n−3 Polyunsaturated Fatty Acids Suppress Mitochondrial Translocation to the Immunologic Synapse and Modulate Calcium Signaling in T Cells

Abstract

Recent studies indicate that the process of Ag presentation induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the immunologic synapse (IS). This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca²⁺ influx and Th cell activation. Recently, we demonstrated that n−3 polyunsaturated fatty acids (PUFAs) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n−3 PUFAs suppress CD4⁺ T cell mitochondrial translocation during the early stages of IS formation and downmodulate Ca²⁺-dependent Th cell activation. CD4⁺ cells derived from fat-1 mice, a transgenic model that synthesizes n−3 PUFA from n−6 PUFA, were cocultured with anti-CD3–expressing hybridoma cells (145-2C11) for 15 min at 37°C, and mitochondrial translocation to the IS was assessed by confocal microscopy. Fat-1 mice exhibited a significantly (p < 0.05) reduced percentage of T cells with mitochondria which translocated to the IS; fat-1 (30%) versus wild type control (82%). Regarding the effect on the mitochondrial-to-cytosolic Ca²⁺ ratio, wild type cells showed significant increases at the IS (71%) and total cell (60%) within 30 min of IS formation. In contrast, fat-1 CD4⁺ T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca²⁺ ratio was observed in wild type cells that were coincubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca²⁺ (CRAC) channels, BTP2. These observations provide evidence that n−3 PUFAs modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca²⁺entry.

T cell activation requires contact with APCs to bring the TCR and MHC–peptide complex together. In general, contact is defined by the size of the TCR and MHC–peptide complex which, at ~10–15 nm, requires extensive interdigitation of the plasma membrane of the T cell and APCs. T cells are typically activated via formation of a stable T cell–APC junction, referred to as an immunologic synapse (IS) (1–4).

When the TCR is activated by Ag presentation, mitochondria redistribute close to the site of the IS, where they promote influx of Ca²⁺ (5). The actin cytoskeleton-based recruitment of mitochondria in the vicinity of active synapses is required for Ca²⁺ influx through calcium release-activated calcium (CRAC) channels and activation of key downstream transcription factors such as NFAT. The translocation of the mitochondria to the IS is typically observed within 15 min after focal TCR cross-linking (5) with a sustained peak at ~30 min (6). Reduction in the T cell Ca²⁺ concentration close to the channel serves as a feedback inhibitory mechanism responsible for adjusting channel activity (5). Any agent that inhibits the movement of mitochondria to the IS and modulates intracellular Ca²⁺ homeostasis can prevent sustained IS formation, thereby suppressing T cell activation and function.

The literature supports the contention that dietary ω-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA; 20:5n−3) and docosahexaenoic acid (DHA, 22:6n−3) in particular, are important modulators of a host’s inflammatory and immune responses (7–9). These studies demonstrate that EPA and DHA reduce proinflammatory responses, in part, by diminishing T cell proliferative capacity in response to both mitogenic and antigenic stimulation (10). In previous studies from our laboratory, we demonstrated that n−3 PUFA modulate CD4⁺ T cell immune response via reduction in Th1 effector cell clonal expansion mediated, in part, by diminished IL-2 secretion and IL-2R α-chain mRNA transcription (11, 12). However, the molecular mechanisms by which n−3 PUFA suppress CD4⁺ T cell function are not fully understood. Recently, we demonstrated that n−3 PUFA are capable of suppressing the localization and activation of signaling proteins at the IS in mouse T cells (12, 13).

Mammals cannot produce n−3 PUFA from the major n−6 PUFA found in the diet because of a lack of Δ15-desaturase activity. Therefore, it is necessary to enrich the diet with EPA and/or DHA to assess their biologic properties in vivo. However, transgenic mice expressing the fat-1 gene encoding an n−3 fatty acid desaturase cloned from Caenorhabditis elegans can catalyze the conversion of n−6 PUFA to n−3 PUFA by introducing a double bond into fatty acyl chains (14). Therefore, the fat-1 mouse model facilitates the investigation of the biologic properties of n−3 PUFA in T cells without having to incorporate these fatty acids into the diet.

