Myelin basic protein priming reduces the expression of Foxp3 in T cells via nitric oxide

Abstract

Regulatory T cells (Tregs) play a vital role in autoimmune disorders. Among several markers, forkhead box p3 (Foxp3) is the most specific with regard to Treg activity. Therefore, understanding mechanisms that regulate Foxp3 expression is a critical step for unraveling the complicacy of autoimmune pathophysiology. The present study was undertaken to investigate the crosstalk between NO and Tregs. Interestingly, after myelin basic protein (MBP) priming, the expression of Foxp3 decreased in MBP-primed T cells. However, blocking NO either by inhibiting inducible NO synthase with l-N(6)-(1-iminoethyl)-lysine hydrochloride or through scavenging with PTIO or by pharmacological drugs, such as pravastatin, sodium benzoate, or gemfibrozil, restored the expression of Foxp3 in MBP-primed T cells. However, this restoration of Foxp3 by pharmacological drugs was reversed by S-nitrosoglutathione, an NO donor. Similarly, NO also decreased the populations of Tregs characterized by CD4(+)CD25(+) and CD25(+)FoxP3(+) phenotypes. We have further confirmed this inverse relationship between NO and Foxp3 by analyzing the mRNA expression of Foxp3 and characterizing CD25(+)FoxP3(+) or CD4(+)Foxp3(+) phenotypes from inducible NO synthase knockout mice. Moreover, this inverse relation between NO and Foxp3 also was observed during priming with myelin oligodendrocyte glycoprotein, another target neuroantigen in multiple sclerosis, as well as collagen, a target autoantigen in rheumatoid arthritis. Finally, we demonstrate that NO inhibited the expression of Foxp3 in MBP-primed T cells via soluble guanylyl cyclase-mediated production of cGMP. Taken together, our data imply a novel role of NO in suppressing Foxp3(+) Tregs via the soluble guanylyl cyclase pathway.

FIGURE 1

NO negatively regulates the expression of Foxp3 and CD25 in MBP-primed T cells. Splenocytes isolated from MBP-immunized female SJL/J mice were stimulated with MBP in the presence or absence of different doses of l-NIL, PTIO, or GSNO. After 48 h of stimulation, mRNA expression levels of Foxp3, CD25, CD4, and IFN-γ were monitored by semiquantitative RT-PCR (A), and the mRNA expression level of Foxp3 was further confirmed by quantitative real-time PCR (B). After 48 h, supernatants were used for the nitrite assay (C) as described in Materials and Methods. Data are mean ± SD of three different experiments. ^ap < 0.001 versus control; ^bp < 0.001 versus MBP only.

FIGURE 2

NO negatively regulates CD4⁺CD25⁺ as well as CD25⁺Foxp3⁺ T cell populations in MBP-primed T cells. Splenocytes isolated from MBP-immunized female SJL/J mice were stimulated with MBP in the presence or absence of l-NIL or PTIO. After 96 h of stimulation, T cells were incubated with appropriately diluted PE-conjugated anti-CD4 and FITC-conjugated anti-CD25 Abs (A) or PE-conjugated anti-Foxp3 and FITC-conjugated anti-CD25 Abs (D) as described in Materials and Methods followed by FACS analysis. Results represent three independent experiments. MFIs were calculated based on gated populations of CD4⁺CD25⁺ (B), CD4⁻CD25⁺ (C), and total Foxp3⁺ (E) cells by using CellQuest software (BD Biosciences). Data are mean ± SD of three different experiments. ^ap < 0.001 versus control; ^bp < 0.001 versus MBP only.

FIGURE 3

Pharmacological inhibitors of iNOS upregulate the expression of Foxp3 and CD25 in MBP-primed T cells. Splenocytes isolated from MBP-immunized female SJL/J mice were stimulated with MBP in the presence or absence of NaB (1.0 mM), pravastatin (10 μM), or gemfibrozil (200 μM) only or along with different doses of GSNO. After 48 h of stimulation, mRNA expression levels of Foxp3, CD25, and CD4 were monitored by semiquantitative RT-PCR (A), and supernatants were used for the nitrite assay (B). Data are mean ± SD of three different experiments. ^ap < 0.001 versus control; ^bp < 0.001 versus MBP only.

FIGURE 4

Pharmacological inhibitors of iNOS enrich CD4⁺CD25⁺ T cell populations in MBP-primed T cells. Splenocytes isolated from MBP-immunized female SJL/J mice were stimulated with MBP in the presence or absence of NaB (1.0 μM) or pravastatin (10 μM) only or along with GSNO (500 μM) or DETA-NONOate (50 μM). After 96 h of stimulation, T cells were incubated with appropriately diluted PE-conjugated anti-CD4 and FITC-conjugated anti-CD25 Abs (A) as described in Materials and Methods followed by FACS analysis. Results represent three independent experiments. MFIs were calculated based on gated populations of CD4⁺CD25⁺ (B) and CD4⁻CD25⁺ (C) cells by using CellQuest software (BD Biosciences). Data are mean ± SD of three different experiments. ^ap < 0.001 versus MBP; ^bp < 0.001 versus MBP and NaB.

