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© 2006 American Society for Nutrition J. Nutr. 136:2391-2398, September 2006

Nutritional Immunology

Dietary Fish Oil Inhibits Antigen-Specific Murine Th1 Cell Development by Suppression of Clonal Expansion1,2

Ping Zhang3, Wooki Kim3, Lan Zhou4, Naisyin Wang4,5, Lan H. Ly6, David N. McMurray3,5,6 and Robert S. Chapkin3,5,*

3 Faculty of Nutrition, 4 Department of Statistics, and 5 Center for Environmental and Rural Health, Texas A&M University, College Station, TX and 6 Department of Microbial and Molecular Pathogenesis, Texas A&M University System Health Science Center, College Station, TX

* To whom correspondence should be addressed. E-mail: r-chapkin{at}tamu.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
To determine the mechanisms by which dietary fish oil (FO) affects antigen-stimulated Th1 cell development, DO11.10 Rag 2–/– T cell receptor transgenic mice were fed a control diet (5% corn oil (CO) or a FO diet (1% CO + 4% FO, (n-3) PUFA) for 2 wk. CD4+ T cells were cultured under neutral or Th1 polarizing conditions. FO feeding suppressed (P < 0.05) ovalbumin peptide–induced proliferation of nonpolarized CD4+ T cells. Differentiation in vitro to Th1 cells was not affected by dietary FO, as evidenced by similar percentages of KJ1–26+, IFN-{gamma}+, IL-4– Th1 cells in cultures from CO-fed (99%) and FO-fed (97%) mice. However, the absolute number of viable Th1 cells in polarized cultures from FO-fed mice was less than half that observed in CO-fed mice (P < 0.05), indicating that FO inhibits in vitro Th1 clonal expansion. The reduced number of Th1 cells in FO cultures was not a result of increased apoptosis, because similar percentages of apoptotic Th1 cells were observed in cultures from FO- and CO-fed mice. IL-2–induced cell proliferation was significantly decreased in polarized Th1 cells from the FO group; however, the suppressed proliferation was not linked to reduced CD25 surface expression on antigen-stimulated CD4+ T cells. Adoptively transferred CFSE-labeled DO11.10 CD4+ cells into immunized mice (Th1 polarizing agents) showed that dietary FO reduced (P < 0.05) the number of cell divisions in vivo. These studies suggest that the attenuated inflammatory response which accompanies FO feeding may be explained, at least in part, by suppression of Th1 clonal expansion.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Antigen stimulation elicits clonal expansion of CD4+ T cells, during which the cells may differentiate into 2 distinct types of effector cells, Th1 or Th2, based on cytokine secretion profiles. Defining the mechanisms by which nutrients modulate Th1/Th2 development could facilitate the use of diet as adjunct therapy for treating T cell–mediated autoimmune diseases.

Among dietary factors, (n-3) PUFA found in fish oil (FO)7 have been shown to attenuate many T cell–mediated inflammatory diseases (1–4). The primary effector molecules are considered to be eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Consumption of diets rich in (n-3) PUFA is associated with a reduced risk for a number of autoimmune or inflammatory diseases (5–8). Clinical dietary intervention trials demonstrated a protective effect of FO in rheumatoid arthritis, Crohn's disease, and ulcerative colitis (9). Dietary FO has also been shown to have beneficial effects in various animal models of human chronic diseases, including decreased joint inflammation in rodents with collagen-induced arthritis (10), reduced inflammation in rat models of colitis (11,12), and decreased proteinuria in mice with autoimmune glomerulonephritis (13,14).

We and others have recently demonstrated that dietary (n-3) PUFA affect a variety of immune responses by modulating T cell function (1,15,16). Specifically, FO suppressed proliferation of splenocytes and purified CD4+ T cells in response to mitogen, reduced IL-2 secretion, IL-2 receptor (IL-2R) mRNA, increased activation-induced cell death of CD4+ T cells and polarized Th1 cells, and suppressed T cell signaling via changes in lipid rafts (17–21). However, studies investigating the effects of dietary (n-3) PUFA on Th1 and Th2 cytokines are limited. Wallace et al. reported decreased IFN-{gamma} protein secretion in splenocytes from FO-fed mice (22), and IFN-{gamma} mRNA expression was reduced in the Peyer's patches of FO-fed Bio Breeding (BB) rats (23). Similarly, in humans with multiple sclerosis, FO supplementation reduced IL-2 and IFN-{gamma} secretion by peripheral blood mononuclear cells (PBMCs) (24). In addition, FO supplementation of healthy human volunteers decreased Th1 cell-mediated delayed type hypersensitivity responses (2,25). Consistent with human studies, FO-fed mice showed reduced delayed type hypersensitivity responses when compared with those fed control diets lacking (n-3) PUFA (15). Taken together, these studies strongly suggest that FO may suppress inflammation by inhibiting proinflammatory cytokine production by Th1 cells.