In this study, we investigated the effect of n−3 PUFA on T cell–APC interactions in fat-1 mice. Specifically, the effect of n−3 PUFA on the recruitment of mitochondria to the IS during the initial activation phase of T cells in contact with APCs and the consequent changes in Ca²⁺ signaling in T lymphocytes was investigated. We demonstrate for the first time that n−3 PUFA modulates the early Ca²⁺ mediated events following the interaction of T cells with APCs. These results are consistent with the ability of n−3 PUFA to inhibit downstream signaling and suppress T cell activation.

Materials and Methods

Materials

RPMI 1640 medium and sodium pyruvate were purchased from Cellgro (Manassas, VA). FBS was purchased from Irvine Scientific (Santa Ana, CA). DMEM 1× medium, Leibovitz medium, penicillin, streptomycin, and glutamax were purchased from Life Technologies (Carlsbad, CA). Fluo-4 AM, Rhod-2 AM, and MitoTracker Green FM were purchased from Molecular Probes (Eugene, OR). RU-360 and BTP2 were purchased from Calbiochem (San Diego, CA). Two-well Lab-Tek chambered cover glass slides were purchased from Nunc (Naperville, IL). Poly-L-lysine solution was purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions of mitotracker green, Fluo-4 AM, Rhod-2 AM and BTP2 were prepared in DMSO and diluted with medium to 100 nM, 1.0 μM, 1.5 μM, and 10 μm, respectively (final concentration of the vehicle DMSO was maintained at 0.1–0.3% in culture). Stock solutions of RU-360 was prepared in degassed water and diluted with medium to a final concentration of 10 μM.

Cell culture

Hybridoma cells expressing hamster mAbs specific for either murine CD3 (clone 145-2C11) or an irrelevant Ag, DNP (clone UC8-1B9, anti-DNP hybridoma) were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM-1× with 10% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids, and 10⁵ U/l penicillin and 100 mg/l streptomycin at 37°C and 5% CO₂. In addition to the presence of CD3 engagement, both types of hybridoma also display significant surface levels of the costimulatory ligands B7 and ICAM-1, which are capable of engaging their counterparts on the T cell (15). This model has been extensively used to examine T cell–APC interaction (16).

Animals and CD4⁺ T cell isolation

Fat-1 transgenic mice were generated and backcrossed onto a C57BL/6 background as described previously (14, 17). All procedures followed guidelines approved by Public Health Service and the Institutional Animal Care and Use Committee at Texas A&M University. The colony of fat-1 mice used for this study was generated by breeding heterozygous mice. Mice were genotyped using tail DNA. To confirm the phenotype, total lipids were isolated from splenic T cells, and the fatty acid profile was characterized by gas chromatography as described previously (17, 18). Specific pathogen-free animals were maintained under barrier conditions and were fed a 10% safflower oil diet (n−6 PUFA rich, Research Diets, New Brunswick, NJ) ad libitum with a 12 h light/dark cycle. The diet contained 40 (g/100 g diet) sucrose, 20 casein, 15 corn starch, 0.3 DL-methionine, 3.5 AIN 76A salt mix, 1.0 AIN 76A mineral mix, 0.2 choline chloride, 5 fiber (cellulose), 10 safflower oil. CD4⁺ T cells from fat-1 or wild type mice were isolated from spleens by a magnetic microbead positive selection method (13).

CD4⁺ T cell mitochondria labeling using Mitotracker Green FM (mitochondrial localization probe)

Purified (>92.0% determined by flow cytometry) CD4⁺ T cells (2 × 10⁶ cells/ml) were labeled with Mitotracker Green FM (Molecular Probes, Eugene, OR) for 15 min at 37°C in RPMI 1640 (complete medium with 10% FBS). Mitotracker Green is a green-fluorescent mitochondrial stain that localizes in the mitochondria regardless of membrane potential (19). This probe is essentially nonfluorescent in aqueous solution, becoming fluorescent only when it accumulates in the lipid environment of the mitochondria.

CD4⁺ T cell mitochondria Ca²⁺ labeling using Rhod-2 AM (mitochondrial Ca²⁺ indicator)

Isolated CD4⁺ T cells (2 × 10⁶ cells/ml) were labeled with red fluorescent dye Rhod-2 AM for 30 min at 37°C. Cells were subsequently washed to remove cytosolic Rhod-2 and resuspended in serum-free Leibovitz medium. Mitotracker Green FM was used to confirm that Rhod-2 fluorescence corresponded to mitochondrial Ca²⁺ accumulation (19).

