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Published online November 14, 2007
Diabetes 57:484-493, 2008
DOI: 10.2337/db07-0855
© 2008 by the American Diabetes Association
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Sphingosine-1-Phosphate Reduces CD4+ T-Cell Activation in Type 1 Diabetes Through Regulation of Hypoxia-Inducible Factor Short Isoform I.1 and CD69

Suseela Srinivasan1, David T. Bolick1, Dmitriy Lukashev2, Courtney Lappas3, Michail Sitkovsky2, Kevin R. Lynch3, and Catherine C. Hedrick1,3

1 Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia
2 New England Inflammation and Tissue Protection Institute, Northeastern University, Boston, Massachusetts
3 Department of Pharmacology, University of Virginia, Charlottesville, Virginia

Address correspondence and reprint requests to Catherine C. Hedrick, PhD, Cardiovascular Research Center, University of Virginia, P.O. Box 801394, 415 Lane Rd., MR5, Rm. G123, Charlottesville, VA 22908. E-mail: cch6n{at}virginia.edu

Abbreviations: ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; HIF, hypoxia-inducible factor; HIFBS, heat-inactivated fetal bovine serum; IFN-{gamma}, {gamma}-interferon; IL, interleukin; mAb, monoclonal antibody; S1P, sphingosine-1-phosphate; TCR, T-cell receptor; TNF-{alpha}, tumor necrosis factor-{alpha}; UVA, University of Virginia


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVES—Non-obese diabetic (NOD) mice develop spontaneous type 1 diabetes. We have shown that sphingosine-1-phosphate (S1P) reduces activation of NOD diabetic endothelium via the S1P1 receptor. In the current study, we tested the hypothesis that S1P could inhibit CD4+ T-cell activation, further reducing inflammatory events associated with diabetes.

RESEARCH DESIGN AND METHODS—CD4+ T-cells were isolated from diabetic and nondiabetic NOD mouse splenocytes and treated in the absence or presence of S1P or the S1P1 receptor-specific agonist, SEW2871. Lymphocyte activation was examined using flow cytometry, cytokine bead assays, and a lymphocyte:endothelial adhesion assay.

RESULTS—Diabetic T-cells secreted twofold more {gamma}-interferon (IFN-{gamma}) and interleukin-17 than nondiabetic lymphocytes. Pretreatment with either S1P or SEW2871 significantly reduced cytokine secretion by ~50%. Flow cytometry analysis showed increased expression of CD69, a marker of lymphocyte activation, on diabetic T-cells. Both S1P and SEW2871 prevented upregulation of CD69 on CD4+ cells. Quantitative RT-PCR showed that lymphocytes from diabetic NOD mice had 2.5-fold lower hypoxia-inducible factor (HIF)-1{alpha} short isoform I.1 (HIF1{alpha}I.1) mRNA levels than control. HIF1{alpha}I.1 is a negative regulator of lymphocyte activation. S1P significantly increased HIF1{alpha} I.1 mRNA levels in both control and diabetic groups. IFN-{gamma} production and surface CD69 expression was significantly increased in lymphocytes of HIF1{alpha}I.1-deficient mice. S1P did not reduce either CD69 or IFN-{gamma} expression in lymphocytes from HIF1{alpha}I.1-deficient mice.

CONCLUSIONS—S1P acts through the S1P1 receptor and HIF1{alpha} I.1 to negatively regulate T-cell activation, providing a potential therapeutic target for prevention of diabetes and its vascular complications.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that functions as an extracellular mediator and as an intracellular second messenger. S1P is synthesized by a wide variety of cell types, including lymphocytes, platelets, and macrophages in response to growth factors and cytokines (1). S1P evokes diverse cellular responses by binding to a group of five G-protein–coupled receptors of the endothelial differentiation gene (Edg) family. S1P receptor expression varies among vascular cell types, with T-cells expressing only S1P1 and S1P4 (2). Recently, we reported an anti-inflammatory role for S1P in aortic endothelial cells, most likely through S1P1 action (3). Studies have recently shown that S1P receptor agonists, such as FTY720, reduce renal (4) and hepatic ischemic injury (5). FTY720 is currently being used in phase III clinical trials for multiple sclerosis (6) and has been shown to prevent onset of autoimmune diabetes in mouse models in vivo (7). Nofer et al. (8) have recently reported that FTY720 reduces atherosclerosis in LDL receptor–deficient mice. Other studies have shown the importance of S1P and the S1P1 receptor in modulating T-cell proliferation and homing to lymph nodes (9,10). Furthermore, S1P has recently been shown to regulate lymphocyte egress from peripheral lymph nodes through interaction with the adaptor protein DOCK2 (11). FTY720 and SEW2871, two well-characterized S1P receptor agonists that act on the S1P1 receptor, cause dramatic lymphopenia in mice when used in vivo. Goetzl and colleagues (12,13) have shown that S1P regulates CD4+ T-cell chemotaxis and proliferation through action on S1P1. Thus, S1P analogs have the potential to be important drug therapies for inflammation and immune responses in a wide range of diseases.