Our laboratory recently demonstrated that purified mouse CD4+ T cells stimulated in vitro with various agonist combinations display distinct cytokine profiles that correspond to Th1 and Th2 cells (20). Splenic CD4+ T cells cultured with phorbol ester (PMA) expressed a Th1-like cytokine profile whereas those activated with anti-CD3/PMA exhibited a Th2-like profile. The suppressed IL-2 secretion of Th1-like cells in mice fed FO compared with corn oil (CO) suggested that FO may alter Th1 development. Indeed, we recently confirmed that the antiinflammatory properties of dietary FO were due in part to a direct effect on Th1 development as opposed to the indirect regulation of Th2 activation (26). Unfortunately, previous studies of (n-3) PUFA on T-cell function have used nonphysiologic, mitogenic stimuli such as concanavalin A (ConA) or anti-CD3/CD28 (15,20). Little is known regarding how dietary (n-3) PUFA affect antigen-induced T cell activation and antigen-stimulated Th1 development. Therefore, in this study, we investigated the effects of dietary (n-3) PUFA on antigen-specific CD4+ T cell activation and differentiation into Th1 cells using the DO11.10 Rag 2–/– transgenic mouse system, a widely used model, to study antigen-specific T cell activation and differentiation. The DO11.10 mouse expresses a transgenic T cell receptor (TCR), which recognizes only an ovalbumin (OVA) peptide, OVA 323–339 and which can be identified using the clonotypic monoclonal antibody (mAb) KJ1–26 (27). Besides antigen-specificity, this model has another advantage due to the rag2 gene deletion; i.e., virtually all peripheral CD4+ T cells from these mice are TCR transgenic, whereas there are ~25% TCR positive CD4+ T cells in the periphery of the conventional DO11.10 mouse (28).


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Animals and diets. All experimental procedures using laboratory animals were approved by the Texas A&M University Laboratory Animal Care Committee. Pathogen-free, female, BALB/c mice (4–6 wk of age) were purchased from Frederick National Cancer Research Facility, Frederick, MD. B10.D2 and syngeneic TCR transgenic DO11.10 Rag2–/– mice were purchased from Taconic Farms, bred, and maintained at Texas A&M University. At the onset of the study, B10.D2 and DO11.10 Rag2–/– mice (8–10 wk of age) were fed a control semi-purified diet containing 5% CO by weight during the 7 d acclimation period, followed by a 2 wk feeding period with the same control diet or a FO diet (1% CO + 4% FO). B10.D2 mice were maintained on the same diets following adoptive transfer and immunization. All diets met the NRC nutrition requirements and varied only in lipid content (17). The basic diet composition, expressed as g/100 g was as follows: casein, 20; sucrose, 42; cornstarch, 22; cellulose, 6; AIN-76 mineral mix, 3.5; AIN-76 vitamin mix, 1; DL-methionine, 0.3; choline chloride, 0.2; Tenox 20A, 0.1; and oil, 5 (17). See supplemental Table 1 for fatty acid composition analyses. BALB/c mice were fed a standard mouse semipurified diet (Teklad 9F rodent diet). After feeding, mice were killed by CO2 asphyxiation.

    T Cells. Spleens and lymph nodes (brachial and mesenteric) were removed from DO11.10 Rag2–/– mice and placed in RPMI complete medium (RPMI 1640 medium with 25 mmol/L HEPES (Irvine Scientific), supplemented with 5% fetal bovine serum (FBS) or homologous mouse serum (HMS 2.5% + 2.5% FBS), 105 U/L penicillin and 100 mg/L streptomycin (Irvine Scientific), 2 mmol/L L-glutamine, and 10 µmol/L 2-mercaptoethanol). T cells were purified by a negative selection column method as previously described (20). HMS was collected according to the method of Pompos et al. (28). In general, spleens from DO11.10 Rag2–/– mice were smaller than conventional mice (data not shown), and the number of lymphocytes obtained was much lower (1.7 x 106 vs. 20 x 106). Approximately 40–70% of the cells were TCR positive in the whole spleens and >90% of cells from lymph nodes were CD4 and TCR positive (data not shown).