CD4⁺ T cell mitochondrial-to-cytosolic calcium labeling using Rhod-2 AM and Fluo-4 (cytosolic Ca²⁺ indicator)

Isolated CD4⁺ T cells (2 × 10⁶ cells/ml) were incubated with green fluorescent dye, Fluo-4, and red fluorescent dye Rhod-2 for 1 h at 37°C. Fluo-4 is a visible wavelength nonradiometric cytosolic Ca²⁺ indicator that exhibits a 40-fold increase in fluorescence intensity with Ca²⁺ binding (20). After 1 h incubation with the probe, cells were washed with Leibovitz medium and imaged. The ratio of the cytosolic-to-mitochondrial Ca²⁺ level was subsequently calculated. In addition, experiments were performed in cultures incubated with RU-360, an inhibitor of mitochondrial uniporter, for 30 min prior to the calcium labeling with Rhod-2 and Fluo-4, following which the mitochondrial-to-cytosolic Ca²⁺ ratio was determined. In separate experiments, cells were incubated with BTP2, a pharmacologic inhibitor of CRAC/Orai1 channel activity (21, 22), for 1 h prior to the calcium labeling with Rhod-2 and Fluo-4, after which the mitochondrial-to-cytosolic Ca²⁺ ratio was determined. The mitochondrial-to-cytosolic Ca²⁺ ratio represents the relative fluorescence intensities of Rhod-2 and Fluo-4.

CD4⁺ T cell activation and fluorescence microscopy

For mitogenic CD4⁺ T cell activation, an anti-CD3 expressing hybridoma (clone 145-2C11, ATCC) was used. Mitotracker/Rhod 2-labeled CD4⁺ T cells were coincubated with hybridoma cells at a 5:1 ratio. Cell mixtures were seeded onto poly-L-lysine precoated Lab-tek two-well chambered coverglass slides (2 × 10⁶ CD4⁺ T cells per chamber). Cell cocultures were incubated for 15 min at 37°C in 5% CO₂. For quantification of Rhod-2 and Fluo-4 fluorescence, excitation wavelengths of 488 and 550 nm were used, and fluorescence emission was monitored at 530 and 590 nm, respectively. Ten to 15 areas with 20–25 cells per area and 4–8 wells per treatment were imaged with a 510 META NLO laser scanning microscope (Carl Zeiss Microimaging, Thornwood, NY) equipped with an argon laser and LSM software. Images were collected using a ×63 objective (1.3 numerical aperture). To validate IS formation, T cells were incubated with irrelevant DNP-specific Ab expressing hybridoma cells (clone UC8-1B9, anti-DNP hybridoma) as a negative control (13, 16).

Image processing and quantification of mitochondrial-to-cytosolic Ca²⁺

All images were acquired by confocal microscopy and were converted to an 8-bit/channel TIFF format, and the percentage of cells with mitochondrial translocation to the IS was determined. To assess mitochondria-to-cytosolic calcium at the synapse and whole cell, polygons were drawn to designate the IS and the contact whole cell area. The normalized Rhod-2 intensity, an indicator of mitochondrial calcium, and normalized Fluo-4 intensity, an indicator of cytosolic calcium, was calculated by using mean fluorescence intensity (MFI) according to the formula: (Rhod-2 MFI/Fluo-4 MFI at the synapse and contact whole cell). Calcium measurements were determined in individual cells for each experiment, and comparisons were made with same day controls, including inhibitors. Quantitative analysis of MFI was performed using Adobe Photoshop CS3 for Windows (Adobe Systems, San Jose, CA).

Statistical analysis

The effect of the independent variable (fat-1 versus wild type) was assessed using SPSS (Chicago, IL) 15.0 for Windows. Data are expressed as mean ± SE. More than three independent experiments were performed for each experimental condition. Differences between means were tested using t/F-type tests of contrast (two-tailed). Values of p < 0.05 were considered to be statistically significant.