Hypoxia-inducible factor (HIF)-1{alpha} is a basic helix-loop-helix transcription factor that is induced under hypoxic conditions. HIF1{alpha} can also be induced during normoxic conditions by vascular endothelial growth factor and other growth factors, various lipids, thrombin, and angiotensin II (14). The murine HIF1{alpha} gene contains two different first exons, termed I.1 and I.2 (15). Expression of HIF1{alpha}I.1 and I.2 is regulated via two distinct promoters, which give rise to these two mRNA isoforms (15). HIF{alpha}I.1 is inducible and differentially expressed, whereas the longer, classical isoform, HIF1{alpha}I.2, is constitutively expressed. The isoform HIF1{alpha} I.1 is an immediately early response gene in T-cells that is differentially upregulated upon T-cell receptor (TCR) activation of T-cells (16). Rapid accumulation of HIF1{alpha}I.1 in activated T-cells has been observed in vivo during cytokine-mediated inflammation (16). The first indications that HIF1{alpha} had both immunosuppressive and tissue-protective roles were provided by in vivo studies showing dramatically increased autoimmunity and tissue damage in chimeric mice that possessed genetic deficiency of HIF1{alpha} in their T- and B-cells (17). Direct evidence that both isoforms of HIF1{alpha} are immunosuppressive in activated T-cells was provided by studying T-cells from mice with complete genetic knockout of HIF1{alpha} isoforms (18). Sitkovsky and colleagues (18) have shown that the HIF1{alpha}I.1 isoform is anti-inflammatory in T-cells, thereby preventing T-cell activation in vivo. Interactions between the immunosuppressive A2A adenosine receptor and HIF1{alpha} in T-cells was first suggested by Sitkovsky and colleagues (19,20). These studies led us to hypothesize that the immunosuppressive effects of S1P could be related to HIF1{alpha} function in lymphocytes.

Despite several reports showing a protective role for S1P and S1P receptor agonists in inflammatory and immune diseases, the precise mechanisms for S1P action in inhibiting inflammation and tissue damage are unclear. In the current study, we report for the first time that S1P induces HIF1{alpha}I.1 expression and activity to modulate T-cell activation in NOD diabetic mice. We now show that regulation of HIF1{alpha}I.1 expression in lymphocytes is a primary mechanism by which the S1P-S1P1 axis regulates lymphocyte activation. Thus, the action of pharmacological agonists of the S1P1 receptor should be beneficial to reduce inflammatory events associated with several vascular diseases, including atherosclerosis and diabetes.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Female NOD/LtJ mice (stock no. 001976) were purchased from the Jackson Laboratories and were maintained in a pathogen-free barrier facility. All animal studies were performed following the approved guidelines of the University of Virginia (UVA) Animal Care and Use Committee. Mice were monitored for hyperglycemia by weekly measurement of blood glucose levels using a One Touch Ultra glucometer (LifeScan). Mice were considered diabetic when two consecutive blood glucose levels were >13.8 mmol/l. Mice lacking HIF1{alpha}I.1 were generated as described previously (18).

Reagents.
S1P was purchased from Cayman Chemicals. Fluorescein isothiocyanate (FITC)-labeled anti-mouse CD69 (clone H1.2F3), phycoerythrin-labeled anti-mouse CD4 (clone GK 1.5), and isotype control antibodies rat IgG2b-PE and hamster IgG-FITC were all purchased from eBiosciences. Enzyme-linked immunosorbent assay (ELISA) kits for murine interleukin (IL)-17, tumor necrosis factor-{alpha} (TNF-{alpha}), and IL-10 were purchased from eBiosciences. Mouse CD4 subset columns were obtained from R&D Systems. Anti-CD3 monoclonal antibody (mAb)–coated plates and Th1/Th2 cytokine Cytometric Bead Arrays were purchased from BD Biosciences. HIF1{alpha} smart pool siRNA was purchased from Dharmacon. Pertussis toxin was provided by Dr. Erik Hewlett (UVA).

In vivo studies of SEW2871 action.
Diabetic and nondiabetic NOD mice were bled retro-orbitally to obtain a prebleed sample. Mice were then injected intravenously with 2 mg/kg body wt SEW2871. After 4 h, blood was collected for measurement of plasma cytokine levels. Cytokines (IL-4, IL-10, and {gamma}-interferon [IFN-{gamma}]) were measured using ELISA.

CD4+ T-cell isolation and activation.
Spleens were harvested from female diabetic and age-matched nondiabetic NOD mice, and the tissue was gently disrupted in PBS containing 5% heat-inactivated fetal bovine serum (HIFBS) on ice. Splenocytes were passed through a 40-µm nylon cell strainer (BD Biosciences) and collected in PBS. Erythrocytes were removed using lysing buffer (Sigma-Aldrich). CD4+ T-cells were isolated with mouse CD4 subset column kit following the manufacturer's protocol. The purity of CD4+ T-cells after isolation was >95% as measured by flow cytometry using anti-CD4 antibody. Cells were washed and resuspended in RPMI 1640 containing 10% HIFBS and 1% antibiotic-antimycotic solution. T-cells were activated in 96-well plates coated with 2–10 µg/ml immobilized anti-CD3 mAb in the presence and absence of 500 nmol/l S1P, 1 µmol/l S1P, or 1 µmol/l SEW2871 at 37°C in 5% CO2 for 16 h. In some studies, cells were incubated in the presence of 100 ng/ml pertussis toxin or 10 µmol/l VPC23019 for 16 h to inhibit S1P1 receptor signaling.

Cytokine measurements.
Splenic CD4+ T-cells were isolated and activated as described above. IFN-{gamma}, IL-4, IL-17, and IL-10 levels secreted into the media were measured by ELISA of cell supernatants after 16 h of treatment with 500 nmol/l or 1 µmol/l S1P, 1 µmol/l SEW2871, or in some cases 100 ng/ml pertussis toxin or 10 µmol/l VPC23019.