    Th1 polarization. CD4+ T cells (5 x 108/L) were cultured in 24 well plates with OVA peptide (0.3 µmol/L) in the presence of irradiated (2600 rads) BALB/c splenocytes (5 x 106), combined with rIL-12 (5 µg/L), anti-IL-4 (10 mg/L), and rIL-2 (20 µg/L) in complete medium containing HMS (2.5% + 2.5% FBS). After culture at 37°C with 5% CO2 for 2 d, the cells were expanded with 20 µg/L rIL-2 and 5 µg/L rIL-12 for an additional 3 d.

    Intracellular cytokine staining. Intracellular cytokine staining was quantified as described previously (29). After staining with Fc block, incubation with KJ1–26-TC (Caltag), and permeabilization at 4°C in Perm/Fix (BD PharMingen), cells were washed in Perm/Wash (BD PharMingen), followed by staining with PE-labeled mAb to murine IL-4 and fluorescein isothiocyanate (FITC)-labeled mAb to murine IFN-{gamma} in Perm/Wash. Preliminary experiments using isotype controls are described elsewhere (30).

    IL-2R assay. CD4+ T cells (5 x 109/L) from diet-fed DO11.0 Rag2–/– mice were cultured in 24 well plates with OVA peptide (0.3 µmol/L) in the presence of irradiated (2600 rads) BALB/c splenocytes for 24 h. For quantitative surface IL-2R staining, 1 x 106 cells were stained with 7-amino-actinomycin D (7-AAD) and anti-CD25-PE (BD PharMingen). Phycoerythrin (PE) fluorescence was collected through the 585/42-nm band pass filter, and 7-AAD fluorescence was collected through the 670-nm long pass filter on a FACSCalibur (Becton Dickinson Immunocytometry Systems) flow cytometer, equipped with a 15 mW air-cooled argon laser, using CellQuest (Becton Dickinson) acquisition software. List mode data were acquired on a minimum of 10,000 events defined by light scatter gates. Surface protein expression was quantified using QuantiBrite PE Beads (BD Biosciences), conjugated with 4 levels of PE. Data analysis was performed in FlowJo (Tree Star), using forward and side light scatter to gate on the lymphocyte population and 7-AAD fluorescence to exclude nonviable cells from analysis. The Calibration Platform in FlowJo was used to convert the fluorescence intensity scale into the absolute number of PE molecules. For each sample stained with anti-CD25 mAb, the median fluorescence intensity, expressed as the equivalent number of PE molecules, was determined to be the median antibody binding capacity for the cell population. By using a known ratio of PE to antibodies (1:1), PE molecules per cell were then converted to antibodies per cell or antibody binding capacity (29).

    T cell proliferation. To determine proliferation of CD4+ T cells under neutral conditions, CD4+ T cells were cultured with OVA peptide (0.3 µmol/L) in the presence of irradiated (2600 rads) BALB/c splenocytes in 96-well round-bottomed microtitre plates (Falcon, Becton-Dickinson). Cells were labeled with 1.0 µCi [3H]-thymidine, cultured for 5 h at 37°C, and counted as described previously (20).

    Apoptosis. CD4+ T cells from diet-fed DO11.0 Rag2–/– mice were Th1 polarized as described above. At d 4 and d 5 in culture, cells were harvested and stained with Annexin-V and PI (BD Biosciences) and analyzed by flow cytometry as previously described (31).

    Adoptive transfer and immunization. CD4+ T cells were purified from spleens and lymph nodes (brachial, axillary, and mesenteric) of DO11.10 donor mice, as described above, and labeled using carboxyfluorescein diacetate succinimidyl ester (CFSE). Briefly, purified CD4+ T cells were mixed with 10 µmol/L CFSE in PBS containing 0.1% bovine serum albumin and incubated at 37°C. After 10 min, 10% FBS RPMI was added and the cells were placed on ice for 5 min to quench excessive CFSE in medium. Cells were adjusted to 3 x 1010 cells/L, loaded into 30-guage insulin syringes, and injected (100 µL) via tail vein into B10.D2 recipient mice that were maintained on the same diet as donor mice. One day after adoptive transfer, recipient mice were immunized with OVA peptide (300 µg/mouse) and CFA via subcutaneous injection at 2 dorsal sites on the back, according to the protocol of Lee et al. (32). Unimmunized animals served as a control.