Results

n−3 PUFAs inhibit mitochondrial translocation to the vicinity of the IS

Both fat-1 transgenic and wild type control offspring were fed a 10% safflower diet enriched in n−6 PUFA throughout the duration of the study. To investigate the effect of endogenous n−3 PUFA on mitochondria translocation to the IS, Mitotracker green-labeled CD4⁺ T cells were cocultured with anti–CD-3 hybridoma cells at 37°C in 5% CO₂ for 0–30 min. Images of conjugate CD4⁺ T cells and hybridoma cells were captured, whereas noncontact CD4⁺ T cells served as negative controls. The DNP-specific Ab expressing hybridoma was used as a negative control and failed to exhibit direct mitochondrial translocation to the immediate vicinity of the IS formation (Fig. 1A–C). Typically, >75% of wild type (control) CD4⁺ cells exhibited a directed movement of mitochondria to the IS by 15 min (Fig. 1C, 1D). In contrast, CD4⁺ T cells from fat-1 mice had a significantly (p < 0.05) reduced percentage of cells with mitochondria translocation to the IS (Fig. 2). Typically, mitochondria in T cells are localized preferentially in one area of the cell (5). This apparent polarized mitochondrial localization may be caused by the rather large nucleus in T cells, and explains why a localized mitochondrial distribution was observed (Fig. 1B).

FIGURE 1.

Focal stimulation of TCR activates mitochondrial translocation to the IS. A, Schematic of IS (positive control) formation (left) and noncontact (negative control) IS (right). B, Immunofluorescence images (original magnification ×63) from MitoTracker Green/AM loaded CD4⁺ T cells cocultured with (left) anti CD3 (positive control) and (right) anti-DNP (negative control) hybridoma. Scale bar, 5 μm. C, Summary image analysis of subplasma membrane localization of mitochondria in wild type CD4⁺ T cells after 15 min costimulation with anti-CD3 expressing hybridoma 145-2C11 (positive control) and irrelevant expressing Ab Hybridoma UC8-1B9 (negative control); mean ± SEM of n = 21–31 cells. D, Confocal fluorescence images (original magnification ×63) from a single Mitotracker Green/AM-loaded CD4⁺ T cell forming an IS with an APC. Image kinetics were measured from time 0 to 30 min following coincubation of Mitotracker-loaded (bright green) CD4⁺ T cells (from a wild type/control mouse) with APC (Hybridoma 145-2C11). Images in each panel represent the same coincubated cells (7–26 min). Green patching represents mitochondria location in the T cell. Scale bar, 5 μm.

FIGURE 2.

n−3 PUFA suppresses mitochondria translocation to the IS. Subplasma membrane localization of mitochondria to the IS was quantified in CD4⁺ T cells (n = 140 cells from five fat-1 mice) after a 15 min co-stimulation with Hybridoma 145-2C11 APCs. Percentage of cells exhibiting mitochondrial translocation, fat-1 versus wild type comparison. *p < 0.05.

In independent experiments in our laboratory, total lipid fatty acid compositional analyses of the fat-1 CD4⁺ T cell revealed an increase in n−3 PUFA, EPA (20:5n−3), docosapentaenoic acid (22:5n−3) and DHA (22:6n−3), and decrease in n−6 PUFA, arachidonic acid (20:4n−6), adrenic acid (22:4n−6), and docosapentaenoic acid (22:5n−6) (13). In addition, the ratio of n−6 to n−3 PUFA was significantly (p < 0.05) suppressed in fat-1 compared with wild type cells. This finding indicates that an appropriate activity of n−3 fatty acid desaturase was present and that T cells from fat-1 mice were enriched in n−3 PUFA. These data demonstrate that n−3 PUFA suppress mitochondrial localization to the IS.

n−3 PUFA reduce Ca²⁺ uptake by mitochondria at the IS

Mitochondrial modulation of CRAC channel activity is involved in mediating the translocation of mitochondria toward the IS at the plasma membrane. Translocation to the IS allows mitochondria to take up a large amount of extracellular Ca²⁺ directly beneath the mouth of the channels, thereby reducing channel inactivation (5). Therefore, we examined the effect of n−3 PUFA on mitochondrial Ca²⁺ levels using Mitotracker Green and Rhod-2 AM probes. In this double-labeling experiment, Rhod-2 at the synapse is an indicator of mitochondrial Ca²⁺ because of the much higher colocalization of both dyes. Fig. 3A shows representative images documenting differential intracellular localization of Ca²⁺ in mitochondria (yellow pixels). In addition, mitochondrial Ca²⁺ was measured within the region of interest near the IS (Fig. 3B). In cells exhibiting mitochondrial translocation to the IS, no significant differences in Ca²⁺ concentration between the wild type and fat-1 T cells was observed (Fig. 3C). In contrast, there was a significant reduction in Ca²⁺ levels (p <0.05) at the IS in fat-1 T cells when both translocated and nontranslocated mitochondria data were combined (Fig. 3D). These results indicate that T cell Ca²⁺ signaling at the IS is suppressed in CD4⁺ T cells enriched with n−3 PUFA. The higher intramitochondrial Ca²⁺ at the IS is probably caused by the intimate contact between mitochondria and the IS, which exposes mitochondria to higher micro-environments of Ca²⁺.