T-cell:endothelial adhesion assay.
Aortic endothelial cells were isolated and cultured as described previously (21) from nondiabetic NOD mice. Activated CD4+ T-cells were labeled with Calcein-AM (Molecular Probes) according to the manufacturer's instructions. Fluorescently labeled CD4+ T-cells (50,000 per well) were added to a monolayer of endothelial cells in a 48-well tissue culture dish. After 45 min of incubation at 37°C, unbound T-cells were rinsed off, cells were fixed in 1% glutaraldehyde, and bound T-cells were counted within a 10 x 10 grid using epifluorescence microscopy.

Flow cytometry studies.
T-cells (0.5 x 106) in 100 µl PBS with 1% HIFBS (fluorescence-activated cell sorting [FACS] buffer) were labeled with 0.25 µg each of FITC-labeled anti-mouse CD69 and PE-labeled anti-mouse CD4 antibodies or FITC- and PE-conjugated isotype control antibodies at 4°C for 30 min. Stained cells were washed with 10-fold excess volume of FACS buffer and resuspended in PBS containing 1% paraformaldehyde. The fluorescence intensity was measured by collecting a minimum of 10,000 events using a BD Biosciences FACS-Calibur dual laser benchtop flow cytometer using CellQuest software (Becton Dickinson) and analyzed using FlowJo software (Treestar, San Carlos, CA).

RNA isolation and RT-PCR.
CD4+ T-cells were harvested, and activated using anti-CD3 mAb as described above. Total RNA was isolated using an RNeasy minikit (Qiagen). Quantitative mRNA analysis was performed by real-time PCR using SYBR Green PCR master mix (Bio-Rad) in a Bio-Rad MyIQ icycler. Primer sequences used are listed in Table 1. Data were analyzed and presented as relative expression of mRNA of interest normalized to cyclophylin using the {Delta}Ct method (22).


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  		TABLE 1 Quantitative real-time PCR primers used in this study

 
HIF1{alpha} siRNA transfection.
CD4+ T-cells were isolated from nondiabetic mouse spleens as described above. Four million cells were transfected with 400 pmol HIF1{alpha} siRNA or scrambled siRNA by nucleofection using a mouse T-cell nucleofector kit (Amaxa Biosystems). After 2 h, transfected cells were incubated with 1 µmol/l S1P, and the cells were activated using anti-CD3 mAb as described above. After 24 h, medium was collected for IFN-{gamma} ELISA. Cells were used for FACS analysis of CD69 and mRNA expression of IFN-{gamma}, CD69, and HIF1{alpha}.

Lymphocyte studies using HIF1{alpha}I.1-deficient mice.
CD4+ T-cells were isolated from the spleens of HIF1{alpha}I.1–/– and wild-type mice as described above and were activated in the presence and absence of 1 µmol/l S1P for 16 h. Medium was collected for IFN-{gamma} measurement by ELISA. CD69 surface expression was analyzed by flow cytometry.

Statistical analyses.
Data were analyzed by ANOVA and Fisher's protected least significant difference test using StatView 6.0 software program. Results are expressed as the means ± SE of eight mice per group unless otherwise noted in the figure legends.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Elevated levels of IFN-{gamma} in the plasma of diabetic NOD/LtJ mice.
Recently, we reported that aortic endothelial cells from diabetic NOD mice were highly activated and that monocyte:endothelial interactions were dramatically increased in diabetic mice (3). In the current study, we explored T-cell activation in this diabetic NOD mouse model. Plasma from hyperglycemic NOD mice and normoglycemic littermate controls was analyzed for Th1/Th2 cytokines (Fig. 1). Plasma IFN-{gamma} was approximately twofold higher in the diabetic mice (15.95 ± 0.19 pg/ml) compared with their nondiabetic counterparts (8.54 ± 0.19 pg/ml). Plasma levels of TNF-{alpha}, IL-2, IL-4, and IL-5 were not significantly different between groups. However, diabetic NOD mice showed a 50% reduction in plasma IL-10 levels (P < 0.01).


Figure 1
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  		FIG. 1. Plasma cytokine levels are elevated in diabetic NOD mice. Blood was collected from nondiabetic (CT) and diabetic (Diab) NOD mice. Plasma levels of IFN-{gamma}, IL-4, TNF-{alpha}, IL-5, and IL-2 were measured using a Cytometric Bead Array, and IL-10 was measured by ELISA. Diabetic NOD mice had significantly higher levels of circulating IFN-{gamma} (*P < 0.005). IL-10 levels were significantly lower in diabetic NOD when compared with control mice (#P < 0.01). Data are shown as mean ± SE of six mice per group.