    Immune cell collection and flow cytometry. Draining (axillary, brachial, and inguinal) lymph nodes were collected from individual recipient mice 3 d after immunization. Approximately 2 x 106 cells were collected and labeled using KJ1–26 mAb specific for detection of DO11.10 transgenic TCR. Briefly, cells were incubated with Fc block (5 mg/L) at 4°C for 15 min, washed with buffer (PBS with 1% heat-inactivated FBS and 0.09% sodium azide, pH 7.4), stained using 2 mg/L KJ-126 mAb at 4°C for 30 min, and analyzed using flow cytometry. Green fluorescence from CFSE was collected through a 530-nm band pass filter and red fluorescence from phycoerythrin-cyanine 5 (PE-Cy5) through a 670-nm long pass filter. List mode data were acquired on at least 20,000 KJ1–26-positive events or until the sample was consumed. Data analysis was performed using ModFitLT (Verity Software House). A lymphocyte region was established using forward and side light scattering properties, and a region for KJ1–26-positive events within the lymphocyte region was defined. Using the Proliferation Wizard, KJ1–26-positive lymphocytes from unimmunized mice were used to establish the position of nondividing cells, and the data for cells from immunized mice were modeled using that point for the parent generation. Parent generations were established independently for each set of CFSE-stained, adoptively transferred lymphocytes.

    Statistical analysis. Significance of main treatment effects was assessed using PROC GLM in SAS. The differences among means were determined by Tukey's test. A 95% level of probability was accepted as being statistically significant. To elucidate the effects of diet, mean proliferation percentages of antigen-specific T cells were determined as described elsewhere (33). Each subdata set of every 4 consecutive generations was analyzed using a quadratic random effect model that incorporated the correlation structure and random effects of individual samples (34). We determined that the quadratic model performed well for data from 4 consecutive generations. Individual samples were preserved as the experimental units throughout the correlation structure of the data. The model was fit using restricted maximum likelihood (35). Wald tests (36) were performed for any main effects or interactions. Analyses on the subdata sets were performed using R (version 1.9.1, R Foundation for Statistical Computing). Data are expressed as mean ± SEM.

For an extended and detailed description of reagents, see the Supplemental Materials online.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    (n-3) PUFA suppress antigen-specific CD4+ T cell proliferation. To determine how dietary (n-3) PUFA affect antigen specific CD4+ T cell proliferation, CD4+ T cells from control (CO-fed) and FO-fed DO11.10 Rag2–/– mice were activated with OVA peptide in the presence of irradiated splenocytes from BALB/c mice fed a semipurified diet. Preliminary studies demonstrated that CD4+ T cells from DO11.10 Rag2–/– mice proliferate in a manner dependent both on the dose of OVA peptide and number of antigen-presenting cells (APCs) (30). The optimum dose was in the range of 0.1–1 µmol/L OVA peptide, and 2 APC ratios were selected to examine optimal and suboptimal stimulation of CD4+ T cells. We have previously demonstrated the need to incorporate HMS in long-term cultures to preclude the loss of (n-3) PUFA-induced T-cell membrane alterations (26,31). When cultured in the presence of HMS, antigen-stimulated proliferation of CD4+ T cells from transgenic mice fed FO was significantly suppressed (P < 0.05) at both APC ratios (Fig. 1). However, in the presence of FBS, there were no significant differences (P > 0.05) in cell proliferation between CO- and FO-fed mice under either optimal or suboptimal conditions.


Figure 1
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  		Figure 1  Effect of diet on antigen-specific CD4+ T cell proliferation. Purified CD4+ T cells from DO11.10 Rag2–/– mice were cultured with OVA peptide and irradiated BALB/c splenocytes in the presence of either (A) FBS (5%) or (B) HMS (HMS 2.5% + 2.5% FBS). Values represent mean ± SEM of the net thymidine uptake (DPM); n = 4 observations/diet group, and 5 mice were pooled per analysis. Different letters indicate differences (P < 0.05) found between the 2 diet groups.

 
    (n-3) PUFA do not affect Th1 cell differentiation. To determine whether dietary (n-3) PUFA influences in vitro antigen stimulated Th1 differentiation, we polarized CD4+ T cells from CO- and FO-fed DO11.10 Rag2–/– mice, as described in the Materials and Methods. The cytokine patterns of differentiated Th1 cells from both CO- and FO-fed mice were similar; i.e., virtually all cells (>97%) were IFN-{gamma} positive, IL-4 negative (Fig. 2). This suggests that only Th1 cells and no Th2 cells were present. Thus, FO feeding did not alter in vitro Th1 cell differentiation.


Figure 2
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  		Figure 2  Effect of diet on antigen specific Th1 differentiation. Purified CD4+ T cells from DO11.10 Rag2–/– mice were cultured with OVA peptide in the presence of irradiated BALB/c splenocytes with IL-12, IL-2, anti-IL-4. A and B, Representative dot plots of cultures derived from CO- and FO-fed mice, respectively. The numbers in the lower right quadrant of the dot plot represent the percentages of IFN-{gamma}-producing cells (Th1) and the numbers in the upper left quadrant are the percentages of IL-4-producing cells (Th2). The plots represent the cytokine expression profiles for 10,161 (CO) and 10,209 (FO) CD4+ T cells. C, Bars represent the percentages of Th1 cells generated under the culture conditions. Values represent mean ± SEM; n = 4 observations per diet group, and 5 mice were pooled per analysis.