FIGURE 3.

Measurement of Ca²⁺ uptake at the IS. A, The region of interest where calcium signal is observed in confocal microscopy images (original magnification ×63) of Rhod-2 and Mitotracker-labeled cells: T cell with mitochondrial translocation to the IS (1); cell exhibiting a failure to translocate mitochondria to the IS (2); and T cell with no contact (3); therefore, no IS is formed. B, Representative confocal microscopy images (original magnification ×63) of Rhod-2 and Mitotracker-labeled cells RGB (1), green (2), red channels (3), respectively. ROIs at the T cell immune synapse were selected by drawing an oval or polygon as shown in dotted lines. Mean intensities from red and green channels were recorded to calculate Ca²⁺ values as described in the Materials and Methods. Scale bar, 5 μm. C, Effect of n−3 PUFA on mitochondrial Ca²⁺ at the IS only in cells with mitochondrial translocation to the IS. D, Comparison of mitochondrial Ca²⁺ measurements in CD4⁺ T cells in wild type (n = 26 cells from three mice) and fat-1 (n = 18 cells from three mice). *p < 0.05. ROI, region of interest.

n−3 PUFAs modulate intracellular calcium signaling by altering the mitochondrial-to-cytosolic Ca²⁺ ratio

Because mitochondria are key organelles involved in the regulation of intracellular Ca²⁺ homeostasis, we measured the ratio of mitochondrial-to-cytosolic Ca²⁺ ratio in individual cells. Fluo-4, a visible wavelength nonratiometric cytosolic indicator, was loaded along with Rhod-2 AM to evaluate the mitochondrial-to-cytosolic Ca²⁺ ratio. The relative fluorescence intensities of Rhod-2 to Fluo-4 were measured to determine the mitochondrial-to-cytosolic ratio in living CD4⁺ T cells from fat-1 and wild type mice. Because IS formation, mitochondrial translocation, and Ca²⁺ influx peaks by ~30 min (23), mitochondrial-to-cytosolic ratio data were collected over a 15–30 min time interval. Fig. 4A shows representative images of Fluo-4 and Rhod-2 labeled cells. In wild type cells, an increasing trend of mitochondrial-to-cytosolic ratio is observed over time at both the IS and the whole cell (Fig. 4B, 4C). In contrast, mitochondrial-to-cytosolic ratio shows no change after the IS formation in the fat-1 cells, both at the IS and the whole cell (Fig. 4D, 4E). Comparison between the mitochondrial-to-cytosolic ratio of wild type and fat-1 cells at 15 min was not significant (p > 0.05) at both the IS and the whole cell level. In contrast, at 30 min, significant differences (p < 0.05) were observed in the mitochondrial-to-cytosolic ratio of wild type versus fat-1 cells at the IS and the whole cell (Fig. 4E). These data are consistent with an inhibitory effect of n−3 PUFA on the uptake of calcium by mitochondria after IS formation.

FIGURE 4.

Effect of n−3 PUFA on intracellular Ca²⁺. CD4⁺ T cells were coincubated with Fluo-4 AM (3 μM) and Rhod-2 AM (2.5 μM) for 30 min, and the mitochondrial-to-cytosolic Ca²⁺ ratio was determined. A, Representative confocal microscopy images (original magnification ×63) of Fluo-4 and Rhod-2 labeled cells white (1), RGB (2), green (3), red channels (4), respectively. B, Amplitude of intracellular Ca²⁺ response at the IS in wild type CD4⁺ T cells after coculture with Hybridoma 145-2C11 APCs. Data are from a representative experiment, 15–30 min after coculture; n = 5 independent experiments with 10–20 cells. C, Amplitude of total intracellular Ca²⁺ response in wild type CD4⁺ T cells. D, Amplitude of intracellular Ca²⁺ response at the IS in fat-1 CD4⁺ T cells. E, Amplitude of total intracellular Ca²⁺ response in fat-1 CD4⁺ T cells. F, Comparison of relative mitochondrial-to-cytosolic Ca²⁺ ratio at the IS versus total cell in CD4⁺ T cells from wild type (n = 116 cells from four mice) and fat-1 mice (n = 131 cells from five mice). Measurement at 15 min following the IS formation (left). Measurement at 30 min, the peak of response (right). *p < 0.05.