 
CD4+ T-cells from diabetic NOD mice are highly activated, and S1P reduces T-cell activation.
We tested the hypothesis that S1P would reduce T-cell activation and lymphocyte:endothelial interactions. CD4+ T-cells were isolated from spleens of control and diabetic NOD mice using a mouse CD4 subset column, resulting in a population that was about 95% pure CD4+ T-cells. The remaining 5% of cells were granulocytes, as evidenced by 7/4+ expression (data not shown). Lymphocytes were studied either as naïve cells or after stimulation with CD3 antibody. Naïve T-cells from diabetic mice showed a significant increase in IFN-{gamma} secretion compared with nondiabetic littermates (Fig. 2A). Stimulation of cells with anti-CD3 antibody increased IFN-{gamma} release ~100-fold in both nondiabetic control and diabetic T-cells (Fig. 2, compare ST bars in B with A). After CD3 stimulation, there was a significant threefold increase in the amount of IFN-{gamma} produced by diabetic T-cells (see ST bar in Fig. 2B) compared with nondiabetic control lymphocytes. S1P at both 500 nmol/l and 1 µmol/l concentrations and the S1P1 receptor-specific agonist SEW2871 at 1 µmol/l had profound impacts in reducing IFN-{gamma} secretion by diabetic T-cells (Fig. 2B). S1P and SEW2871 also reduced IFN-{gamma} secretion by nondiabetic lymphocytes (Fig. 2B). We used S1P at 1 µmol/l for all remaining experiments for comparison with SEW2871 because we used SEW2871 at 1 µmol/l in vitro. The results using SEW2871 strongly suggest that the effects of S1P are through action on the S1P1 receptor. The S1P1 receptor couples solely through G{alpha}i, so to confirm the role of S1P1, CD3 antibody-stimulated lymphocytes from nondiabetic and diabetic NOD mice were incubated with S1P or SEW2871 in the presence of either pertussis toxin to inhibit G{alpha}i coupling of S1P1, or with VPC23019, a specific antagonist of S1P1 (23). As shown in Fig. 2B, both pertussis toxin and VPC23019 inhibit the effects of S1P on downregulation of IFN-{gamma} secretion. Taken together, these data support the notion that S1P acts through the S1P1 receptor on T-cells to modulate IFN-{gamma} secretion. We also examined IL-17 secretion, which is indicative of a Th17 response, and IL-4 and IL-10 secretion, which are indicative of a Th2 response. As shown in Fig. 2C, IL-17 levels were increased approximately twofold in CD3 antibody-stimulated diabetic NOD mouse T-cells compared with antibody-stimulated nondiabetic lymphocytes, and both S1P and SEW2871 significantly reduced IL-17 secretion. In contrast, we observed no changes in either IL-4 or IL-10 expression in the diabetic NOD T-cells (Fig. 2C). S1P and SEW2871 had no effect on secretion of IL-4 or IL-10. These data indicate that both Th17 and Th1 lymphocyte responses are activated in type 1 diabetic mice, and that the S1P-S1P1 receptor axis impacts both types of lymphocyte responses. In addition to inhibiting cytokine secretion, S1P and SEW2871 also decreased the expression of CD69, a marker of T-cell activation. Surface CD69 expression was significantly increased in diabetic lymphocytes compared with nondiabetic control lymphocytes, averaging 72 ± 2 and 61 ± 2%, respectively (Fig. 3). Both S1P and SEW2871 reduced CD69 expression in diabetic lymphocytes to levels equal to or below those of control, with SEW2871 having a dramatic impact on CD69 expression in both control and diabetic lymphocytes (Fig. 3). Both pertussis toxin and the S1P1 receptor antagonist VPC23019 prevented S1P action on CD69 expression, again implicating the S1P-S1P1 axis in regulating lymphocyte activation.


Figure 2
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  		FIG. 2. S1P and SEW2871 significantly reduce IFN-{gamma} and IL-17 secretion in diabetic T-cells. CD4+ T-cells from nondiabetic control (CT) and diabetic (Diab) NOD mice were either studied freshly isolated (Naïve) or after incubation on immobilized anti-CD3 mAb (ST) for 16 h in the absence or presence of 500 nmol/l or 1 µmol/l S1P (+S1P) or 1 µmol/l SEW2871 (+SEW2871). Some cells were also incubated in the presence of pertussis toxin (+PTX) or with the S1P1 receptor antagonist VPC23019 (+VPC23019) for 16 h. Medium was collected for the measurement of IFN-{gamma}, IL-4, and IL-17. A: IFN-{gamma} production by naïve lymphocytes. IFN-{gamma} production by freshly isolated naïve lymphocytes from nondiabetic (CT) and diabetic (Diab) NOD mice was measured by ELISA. Diabetic T-cells secreted significantly higher levels of IFN-{gamma} compared with nondiabetic cells (*P < 0.001). B: Effects of S1P-S1P1 axis on IFN-{gamma} production in stimulated lymphocytes. Lymphocytes were isolated from nondiabetic (CT) and diabetic (Diab) NOD mice and stimulated via incubation on anti-CD3–coated plates in the presence or absence of S1P, SEW2871, VPC23019, or PTX. IFN-{gamma} was measured in medium by ELISA. S1P significantly reduced secretion of IFN-{gamma} in nondiabetic (#P < 0.01) and diabetic lymphocytes (*P < 0.0001) compared with CD3-incubated (ST) cells. SEW2871 also reduced IFN-{gamma} secretion by nondiabetic (#P < 0.01) and diabetic T-cells (*P < 0.0001). Incubation of cells with Pertussis toxin (PTX) or with VPC23019 blocked the action of S1P and SEW2871 on lymphocyte IFN-{gamma} secretion. C: Effects of S1P on lymphocyte Th17 and Th2 responses. Lymphocytes were isolated from nondiabetic (CT) and diabetic (Diab) NOD mice and stimulated via incubation on anti-CD3–coated plates in the presence or absence of 1 µmol/l S1P or SEW2871. IL-17, IL-10, and IL-4 were measured in cell supernatants by ELISA. *Significantly lower than CT, P < 0.05; **significantly lower than CT, P < 0.02; #significantly higher than CT, P < 0.001; ##significantly lower than Diab, P < 0.005. There were no significant changes in IL-4 or IL-10 production by the lymphocytes.