FIGURE 5

Knockdown of iNOS prevents the suppression of Foxp3 expression in T cells during MBP priming. Splenocytes isolated from MBP-immunized B6.129 wild-type and iNOS^−/− mice were stimulated with MBP. After 48 h, mRNA expression levels of Foxp3 and CD4 were monitored by semiquantitative RT-PCR (A), and mRNA expression level of Foxp3 was monitored by quantitative real-time PCR (B). Data are mean ± SD of three different experiments. ^ap < 0.001 versus wild-type control; ^bp < 0.001 versus wild-type MBP only. C, Splenocytes isolated from MBP-immunized B6.129 wild-type, stimulated with MBP only, and iNOS^−/− mice were stimulated with MBP in the presence or the absence of DETA-NONOate (50 μM). After 96 h of stimulation, T cells were incubated with appropriately diluted PE-conjugated anti-Foxp3 and FITC-conjugated anti-CD25 Abs followed by FACS analysis. Results represent three independent experiments.

FIGURE 6

l-NIL and pravastatin restore the expression of Treg markers in vivo in the spleens of MBP-immunized mice. Female SJL/J mice immunized with MBP were treated with saline, l-NIL (5 mg/kg body weight), or pravastatin (1 mg/kg body weight) daily for 10 d postimmunization followed by isolation of RNA from spleens and RT-PCR analysis for Foxp3, CD25, iNOS, and CD4 (A). Splenic sections were dual-immunostained for CD3 and Foxp3 (B), iNOS and Foxp3 (C), or iNOS and CD11b (D). DAPI was used to visualize the nucleus. The settings of the microscope remained strictly unaltered during the entire study. Results represent three independent experiments. Original magnification ×40 (B–D).

FIGURE 7

Knockdown of iNOS prevents the suppression of Foxp3 expression invivo in the spleens of MBP-immunized mice: Spleens were isolated from control or MBP-immunized B6.129 wild-type and iNOS knockout mice, and the mRNA expression levels of Foxp3 and CD4 were monitored by semiquantitative RT-PCR (A). The mRNA expression of Foxp3 was estimated by quantitative real-time PCR (B). Data are mean ± SD of three different experiments. ^ap < 0.001 versus wild-type control; ^bp < 0.001 versus wild-type MBP only. Splenic sections were dual-immunostained for CD3 and Foxp3. DAPI was used to visualize the nucleus (C) (original magnification ×40). The settings of the microscope remained strictly unaltered during the entire study. Results represent three independent experiments.

FIGURE 8

Knockdown of iNOS prevents the suppression of Foxp3 expression in T cells during priming with MOG. Splenocytes isolated from MOG-immunized B6.129 wild-type and iNOS^−/− mice were stimulated with MOG. After 48 h, mRNA expression levels of Foxp3 and CD4 were monitored by semiquantitative RT-PCR (A), and mRNA expression level of Foxp3 was monitored by quantitative real-time PCR (B). Data are mean ± SD of three different experiments. ^ap < 0.001 versus wild-type control; ^bp < 0.001 versus wild-type MOG only. C, Splenocytes isolated from MOG-immunized B6.129 wild-type and iNOS^−/− mice were stimulated with MOG. After 96 h of stimulation, T cells were incubated with appropriately diluted PE-conjugated anti-Foxp3 and FITC-conjugated anti-CD4 Abs followed by FACS analysis. Results represent three independent experiments.

FIGURE 9

Immunization with collagen suppresses the expression of Foxp3 in T cells from wild-type but not the iNOS knockout mice. Splenocytes isolated from collagen-immunized B6.129 wild-type and iNOS^−/− mice were stimulated with collagen. After 48 h, mRNA expression levels of Foxp3 and CD4 were monitored by semiquantitative RT-PCR (A), and mRNA expression level of Foxp3 was monitored by quantitative real-time PCR (B). Data are mean ± SD of three different experiments. ^ap < 0.001 versus wild-type control; ^bp < 0.001 versus wild-type collagen only. C, Splenocytes isolated from collagen-immunized B6.129 wild-type and iNOS^−/− mice were stimulated with collagen. After 96 h of stimulation, T cells were incubated with appropriately diluted PE-conjugated anti-Foxp3 and FITC-conjugated anti-CD4 Abs followed by FACS analysis. Results represent three independent experiments.

FIGURE 10

Attenuation of Foxp3 expression in MBP-primed T cells is via the sGC-cGMP pathway. Splenocytes isolated from MBP-immunized female SJL/J mice were stimulated with MBP in the presence or absence of different doses of NS-2028 (A, B), gemfibrozil, or NaB with or without different doses of 8-Br-cGMP (C–F) or MY-5445 (G–J). After 48 h of stimulation, the mRNA expression levels of Foxp3 and CD4 were monitored by semiquantitative RT-PCR (A, C, E, G, I) and quantitative real-time PCR (B, D, F, H, J). Data are mean ± SD of three different experiments. ^ap < 0.001 versus MBP; ^bp < 0.001 versus gemfibrozil; ^cp < 0.001 versus NaB.