Effect of a mitochondrial uniporter inhibitor on intracellular Ca²⁺ levels

To investigate the role of the mitochondrial uniporter in Ca²⁺ uptake, cells were treated with RU-360, a mitochondrial Ca²⁺ uniporter inhibitor (24). Following IS formation, the mitochondrial-to-cytosolic ratio in both the wild type and fat-1 cells coincubated with RU360 showed no change at up to 30 min (Fig. 5A–D). Significant differences (p < 0.05) in the mitochondrial-to-cytosolic Ca²⁺ ratio were observed in wild type cells and fat-1 cells minus RU-360 both at IS and the whole cell at 30 min (Fig. 5E). These data are consistent with the role of mitochondrial uniporter activity in regulating intracellular Ca²⁺ following IS formation.

FIGURE 5.

Effect of mitochondrial uniporter inhibitor (RU-360) on intracellular Ca²⁺. CD4⁺ T cells from wild type and fat-1 mice were pre-incubated with RU-360 (10 μM) for 30 min, followed by washing and coincubation with Fluo-4 AM (3 μM) and Rhod-2 AM (2.5 μM) for 30 min, and the mitochondrial-to-cytosolic Ca²⁺ ratio was determined. A, Amplitude of intracellular Ca²⁺ response at the IS with RU-360 in wild type CD4⁺ T cells after 15 min coculture with Hybridoma 145-2C11 APCs. Data are from one representative experiment, 15–30 min after coculture; n = 2–3 independent experiments with 15–18 cells. B, Amplitude of total intracellular Ca²⁺ response with RU-360 in wild type CD4⁺ T cells after 15 min coculture with Hybridoma 145-2C11 APCs. C, Amplitude of intracellular Ca²⁺ response at the IS with RU-360 in fat-1 CD4⁺ T cells. D, Amplitude of total intracellular Ca²⁺ response with RU-360 in fat-1 CD4⁺ T cells. E, Comparison of relative mitochondrial-to-cytosolic Ca²⁺ ratio at the IS versus total cell in CD4⁺ T cells from wild type (n = 66 cells from two mice) and fat-1 (n = 92 cells from three mice). Measurement with and without RU-360 at 15 min following IS formation (left). Measurement with and without RU-360 at 30 min, the peak of response (right). *p < 0.05.

Effect of CRAC/ORAI1 channel blocker on intracellular Ca²⁺ levels

Because sustained Ca²⁺ influx across the plasma membrane though CRAC channels is required for T cell activation (25), the effects of BTP2, a CRAC/ORAI1 channel blocker, were evaluated in wild type CD4⁺ T cells. As shown in Fig. 6A–C, the mitochondrial-to-cytosolic ratio at the IS and in the whole cell did not show any increase over time. These data suggest that inactivation of CRAC/ORAI1 channels inhibits the influx of extracellular Ca²⁺, preventing the increase in mitochondrial-to-cytosolic Ca²⁺ ratio necessary for downstream effector pathway activation.

FIGURE 6.

Effect of CRAC/Orai1 channel blocker (BTP2) on intracellular Ca²⁺. CD4⁺ T cells of wild type mice were preincubated with BTP2 (10 μM) for 1 h, after which cells were washed and coincubated with Fluo-4 AM (3 μM) and Rhod-2 AM (2.5 μM) for 30 min, and the mitochondrial-to-cytosolic Ca²⁺ ratio was determined. A, Amplitude of intracellular Ca²⁺ response at the IS with BTP2 in wild type CD4⁺ T cells after a 15 min coculture with Hybridoma 145-2C11 APCs (n = 15–19 cells, 15–30 min following IS formation). B, Amplitude of total intracellular Ca²⁺ response with BTP2 in wild type CD4⁺ T cells. C, Comparison of relative mitochondrial-to-cytosolic Ca²⁺ ratio at the IS versus total cell in wild type CD4⁺ T cells (n = 35 cells). *p < 0.05.