 

Figure 3
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  		FIG. 3. Reduction in CD69 expression on murine CD4+ T-cells by S1P and SEW2871. CD4+ T-cells from control (CT) and diabetic NOD (NOD) mice were activated by incubating on anti-CD3 mAb–coated plates in the absence or presence of 1 µmol/l S1P (+S1P), 1 µmol/l SEW2871 (+SEW2871), 100 ng/ml pertussis toxin (+PTX), or 10 µmol/l VPC23019 (+VPC23019). Cell surface expression of CD69 was assessed by flow cytometry. Data are expressed as percentage of CD4+ cells expressing CD69. CD69 was increased on diabetic NOD lymphocytes. Both S1P and SEW2871 reduced CD69 surface expression (*P < 0.05 vs. CT; #P < 0.01 vs. Diab; $P < 0.001 vs. Diab). PTX reversed the effect of S1P (**P < 0.05 vs. CT + S1P; ##P < 0.008 vs. Diab + S1P). VPC23019 reversed the effect of S1P in Diab lymphocytes (##P < 0.008 vs. Diab + S1P). Data represent six mice per experimental condition.

 
As another measure of T-cell activation, we measured T-cell:endothelial adhesion. Our laboratory routinely uses a monocyte:endothelial adhesion assay that uses primary mouse endothelial cells and monocytes (3,21,24). We modified this method to study T-cell:endothelial adhesion. Interestingly, we observed that diabetic T-cells adhered more readily to the endothelium than nondiabetic control T-cells (Fig. 4). We observed a 50% increase in the number of adherent lymphocytes from diabetic mice compared with control mice. In some cases, CD4+ T-cells were pretreated for 4 h with 1 µmol/l S1P or SEW2871 before the lymphocytes were added to the endothelial cells for the adhesion assay. S1P and SEW2871 reduced lymphocyte:endothelial interactions by ~35% (Fig. 4). S1P also slightly reduced nondiabetic mouse lymphocyte adhesion to endothelium, although this did not reach statistical significance. TNF-{alpha} stimulation of EC is shown as a control for maximal adhesion by EC in our assay.


Figure 4
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  		FIG. 4. Diabetic T-cells have increased adherence to endothelium. CD4+ T-cells were isolated from nondiabetic (CT) and diabetic (Diab) NOD mice and activated using anti-CD3 mAb–coated plates in the absence or presence of 1 µmol/l S1P or 1 µmol/l SEW2871. Lymphocytes were labeled with calcein-AM and were incubated with the endothelial cell monolayer for an adhesion assay as described in RESEARCH DESIGN AND METHODS. TNF-{alpha} (10 units/ml) was added to the endothelium as a positive control for maximal adhesion (#P < 0.0001 vs. CT). Diabetic NOD T-cells showed greater adhesion to EC than nondiabetic NOD T-cells (*P < 0.005 vs. CT). S1P and SEW2871 reduced adhesion (**P < 0.05 compared with Diab). Data represent the mean ± SE of four experiments performed in duplicate.

 
SEW2871 reduces the circulating proinflammatory cytokine IFN-{gamma} in vivo.
The successful reduction of IFN-{gamma} production by T-cells in vitro by the S1P1 receptor agonist SEW2871 led us to test the effects of SEW2871 on lymphocyte activation in vivo. SEW2871 was intravenously injected into nondiabetic control and diabetic NOD mice. At 4 h after injection, plasma cytokine levels were measured by ELISA. Diabetic mice showed increased plasma IFN-{gamma} levels and reduced IL-10 levels (similar to those shown in Fig. 1). Plasma IL-4 levels did not change (data not shown). SEW2871 decreased plasma IFN-{gamma} levels in the diabetic mice by 50% to levels equivalent to those found in control mice, P < 0.005 (Fig. 5). Furthermore, SEW2871 treatment in vivo significantly increased plasma IL-10 levels in both nondiabetic and diabetic mice (P < 0.01; Fig. 5). Taken together, these experiments in vitro and in vivo using both S1P and SEW2871 strongly suggest that S1P reduces CD4+ T-cell activation through action on the lymphocyte S1P1 receptor.


Figure 5
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  		FIG. 5. S1P1 receptor agonism in vivo changes the plasma cytokine profile. Nondiabetic (CT) and diabetic (Diab) mice were injected intravenously with 2 mg/kg SEW2871 as described in RESEARCH DESIGN AND METHODS. Plasma expression of cytokines was measured using ELISA. Diabetic NOD mice showed increased IFN-{gamma} expression (**P < 0.0001 vs. CT); SEW2871 reduced IFN-{gamma} expression in NOD mice (*P < 0.005). Diabetic NOD mice had reduced IL-10 secretion in plasma (#P < 0.009 vs. CT); SEW2871 increased IL-10 secretion into plasma of NOD mice ($P < 0.01). Data represent n = 6 mice per group.

