-fat diet, HFD) and ob/ob mice (on a normal chow diet, NCD) treated with ODNR01, negative control ODN1612 or PBS for indicated weeks (one dose/2 weeks).

2. B-E. VAT and SAT fat mass (B), VAT adipocyte sizes (C), serum insulin levels (D), and glucose and insulin tolerance (E) from DIO mice and ob/ob mice at the end of treatment. *p < 0.05, **p < 0.01, n = 7∼8 mice per group.

Figure 4. ODNR01 regulates CD4⁺ T-cell subsets in DIO mice

1. Representative FACS plots of Th1, Th2, Th17 and Treg cells in VAT from DIO mice treated with different ODNs. CD4⁺ T cells were first gated on the SSC/CD4 plots, and then the expression of IFN-γ, IL-4, IL-17 and Foxp3 was analyzed in the gated CD4⁺ T cells.

2. The frequencies of Th1, Th2, Th17 and Treg cells in VAT, SAT and spleen from DIO mice treated with different ODNs.

3. ELISA determined serum IFN-γ, IL-4 and IL-17 concentrations in DIO mice treated with different ODNs.

4. RT-PCR determined mRNA levels of transcription regulators (T-bet, GATA-3, RORγt and Foxp3) in sorted CD4⁺ T cells from VAT, SAT and spleen of DIO mice treated with different ODNs. *p < 0.05, **p < 0.01, ND: not detectable, n = 6∼8 mice per group.

Figure 5. Macrophage accumulation and M1 inflammatory factor expression in VAT from DIO mice treated with or without different ODNs or in vitro cultured macrophages

1. A. Immunostaining of VAT with rat anti-mouse Mac-3 mAb. Graph represents the number of Mac-3⁺ cells in each 10 fields.

2. B-E. RT-PCR determined mRNA levels of (B) M1 markers (TNF-α, IL-6, IP-10 and MCP-1) and (C) M2 markers (Arg1, Mrc1 and IL-10) in VAT after normalizing to GAPDH. Fold change was calculated relative to PBS group. RT-PCR determined mRNA levels (normalized to GAPDH) of M1 markers (TNF-α, IL-6, IP-10 and MCP-1) and M2 markers (Arg1, Mrc1 and IL-10) in untreated BMDMs (D) or BMDMs that were stimulated with LPS (10 ng/ml) and IFN-γ (100 U/ml) for 24 h (E), with or without 5 µM ODNR01 or ODN1612. Fold change was calculated relative to medium group (−). Data represent three independent experiments. *p < 0.05; **p < 0.01, n = 4∼6.

Figure 6. CD4⁺ T-cell-dependent effects of ODNR01 on glucose homeostasis

1. Rag1^−/− mouse body weight at 8 weeks or after consuming 18 weeks of a HFD along with ODNR01 or ODN1612 treatment (one dose/2 weeks), glucose tolerance test (GTT) and insulin tolerance test (ITT) at the end of HFD and ODN treatment.

2. FACS plots to detect CD3⁺CD4⁺ T cells in VAT, SAT and spleen from Rag1^−/− mice that consumed HFD for 18 weeks (no ODN treatments), then received WT naive CD4⁺ T-cell intraperitoneal transfer (5 × 10⁶ mouse⁻¹) at week 26 and consumed HFD for an additional 4 weeks while receiving ODN treatments.

3. Body weight of Rag1^−/− recipients from B.

4. GTT, ITT and serum insulin levels in Rag1^−/− recipients from B.

5. GTT, ITT and serum insulin levels from Rag1^−/− mice that consumed a HFD for 18 weeks (no ODN treatments), received adoptive transfer of in vitro ODN-treated WT CD4⁺ T cells, and consumed a HFD for another 4 weeks. Control: no reconstitution or treatment but HFD for the same time period; PBS: CD4⁺ T-cell-reconstituted mice treated with PBS or mice reconstituted with PBS-treated CD4⁺ T cells. *p < 0.05, **p < 0.01, n = 6∼8.