 
    (n-3) PUFA suppress the accumulation of polarized Th1 cells. Although dietary FO did not affect Th1 differentiation, the dynamics of cell growth was markedly different between the 2 groups (P = 0.0335; supplemental Fig. 1). The total number of viable cells harvested from cultures containing purified CD4+ T cells from FO-fed mice was less than half the number from the cultures from CO-fed mice at d 5; whereas the total numbers were similar initially for both groups. These data suggest that FO feeding suppressed IL-2-induced clonal expansion of differentiated Th1 cells.

    Surface expression of CD25 is not affected by diet. We next examined the proliferative responses ([3H]-thymidine incorporation) of polarized Th1 cells from DO11.10 Rag2–/– mice fed the control CO and FO diets. After 4 and 5 d in culture, FO-fed mice exhibited significantly decreased Th1 proliferation compared with the CO-fed group (supplemental Fig. 2). Suppression was observed at both d 4 (P = 0.042) and d 5 (P = 0.012); both differences were statistically significant. To elucidate the mechanisms by which dietary (n-3) PUFA suppress IL-2–induced proliferation of polarized Th1 cells, the CD25 expression was determined on the surface of activated CD4+ T cells from FO- and CO-fed mice that were cocultured with OVA- pulsed splenocytes from BALB/c mice fed the semipurified diet. As mentioned earlier, rIL-2 is added to the Th1 polarizing cultures during the last 3 d of expansion. Therefore, the decreased proliferation seen in cultures from FO-fed DO11.10 transgenic mice may have been due to reduced IL-2R signaling. These polarizing conditions do not change cell proliferation in similar cultures of antigen-specific CD4+ T cells (37). Indeed, we observed decreased proliferation of CD4+ T cells from FO-fed mice under nonpolarizing conditions (Fig. 1). Therefore, we decided to measure CD25 (IL-2R{alpha} chain) expression after 24 h stimulation with OVA peptide and APCs. Because the culture period was short and diet-derived (n-3) PUFA are lost from membranes during long-term culture (31), we omitted HMS in the culture. CD25 was measured only on living cells, as defined by nonstaining with 7-AAD. There was no difference (P > 0.05) in the surface expression of CD25 between CD4+ T cells from FO- and CO-fed mice (supplemental Fig. 3). Cultures from CO- and FO-fed mice exhibited similar percentages of viable cells, CD25 positive cells, and median fluorescence intensity of CD25 (Table 1). Viable effector cell recovery (~30%) was within expected limits (37).


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  		TABLE 1 Effect of diet on CD25 (IL-2R{alpha}) surface expression in antigen stimulated CD4+ T cells

 
    Lack of effect of (n-3) PUFA on Th1 cell apoptosis. The reduced number of cells in the FO cultures could be attributed either to suppressed proliferation, enhanced apoptosis, or both. To determine whether apoptosis played a role in the decreased number of cells in the Th1 polarized cultures from FO-fed mice, cells were stained using Annexin-V-FITC and propidium iodide. Early apoptotic cells were defined as Annexin-V positive and propidium iodide negative. Flow cytometric analysis revealed no difference in the percentages of apoptotic cells between FO- and CO-fed mice after d 4 and 5 in culture (supplemental Fig. 4).

    (n-3) PUFA suppress proliferation of adoptively transferred Th1 cells in vivo. In complementary experiments, we also determined the ability of (n-3) PUFA to modulate antigen-induced T cell activation in vivo. DO11.10 Rag2–/– donor mice and B10.D2 recipient mice were fed either a control CO diet or a FO diet for 2 wk. CD4+ T cells from lymph nodes of donors were labeled with CFSE to follow cell division. Cells were adoptively transferred into recipient mice fed the same diet as the donor. B10.D2 mice were immunized 1 d after adoptive transfer and, 3 d after immunization, lymph nodes were harvested. Living cells were subsequently counted and stained with mAb KJ1–26, and flow cytometric gating was used to detect cellular division (Fig. 3). The absolute number of viable cells recovered from lymph nodes of FO-fed recipient mice was reduced (P = 0.014) (2.4 ± 0.6 x 106) compared with CO-fed recipients (1.0 ± 0.2 x 107, n = 3). FO-fed mice did not exhibit a significant reduction (P = 0.078) in the percentage of transgenic CD4+ T cells in lymph nodes (FO 1.35 ± 0.34 vs CO 2.12 ± 0.37, n = 6). Consistent with previous studies using peptide-stimulated DO11.10 T cells, flow cytometric analysis showed that the transferred cells of both groups underwent 8 cell divisions in vivo following immunization (33,38). Determination of the number of cell divisions (expressed as generation number) of antigen-specific T cells revealed that mice fed FO exhibited a higher percentage of early generation cells (Fig. 4). Specifically, percentages of cells in generations 3 (P = 0.047) and 6 (P = 0.006) were elevated following FO treatment. In contrast, percentages of cells in generations 8 (P < 0.001) and 9 (P < 0.001) were reduced in the FO vs. CO fed animals. We conclude that antigen-specific CD4+ daughter cells were arrested in their progression through the proliferation cycle in FO-fed mice.