 
S1P inhibits T-cell activation by increasing HIF1{alpha}I.1{alpha} mRNA expression.
HIF1{alpha} is a transcription factor that is induced by both hypoxic and nonhypoxic pathways in T-cells. Two HIF1{alpha} mRNA isoforms are transcribed and translated, giving rise to HIF1{alpha} I.1 and I.2 proteins. The short isoform HIFI.1 negatively regulates lymphocyte activation (18). Based on these findings, we asked whether S1P impacted HIF1{alpha} I.1 expression in T-cells. To examine this, lymphocytes from control nondiabetic littermate and diabetic NOD mice were activated in the absence or presence of either 1 µmol/l S1P or SEW2871. HIF1{alpha} I.1 mRNA expression was quantified by quantitative real-time PCR. Interestingly, naïve diabetic NOD lymphocytes expressed 50% less HIF1{alpha} I.1 mRNA than naïve nondiabetic lymphocytes (Fig. 6A). Similarly, on TCR-stimulated activation, diabetic T-cells expressed 50% less HIF1{alpha}I.1 mRNA compared with nondiabetic T-cells (Fig. 6A). Both S1P and SEW2871 significantly upregulated HIF1{alpha} I.1 mRNA in both control and diabetic lymphocytes, suggesting that S1P and S1P1 agonists could be beneficial for reducing T-cell activation through upregulation of HIF1{alpha} I.1. Figure 6B shows the expression of HIF1{alpha} I.2 mRNA levels. TCR-stimulated control and diabetic lymphocytes had approximately a twofold increase in HIF1{alpha} I.2 mRNA levels over naïve cells. However, no significant differences were observed between control and diabetic lymphocytes. Furthermore, neither S1P nor SEW2871 changed expression of HIF1{alpha} I.2 (Fig. 6B). These data suggest that action of S1P and SEW2871 through binding to the S1P1 receptor specifically regulates expression of anti-inflammatory HIF1{alpha}I.1.


Figure 6
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  		FIG. 6. S1P and SEW2871 increase HIF1{alpha} I.1 mRNA expression in diabetic T-cells. CD4+ T-cells from nondiabetic (CT) and diabetic (Diab) NOD mice were either studied freshly isolated (Naïve) or incubated on immobilized anti-CD3 mAb (ST) in the presence of 1 µmol/l S1P (+S1P) or 1 µmol/l SEW2871 (+SEW2871). A: HIF1{alpha}I.1 mRNA expression. HIF1{alpha} I.1 mRNA expression was measured by quantitative real-time PCR. Diabetic T-cells expressed 50% less HIF1{alpha}I.1 mRNA compared with nondiabetic T-cells (*P < 0.005). Both S1P and SEW2871 significantly upregulated HIF I.1 mRNA expression in control and diabetic lymphocytes, **P < 0.009. B: HIF1{alpha}I.2 mRNA expression. HIF1{alpha}I.2 mRNA expression was measured by quantitative real-time PCR. Expression was increased on lymphocyte stimulation (*P < 0.009 vs. naïve); however, there was no effect of either S1P or SEW2871 on HIF1{alpha}I.2 mRNA expression.

 
HIF1{alpha} negatively regulates the activation of T-cells.
To directly test the contribution of HIF1{alpha} I.1 on T-cell activation, we used siRNA to HIF1{alpha}. The siRNA was not specific for the HIF1{alpha}I.1 isoform in that knockdown of HIF1{alpha} I.1 mRNA was 53% and knockdown of HIF1{alpha}I.2 was 43% in primary CD4+ T-cells from control nondiabetic NOD mice (Fig. 7A). The knockdown of HIF1{alpha} corresponded to a significant 2.7-fold increase in IFN-{gamma} production by lymphocytes (Fig. 7B). S1P reduced IFN-{gamma} production in lymphocytes transfected with scrambled siRNA, but not in lymphocytes transfected with HIF1{alpha} siRNA (Fig. 7B), suggesting that HIF1{alpha} is important for S1P action in lymphocytes. Moreover, knockdown of HIFI{alpha} caused a dramatic induction of surface CD69 expression on lymphocytes, illustrating activation of T-cells when HIF1{alpha} levels are low (Fig. 7C). As shown in the magenta and light blue lines in Fig. 7C, siRNA to HIF1{alpha} increased CD69 expression compared with either scrambled siRNA (dark blue and dark red lines) or untreated nondiabetic lymphocytes (light green and orange lines). An isotype control antibody is used in each case to illustrate specificity of the fluorescent signal. Also shown in Fig. 7C are corresponding dot plots from this representative experiment, as well as a bar graph showing the means ± SE of results using lymphocytes from six mice per condition. These data strongly suggest that HIF1{alpha} expression regulates CD69 activation in T-cells.


Figure 7
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  		FIG. 7. S1P acts through HIF1{alpha} to regulate CD4+ T-cell activation. CD4+ T-cells from nondiabetic NOD mice were transfected with either scrambled, control siRNA or HIF1{alpha} siRNA. Untransfected CD4+ T-cells from nondiabetic NOD mice were used as controls for comparison to rule out any nonspecific siRNA effects. A: HIF1{alpha}I.1 and I.2 mRNA. Expression of HIF1{alpha}I.1 and I.2 mRNAs were measured by quantitative RT-PCR. *Significantly lower than scrambled siRNA and untransfected cells, P < 0.001. B: IFN-{gamma} secretion. CD4+ T-cells from nondiabetic NOD mice were transfected with either scrambled, control siRNA or HIF1{alpha} siRNA and treated in the absence or presence of S1P (+S1P). Untransfected CD4+ T-cells from nondiabetic NOD mice were used as controls. IFN-{gamma} secretion was measured by ELISA. #Significantly lower than scrambled and untransfected, P < 0.05; *Significantly higher than scrambled and untransfected, P < 0.0001. S1P had no effect in reducing IFN-{gamma} secretion in the absence of HIF1{alpha}. C: CD69 surface expression. CD69 surface expression on CD4+ T-cells of nondiabetic NOD mice was quantified by flow cytometry. HIF1{alpha} siRNA significantly increased the percentage of surface CD69 expression (magenta lines), compared with scrambled siRNA (SCsiRNA) control (dark blue lines). Control, untransfected nondiabetic lymphocytes (CT) are shown in green. Isotype controls were also run with each sample: CT + isotype is shown in orange; the scrambled siRNA + isotype is shown in dark red; and the HIF1{alpha} siRNA + isotype is shown in light blue. The right panel shows representative dot plots for the corresponding histogram. The bar graph shows the means ± SE of the percentage of CD4+CD69+ T-cells from six mice per group. *Significantly higher than CT or SCsiRNA, P < 0.008.