Discussion

Autoimmune and/or chronic inflammatory diseases primarily involve an overactive immune response directed toward a particular tissue (26). Improper T cell regulation results in an imbalance between T cell activation and suppression, resulting in chronic inflammation. For example, hyperactivation of CD4⁺ T cells is associated with enhanced susceptibility to autoimmune disorders and chronic inflammation induced diseases (27). Many studies demonstrate that T cell activation is suppressed by n−3 PUFA/DHA, in part, by altering the physical properties of biologic membranes (10–13, 18, 28, 29). To further investigate the immunomodulatory effects of n−3 PUFA, we examined in vivo mitochondrial translocation and calcium homeostasis in T lymphocytes following IS formation. There is evidence that mitochondria translocate toward the IS in CD4⁺ and Jurkat T cell lines (5, 25, 30). Recently, the reorganization of mitochondria at the NK cell synapse has been documented (31).

To our knowledge, this is the first study to investigate the effects of n−3 PUFA on critical early Ca²⁺-mediated events of T cell activation. For this purpose, we used the fat-1 transgenic mouse model to test the hypothesis that n−3 PUFA inhibits mitochondrial translocation and, therefore, suppress sustained Ca²⁺ signaling necessary for T cell activation. The fat-1 transgenic mouse model was used because it is capable of converting n−6 PUFA to n−3 PUFA endogenously by introducing a cis-double bond into fatty acyl chains (14, 17). This model allows us to investigate the biologic properties of n−3 PUFA without having to incorporate the fatty acid in the diet.

Recent evidence indicates that upon initial contact between APCs and T cells, mitochondria are recruited to the immediate vicinity of the IS (5). This critical event is followed by a reduction in the local accumulation of Ca²⁺ so as to maintain the robust influx of Ca²⁺ across the plasma membrane through CRAC channels, resulting in the activation of key downstream transcription factors such as NFATs (5). We have previously demonstrated that incorporation of n−3 PUFA into cytofacial leaflet phospholipids alters the lateral composition of lipid rafts in the plasma membrane, thereby altering the IS microenvironment to impact thresholds for the activation of TCR-mediated cell signaling. Thus, n−3 PUFA-enriched CD4⁺ T cells exhibit a suppressed localization of important signaling proteins, such as F-actin, phospholipase C γ-1, and protein kincase C θ, into the IS as assessed by immunofluorescence labeling (13). Therefore, we hypothesized that n−3 PUFA would suppress CD4⁺ T cell mitochondrial translocation during the early stages of IS formation and downmodulate Ca²⁺-dependent signaling. Our data demonstrate that n−3 PUFA suppresses the activation of T cells, in part, by inhibiting mitochondrial translocation to the IS. Specifically, experiments showed that CD4⁺ T cells from fat-1 mice had a significantly reduced percentage of cells that exhibited mitochondrial translocation to the IS as compared with cells isolated from wild type mice.

The sustained activity of Ca²⁺ channels in the plasma membrane requires translocation of mitochondria to the plasma membrane (5). This is attributed to the fact that the decreased distance between mitochondria and the plasma membrane enables mitochondria to take up large amounts of extracellular Ca²⁺, thereby preventing Ca²⁺-dependent inactivation of CRAC channels, and sustaining Ca²⁺-dependent signaling. Because mitochondrial translocation was inhibited in T cells isolated from fat-1 transgenic mice, we further examined the effect of n−3 PUFA enrichment on intracellular Ca²⁺ homeostasis in T cells using a double excitation technique—that is, excitation at two wavelengths (488 and 550 nm) to determine increases in [Ca²⁺]_i. This technique corrects for errors that are caused by the shift in the spectrum from one wavelength to another during increases in intracellular Ca²⁺ (20). As anticipated, the inhibition of mitochondrial translocation in fat-1 T cells was associated with a reduction in mitochondrial Ca²⁺ uptake near the IS. We also noted that the mitochondrial-to-cytosolic Ca²⁺ ratio remained unchanged in n−3 PUFA-enriched (fat-1) T cells following IS formation. In contrast, wild type cells showed an increasing trend with respect to the mitochondrial-to-cytosolic ratio Ca²⁺ over time at both the IS and the whole cell. These data are noteworthy because Ca²⁺ mediated regulation of CRAC channels in T cells enable sustained elevation of intracellular Ca²⁺, an obligate requirement to drive nuclear translocation of NFAT (32). Maintenance of nuclear localization of NFAT is a prerequisite for the expression of IL-2 and other T cell activation responses after exposure to Ag (33, 34). In the absence of mitochondrial translocation and, hence, mitochondrial Ca²⁺ uptake, the local accumulation of Ca²⁺ near the IS inactivates CRAC/ORAI1 channel quickly and reduces Ca²⁺ entry into the cell (5). Our results reaffirm the importance of mitochondrial translocation and demonstrate for the first time that n−3 PUFA are capable of decreasing mitochondrial Ca²⁺ uptake, thereby impairing a universal second messenger pathway important for T cell activation.