Figure 3
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  		Figure 3  Flow cytometric gating used to detect antigen-induced cellular division. CFSE-labeled CD4+ T cells were adoptively transferred into B10.D2 recipient mice fed the same diet as the donor. B10.D2 mice were immunized 1 d after adoptive transfer. Following immunization, lymph nodes were harvested and living cells were counted and stained with mAb KJ1–26. A and B, representative viable immunized/dividing KJ1–26+ cells from CO and FO fed animals, respectively, were gated for CFSE fluorescence; C (CO) and D (FO), percentages of cells at specific rounds of division were calculated using the Proliferation Wizard in ModFitLT; E, viable unimmunized/nondividing KJ1–26+ cells (negative control) were gated for CFSE fluorescence.

 

Figure 4
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  		Figure 4  Dietary (n-3) PUFA modulate T cell clonal expansion in vivo. Values represent data sets derived from consecutive generations and were plotted by expressing the generation number against the difference of the means between the estimated expected FO % and CO % at that generation. The data shown were compiled from a total of 6 mice for each diet group from 3 separate experiments. Values above 0 indicate that in the FO group, the % of cells in a specific generation exceeds the CO group. Likewise, values below 0 indicate that in the CO group, the % of cells in a specific generation exceeds the FO group. The P-value was calculated using the Wald test, (*P < 0.05).

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Among dietary factors, (n-3) PUFA found in fish oil are well known for their immunomodulatory effects (1). Although FO supplementation has been shown to be beneficial in many Th1-mediated autoimmune diseases, the precise mechanisms of action remain unlear. We and others have shown that T cells may be the main target for (n-3) PUFA (16,39,40). To further delineate the immunosuppressive effects of (n-3) PUFA on T cells, we investigated the diet effects on antigen-specific T cell activation and Th1 cell differentiation or proliferation using the DO11.10 Rag2–/– TCR–transgenic mouse. With respect to the physiological relevance of the diets used in our studies, the fish oil diet contained ~1.4 and 1.0 energy % as EPA [20:5(n-3)] and DHA [22:6(n-3)], respectively. As a point of reference, the Japanese typically consume EPA and DHA at 1–2% of energy in the diet (41), whereas most European countries and the U.S. consume ~0.1–0.2% of energy as (n-3) PUFA (42). Therefore, the experimental diets were within the range consumed by humans.

Studying the effects of dietary factors on antigen-specific T cell activation is a relatively new concept. Pompos et al. (2002) found that dietary (n-3) PUFA suppressed OVA stimulated proliferation of splenocytes in DO11.10 TCR transgenic mice (28). To extend this observation, our studies revealed that purified CD4+ T cells from the DO11.10 Rag2–/– mice fed FO had significantly decreased proliferation in response to OVA peptide, when cultured in the presence of HMS. To our knowledge, this is the first study to show that dietary (n-3) PUFA are capable of suppressing proliferation of purified, antigen-specific CD4+ T cells. The effects of serum on antigen-specific T cell activation also confirm our previous findings that including HMS in long-term culture is required to preserve (n-3) PUFA-induced changes in plasma membrane microdomain fatty acyl composition and cell functionality (31).