 
Lymphocytes from HIF1{alpha}I.1 knockout mice do not respond to S1P.
Although we achieved compelling results using siRNA, the siRNA was not specific for the HIF1{alpha}I.1 isoform. To prove that HIF1{alpha}I.1 is the signaling molecule for S1P in lymphocytes, we studied T-cells isolated from HIF1{alpha} I.1-deficient mice. HIF1{alpha}I.1-deficient mice appear normal, and thymocytes and T-cells revealed no phenotypic differences compared with wild-type mice (18). CD4+ T-cells were isolated from littermate and HIF1{alpha}I.1-deficient mice (18), and we found a significant pro-inflammatory induction of IFN-{gamma} secretion (Fig. 8A) and increased CD69 expression (Fig. 8B) in HIF1{alpha}I.1 knockout mice (green line) compared with littermate mice (red line). Importantly, S1P was unable to reduce expression of IFN-{gamma} and CD69 in the absence of HIF1{alpha}I.1 (orange line), although S1P was effective in reducing IFN-{gamma} and CD69 in littermate control lymphocytes (blue line) (Fig. 8A and B). We also confirmed these data at the mRNA level, suggesting that HIF1{alpha}I.1 is a primary transcription factor regulating expression of both IFN-{gamma} and CD69 in lymphocytes (Fig. 8C). These data show a novel link between S1P and HIF1{alpha} I.1 signaling to regulate inflammation. Thus, S1P signaling through S1P1 in T-cells drives an anti-inflammatory phenotype via HIF1{alpha}I.1 activation that may be an important therapeutic target for inflammation associated with diabetes.


Figure 8
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  		FIG. 8. HIF1{alpha} I.1 is required for the anti-inflammatory action of S1P in T-cells. CD4+ T-cells were isolated from littermate controls (WT) and HIF1{alpha} I.1-deficient (HIFI.1KO) mice and incubated with S1P (+S1P) as described in RESEARCH DESIGN AND METHODS. A: IFN-{gamma} secretion. IFN-{gamma} secretion was measured by ELISA. Significantly higher than littermate controls, *P < 0.01. Significantly lower than littermate controls, #P < 0.05. S1P had no effect in the HIF1{alpha}I.1 KO lymphocytes. B: CD69 expression. CD69 surface expression on CD4+ T-cells was analyzed by flow cytometry. HIF1{alpha}I.1-deficient mice had a significant increase in CD69 surface expression (green line) compared with littermate controls (red line). S1P significantly reduced CD69 expression on littermate lymphocytes (blue line) but did not reduce CD69 expression on HIF1{alpha}I.1 lymphocytes (yellow line). The bar graph represents the means ± SE of CD69 expression in eight mice per group. *Significantly lower than littermate controls, P < 0.05; **significantly higher than littermate controls, P < 0.001. C: IFN-{gamma} and CD69 mRNA expression. IFN-{gamma} and CD69 mRNA expression in littermate controls and HIF1{alpha}I.1-deficient lymphocytes was measured by quantitative real-time PCR. S1P did not reduce expression of either IFN-{gamma} or CD69 mRNA in HIF1{alpha}I.1-deficient lymphocytes. *Significantly lower than littermate controls, P < 0.001; **significantly higher than littermate controls, P < 0.0001 by ANOVA.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD4+ T-cells have been shown to contribute to the atherosclerotic burden in mice (25). S1P has been shown to regulate lymphocyte homing to lymph nodes from circulation through action on the lymphocyte S1P1 receptor (12). Studies have suggested that S1P also reduces IFN-{gamma} production by lymphocyte cell lines (26), but studies to clearly define the role of S1P in regulating lymphocyte activation have not been performed to date. In the current study, we examined the role of S1P in reducing T-cell activation in vivo. We show for the first time that S1P reduces T-cell activation in type 1 diabetic mice by reducing IFN-{gamma} and CD69 expression in primary mouse CD4+ T-cells. Furthermore, we identified that S1P acts via the lymphocyte S1P1 receptor to upregulate HIF1{alpha} isoform I.1, an anti-inflammatory transcription factor that regulates IFN-{gamma} and CD69 expression in primary T-cells. Thus, we have identified a novel link between the transcription factor HIF1{alpha} I.1 and the anti-inflammatory lipid S1P in lymphocytes.

We found that T-cells isolated from diabetic NOD mouse spleens were highly activated and secreted approximately twofold more IFN-{gamma} and IL-17 than lymphocytes isolated from control, nondiabetic littermate mice. CD69, a marker of early lymphocyte activation, was highly upregulated in diabetic NOD lymphocytes. Taken together, these data illustrate that CD4+ T-cells are "preactivated " in diabetic NOD mice and are capable of significantly contributing to the inflammatory state in vivo.

We observed significant reductions in plasma IL-10 levels in type 1 diabetic mice (Figs. 1 and 5). In vivo administration of SEW2871, the S1P1 receptor-specific agonist, increased expression of IL-10 (Fig. 5). IL-10 is secreted by CD4+CD25+ T regulatory cells and by macrophages. As shown in Fig. 2C, we observed no changes in IL-10 secretion in diabetic T-cells, and S1P or SEW2871 did not change secretion of IL-10 by these lymphocytes. We have not yet examined IL-10 secretion in response to S1P or SEW2871 by T-cell subsets, including T regulatory cells. In preliminary studies, we found that both S1P and SEW2871 stimulate macrophage secretion of IL-10 through agonism of the S1P1 receptor on macrophages (data not shown). Increased IL-10 levels are indicative of an anti-inflammatory response, again supporting the notion that the S1P-S1P1 axis promotes an anti-inflammatory phenotype in vivo. Experiments to characterize the role of S1P in T-cell subset activation will be important follow-up studies to this current work.