To address the integration and crosstalk between Ca²⁺ and other signaling pathways in our T cell model system, proof-of-principle experiments were also conducted using mitochondrial uniporter (RU-360) and CRAC/ORAI1 channel (BTP2) inhibitors. As anticipated, following IS formation, mitochondrial-to-cytosolic Ca²⁺ ratios in both wild type and fat-1 cells coincubated with RU-360 were similar. In contrast, in the absence of RU360, the ratio was elevated only in wild type cells. In complementary experiments, inactivation of the CRAC/ORAI1 channel prevented the synapse-induced increase in the mitochondrial-to-cytosolic Ca²⁺ ratio. These data corroborate the functional link between mitochondrial regulation of CRAC/ORAI1 and T cell activation. Our results stress the importance of mitochondria in regulating Ca²⁺ channel activity and signal transmission from the plasma membrane to the nucleus during the earliest stages of T cell activation. Therefore, the immunosuppressive effects of n−3 PUFA on T cell function can be attributed, in part, to modulation of mitochondria-dependent intracellular Ca²⁺ signaling.

Although previous studies have examined the effects of diet in regard to immunosuppression (27, 35, 36), it is apparent that n−3 PUFA profoundly reduces the translocation of mitochondria to the immediate vicinity of the IS (Fig. 7). This, in turn, reduces Ca²⁺ uptake by mitochondria, diminishing the local cytosolic Ca²⁺ concentration, which limits Ca²⁺ influx through CRAC/ORAI1 channels. Ultimately, lower intracellular Ca²⁺ inactivates the phosphatase calcineurin and reduces the subsequent nuclear import and assembly of NFAT transcription complexes that are essential for T cell activation (3, 4, 34). Regarding mechanisms that modulate mitochondrial motility, there is evidence that n−3 PUFA can displace F-actin from the IS, where T cells and APCs form a conjunction (13, 28). This finding is noteworthy because mitochondria predominantly interact with the actin cytoskeleton and use actin tracks to coordinate the relative distance between the IS and mitochondria (5, 37, 38). Further work is needed to examine the effects of membrane lipid composition on T cell cytoskeletal tracks.

FIGURE 7.

Effect of n−3 PUFA on Ca²⁺-dependent T cell activation. A, Schematic model showing the role of mitochondria during T cell activation by Ag. Upon formation of the IS between an APC and Th cell, mitochondria translocate to the immediate vicinity of the IS. Once recruited to the IS, mitochondria reduce local Ca²⁺ accumulation, thereby sustaining Ca²⁺ influx through CRAC/ORAI1 channels. Increase in the intracellular calcium [Ca²⁺]_i concentration leads to calcium and calmodulin binding to the calcium-calmodulin–dependent phosphatase, calcineurin. Calcineurin then dephosphorylates transcription factors (e.g., NFAT), which translocate to the nucleus and remain in a transcriptionally active state. B, The incorporation of n−3 PUFA into the fat-1 CD4⁺ T cell plasma membrane increases the formation of lipid rafts at the IS and also alters the composition of mitochondrial membrane. This results in the inhibition of mitochondrial translocation to the IS, thereby reducing Ca²⁺ uptake by mitochondria, limiting the cytosolic Ca²⁺ concentration, and blocking phosphatase activition of calcineurin. Collectively, these events reduce the dephosphorylation and nuclear translocation of NFAT and suppress the transcription of genes necessary for T cell activation.

We have shown that n−3 PUFA suppresses mitochondrial translocation to the IS and perturbs Ca²⁺ signaling, which is critical to T cell activation. These results provide a critical new paradigm in understanding the molecular mechanisms through which n−3 PUFA modulates T cell activation. Additional studies are needed to further elucidate the effect of dietary n−3 PUFA on T cell mitochondrial motility.

Abbreviations used in this paper

CRAC

calcium release-activated calcium

DHA

docosahexaenoic acid

EPA

eicosapentaenoic acid

IS

immunologic synapse

MFI

mean fluorescence intensity

PUFA

polyunsaturated fatty acid

ROI

region of interest