A reduction in IFN-{gamma} production by FO feeding has been reported by several investigators (22–24). Therefore, we hypothesized that FO may suppress Th1 development. With regard to experimental animal models, the genetic background determines the default Th1/Th2 development of naïve Th cells under neutral conditions. Our DO11.10 mouse is on a Th-prone B10.D2 background (43,44) and >90% of the CD4+ cells were Th1 cells after Th1 polarization (Fig. 2). Interestingly, FO did not alter the percentage of Th1 cells generated under stringent Th1 polarizing conditions, suggesting that dietary FO does not affect the Th1 differentiation process. However, the total cell number in culture by d 5 was significantly lower in FO vs. CO (supplemental Fig. 1). Under these culture conditions, it is known that BALB/c splenocytes die (32,37) and CD4+ T cells acquire their Th1 phenotype after 48 h (45). Because only rIL-2 and rIL-12 were added to the cultures after d 2, and rIL-12 does not contribute to cell expansion (46), the difference in absolute cell numbers that we observed may be attributed to the difference in the accumulation of polarized Th1 cells in response to IL-2. This is noteworthy, because an effective immune response depends not only on the acquisition of Th1 and Th2 effector phenotypes, but also on the survival and clonal expansion of differentiated effector cells (47). Autoreactive Th1 cells are major players in some chronic inflammatory diseases (48–50), and inhibition of the expansion of Th1 cells can be one of the mechanisms by which dietary FO exerts an antiinflammatory effect.

Two active processes occur simultaneously in the cell cultures during the 5 d Th1 polarizing protocol used in this study: cell proliferation and apoptosis. Previously, we have shown that FO promotes activation-induced cell death of polarized Th1 cells from conventional mice reactivated with phorbol ester and ionomycin (mitogenic stimuli) (31). Others have demonstrated that apoptosis was enhanced in mouse CD4+ T cells and also in murine T cell lines (51). The apoptotic cells were generated within each cell cycle and the dead cells disappeared from the cultures after d 1. Obviously, if FO enhanced apoptosis, fewer cells would enter the cell cycle and the cumulative effect would be lower cell numbers after 5 d in culture, as we observed (supplemental Fig. 1). We found that the cells from FO- and CO-fed DO11.10 Rag2–/– mice displayed a similar percentage of apoptotic cells (supplemental Fig. 4), indicating that an alteration in IL-2 induced apoptosis could not explain the difference in absolute cell numbers.

The finding of suppressed proliferation of polarized Th1 cells by dietary (n-3) PUFA is novel. Although suppressed T cell proliferation by (n-3) PUFA has been reported (52,53), this is, to our knowledge, the first study that clearly demonstrated the suppression of fully differentiated Th1 cells by diet. Therefore, previous reports of diminished IFN-{gamma} production by (n-3) PUFA could be due to a reduced proliferation, rather than differentiation, of Th1 cells (22,24). We observed suppressed proliferation of both antigen-specific CD4+ T cells (Fig. 1) and fully polarized Th1 cells (supplemental Fig. 2), which suggests that the main effect of dietary (n-3) PUFA was on the molecular machinery for proliferation as opposed to differentiation. In fact, there are similarities between the cultures of CD4+ T cells and polarized Th1 cells. Specifically, there are 2 distinctive stages for the transition of CD4+ T cells to effectors (37): 1) efficient peptide presentation is not limited to the first 1–2 d; and 2) the late stage is an IL-2 driven, antigen-independent, expansion. Therefore, the proliferation of CD4+ T cells that we measured at d 3 is likely a proliferative response to IL-2.

Reduced IL-2 secretion by (n-3) PUFA has been widely reported (20,39,54). EPA added to cultures in vitro decreased IL-2 secretion by human peripheral blood mononuclear cells (54) and the Jurkat human T cell line (39). We have shown that dietary FO decreased IL-2 secretion in CD4+ T cells stimulated with anti-CD3/anti-CD28 (20). In a similar antigen-specific model, Pompos et al. reported that dietary (n-3) PUFA reduced OVA peptide–stimulated splenocyte IL-2 secretion in TCR transgenic mice (28). Although we did not measure IL-2 production, we would expect similar results. Specifically, we would expect that diet-induced suppression of T-cell proliferation could be reversed by the addition of rIL-2. However, this was not the case in Th1 polarized cultures. In the presence of rIL-2 at d 3 in culture, dietary FO continued to suppress the proliferation of polarized Th1 cells (supplemental Fig. 2). This strongly suggests an alternative immunosuppressive mechanism. Thus, we hypothesized that dietary FO may suppress IL-2R signaling.