Lymphocytes express the S1P receptors S1P1 and S1P4. We found that the activation state of CD4+ T-cells was reduced by S1P through action on the S1P1 receptor using the S1P1 receptor-specific agonist SEW2871 both in vivo and in vitro. This is somewhat in contrast to a recent study by Goetzl and colleagues (13) that indicated that S1P4 regulated IFN-{gamma} secretion by lymphocytes. This group overexpressed murine S1P4 in a D10G4.1 T-cell line that lacked endogenous S1P receptors. They found that addition of 1 µmol/l S1P to the S1P4-D10G4.1 cells caused a significant reduction in IFN-{gamma} secretion (13). We used a S1P4/S1P1 dual agonist, VPC23153 (27), and found similar reductions in lymphocyte activation, especially at doses >1 µmol/l (data not shown). We attributed these findings to agonism at S1P1 rather than S1P4 because of the dose required for maximal effect on lymphocyte function (data not shown). SEW2871 is specific for S1P1 and has no action on S1P4, and we found that SEW2871 was quite effective in reducing lymphocyte activation, both in vitro and in vivo. Thus, our data suggest that S1P1 is the primary receptor through which S1P regulates lymphocyte activation, although we cannot completely rule out some contribution of S1P4.

The murine HIF1{alpha} gene contains two different first exons, termed I.1 and I.2 (15). Expression of HIF1{alpha}I.1 and -I.2 is regulated via two distinct promoters, which give rise to these two mRNA isoforms (15). Sitkovsky and colleagues (18) have recently shown that the HIF1{alpha}I.1 isoform negatively regulates T-cell activation. In Fig. 7, we used siRNA to HIF1{alpha} and found approximately a ~50% reduction in HIF1{alpha}I.1 mRNA and HIF1{alpha}I.2 mRNA expression in T-cells. Interestingly, the HIF1{alpha} siRNA completely abolished the action of S1P on IFN-{gamma} production (Fig. 7B). This was somewhat surprising in that we observed a total loss of the S1P effect with only a 50% loss of HIF1{alpha}I.1. There are two possible explanations for this outcome. First, HIF1{alpha}I.2 plays a minor role in regulating T-cell activation as we have shown previously (18). Thus, reductions in expression of both isoforms of HIF1{alpha} may contribute to the loss of the S1P effect. Second, antibodies to accurately quantify protein levels of the two isoforms are not available, so we are unsure as to the level of expression of HIF1{alpha}I.1 protein in the siRNA studies. It could be that there is very little HIF1{alpha}I.1 protein remaining in the cell after HIF1{alpha} siRNA treatment. However, we anticipate that S1P primarily acts through the HIF1{alpha}I.1 isoform to regulate T-cell activation, in that mice deficient solely in the HIF1{alpha}I.1 isoform showed increased T-cell activation, and S1P had no ability to reduce this cell activation (Fig. 8). Levels of HIF1{alpha}I.2 are normal in these knockout mice, allowing us to specifically study the I.1 isoform in vivo. Thus, our HIF1{alpha}I.1 knockout mouse studies indicate that a primary pathway of regulation of T-cell activation by S1P is through HIF1{alpha}I.1.

HIF1{alpha} is known for its regulation by hypoxia but can also be induced by nonhypoxic stimuli (28). Karliner et al. (29) observed that 10 µmol/l S1P protected rat neonatal cardiomyocytes from hypoxic cell death in vitro, although the mechanisms for this protection were not identified. Further evidence for a link between S1P and hypoxia comes from a recent study by Pyne and colleagues (30) that illustrates that hypoxia increased sphingosine kinase 1 expression to produce more S1P in pulmonary smooth muscle cells. Thus, S1P synthesis could be a protective mechanism induced by the SMC when exposed to chronic hypoxic conditions. Taken together, our results on lymphocytes and the hypoxia studies of others show a novel link between HIF1{alpha} and the protective action of S1P.

In summary, we have found that CD4+ T-cells from type 1 diabetic mouse models are "preactivated " in vivo to release pro-inflammatory cytokines. The anti-inflammatory sphingolipid S1P reduces T-cell activation in diabetic mice in vivo. Action of S1P on lymphocyte activation is primarily through action on the lymphocyte S1P1 receptor through downstream target activation of the anti-inflammatory transcription factor HIF1{alpha} I.1. Thus, specific targeting of the S1P1-HIF1{alpha}I.1 axis in lymphocytes may be an important regulator of immune responses in chronic diseases, such as diabetes and atherosclerosis.


   ACKNOWLEDGMENTS
 
C.C.H. has received research grants from the Juvenile Diabetes Research Foundation and the National Institutes of Health (HL079621).

We thank Joanne Lannigan and Mike Solga in the UVA Flow Cytometry Core Facility for their technical assistance, Dr. Joel Linden (UVA) for helpful discussions, and Dr. Margaret A. Morris (UVA) for providing some of the NOD mice.


   FOOTNOTES
 
Published ahead of print at http://diabetes.diabetesjournals.org on 14 November 2007. DOI: 10.2337/db07-0855.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication June 23, 2007 and accepted in revised form October 31, 2007


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