When added in vitro, EPA has been shown to suppress CD25 expression 24 h after stimulation with phytohemagglutinin in human peripheral blood mononuclear cells (54), and it also suppressed CD25 expression in human Jurkat cells compared with saturated fatty acids (39). In addition, both dietary EPA and DHA reduced CD25 mRNA levels in ConA-stimulated mouse lymphocytes (21). In contrast to these findings, we did not find any difference in cell surface expression of CD25 in antigen-stimulated CD4+ T cells isolated in CO- vs. FO-fed mice (Table 1). We believe this is the first study to examine the effect of dietary FO on the CD25 expression levels on antigen-stimulated CD4+ T cell activation, whereas all previous studies have used mitogenic stimuli. The reported decrease in CD25 expression in other studies may be attributed in part to reduced levels of IL-2 production. The suppression of CD25 signaling can occur both at the transcriptional and posttranscriptional levels (55). Because there was no difference in the surface expression of CD25 on CD4+ T cells from FO- or CO-fed mice, it is unlikely that diet alters IL-2R expression at the transcriptional level. Alternatively, dietary (n-3) PUFA may modify the function of CD25 by altering the composition of lipid rafts. Recently, the functional role of lipid rafts in the regulation of IL-2R signaling was demonstrated (56). Therefore, further investigation of the effect of FO on antigen-stimulated CD4+ T cell IL-2R signaling is warranted.

We have previously demonstrated that dietary FO does not affect proliferation of Th2 polarized cells (26). Considering that activated Th1 and Th2 effector cells exhibit distinct patterns of membrane compartmentalization into lipid rafts (57), it is possible that dietary (n-3) PUFA differentially modulate membrane microdomains in Th1 and Th2 cells, and therefore selectively alter Th1 activation and clonal expansion. It is also possible that serum-containing constituents may convert EPA and/or DHA to recently described metabolically active eicosanoids, which could potentially influence T cell function (58). However, we believe this to be an unlikely scenario in view of the fact that novel products of DHA-derived pathways are produced exclusively by Th2 polarized cells and not Th1 effector cells (59). Clarification of the contribution of the EPA- and DHA-derived metabolic processes with regard to Th1 clonal expansion must await further studies.

To extend our in vitro observations on CD4+ clonal expansion to antigen-stimulated T cells in a whole animal, we use the vital dye CFSE within the context of an adoptive transfer model to examine antigen-specific T cell activation and Th1 proliferation in vivo. In agreement with our in vitro findings, we observed fewer antigen-specific CD4+ T cells in FO-fed recipient mice following immunization with OVA mixed with a Th1 polarizing adjuvant. This, combined with a reduction in daughter cell accumulation (Fig. 4), conclusively demonstrates that dietary FO suppresses Th1 development in vivo. The lower number of transgenic T cells in the lymph nodes of FO-fed recipient mice may therefore be explained by an effect of (n-3) PUFA on T cell trafficking into and out of the nodes and/or on the extent of local lymphoproliferation.

In conclusion, we investigated how dietary (n-3) PUFA affect OVA stimulated CD4+ T cell activation and differentiation into Th1 effector cells. We showed that dietary (n-3) PUFA inhibited Th1 development by suppressing the clonal expansion of polarized Th1 cells, both in culture and in vivo. We also demonstrated that dietary (n-3) PUFA inhibited the proliferation of both antigen-stimulated CD4+ T cells and the IL-2-induced proliferation of antigen-specific polarized Th1 cells. We hypothesize that these suppressive effects are due to modifications in lipid raft composition, which may subsequently alter IL-2 receptor downstream signaling. The exact mechanisms of involvement of lipid rafts in inducing these changes will be the subject of further investigation and will provide insight into how dietary (n-3) PUFA can be beneficial in Th1-mediated autoimmune diseases.


   ACKNOWLEDGMENTS
 
We thank Dr. Michael A. Walker and Vickie Weir for irradiation of BALB/c splenocytes, and Dr. Yang Yi Fan and Evelyn Callaway for their assistance with the collection and purification of T cells and establishment of in vitro cultures. We also thank Dr. Robert Rose, Andrea Taylor, and Stacy Galaviz for assistance with tail injection. Jane Crowther (Omega Protein) kindly donated the Menhaden fish oil used in this study.


   FOOTNOTES
 
1 Supported in part by NIH DK071707 U.S. Department of Agriculture grant 2003-35200-13338, and by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture under Agreement No. 2005-34402-16401, "Designing Foods for Health" through the Vegetable & Fruit Improvement Center. Back

2 Supplemental Figures 1–4, Supplemental Table 1, and Supplemental Materials are available with the online posting of this paper at jn.nutrition.org. Back

7 Abbreviations used: 7-AAD, 7-amino-actinomycin D; APC, antigen presenting cell; CD, cluster of differentiation; CFSE, carboxyfluoroscein succinimidyl ester; CO, corn oil; ConA, concanavalin A; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; FO, fish oil; HMS, homologous mouse serum; IL-2R, interleukin-2 receptor; mAb, monoclonal antibody; OVA, ovalbumin; PE, phycoerythrin; PMA, phorbol ester; TCR, T cell receptor. Back

Manuscript received 19 April 2006. Initial review completed 4 May 2006. Revision accepted 6 June 2006.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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