Increased levels of invariant natural killer T lymphocytes worsen metabolic abnormalities and atherosclerosis in obese mice.

Obesity is a chronic inflammatory state characterized by infiltration of adipose tissue by immune cell populations, including T lymphocytes. Natural killer T (NKT) cells, a specialized lymphocyte subset recognizing lipid antigens, can be pro- or anti-inflammatory. Their role in adipose inflammation continues to be inconclusive and contradictory. In obesity, the infiltration of tissues by invariant NKT (iNKT) cells is decreased. We therefore hypothesized that an excess iNKT cell complement might improve metabolic abnormalities in obesity. Vα14 transgenic (Vα14tg) mice, with increased iNKT cell numbers, on a LDL receptor-deficient (Ldlr−/−) background and control Ldlr−/− mice were placed on an obesogenic diet for 16 weeks. Vα14tg.Ldlr−/− mice gained 25% more weight and had increased adiposity than littermate controls. Transgenic mice also developed greater dyslipidemia, hyperinsulinemia, insulin resistance, and hepatic triglyceride accumulation. Increased macrophage Mac2 immunostaining and proinflammatory macrophage gene expression suggested worsened adipose inflammation. Concurrently, these mice had increased atherosclerotic lesion area and aortic inflammation. Thus, increasing the complement of iNKT cells surprisingly exacerbated the metabolic, inflammatory, and atherosclerotic features of obesity. These findings suggest that the reduction of iNKT cells normally observed in obesity may represent a physiological attempt to compensate for this inflammatory condition.

were incubated with erythrocyte lysis buffer for 5 min, centrifuged, and suspended in fl ow buffer.
Cell suspensions (1 × 10 6 cells/sample) were preincubated with CD16/32 (FcR Block, BD Biosciences, San Jose, CA) for 15 min at 4°C, then stained with fl uorescent-labeled antibodies or IgG isotype controls for 30 min at 4°C. Antibodies used for lymphocyte phenotyping were as follows: FITC-conjugated anti-CD3e, APC-conjugated anti-NK1.1 (both eBioscience, San Diego, CA); PerCP-conjugated anti-CD4 (Biolegend, San Diego, CA); and PEconjugated CD1d tetramer (NIH Tetramer Core Facility). Cells were washed in fl ow buffer twice, resuspended in 0.5% paraformaldehyde, and analyzed using a FACSCanto fl ow cytometer (BD Biosciences, San Jose, CA). Unstained and singly stained control cells were used to set up compensation and gates. Data were analyzed using FACSDiva software. A minimum of 50,000 events were analyzed for each sample.

Analytical procedures
Metabolic variables were measured in blood samples obtained from the retro-orbital sinus after a 5-6 h fast. Cholesterol and triglycerides in plasma and fast-phase liquid chromatography (FPLC) fractions were measured using colorimetric assay kits. Lipoproteins were separated from pooled plasma samples by FPLC. Plasma insulin levels were measured using an ELISA kit (Millipore, Billerica, MA). Alanine aminotransferase (ALT) was measured using an autoanalyzer through the Nutrition and Obesity Research Center at the University of Washington. Tissue lipids were extracted using the Folch technique ( 19 ). Intraperitoneal glucose and insulin tolerance tests were performed after a 5 h fast at weeks 13 and 14 of diet feeding, respectively, as previously described ( 17 ). Insulin resistance index was calculated using the formula fasting glucose (mmol/l) × fasting insulin (mU/l) / 22.5. Body composition was performed on conscious, immobilized mice using quantitative magnetic resonance (EchoMRI whole-body composition analyzer, Echo Medical Systems, Houston, TX).

Real-time quantitative PCR
Total RNA was extracted from ‫ف‬ 100 mg of whole adipose, liver, or aortae using a commercially available RNA extraction kit according to the manufacturer's protocol (Agilent Technologies, Santa Clara, CA). After spectroscopic quantifi cation, 2 µg of RNA was reverse-transcribed, and cDNA thus obtained was analyzed by real-time quantitative PCR. Primers specifi c for individual genes were purchased from Applied Biosystems (Assay-on-Demand, Life Technologies, Carlsbad, CA). GAPDH was used as the control housekeeping gene, levels of which did not change with diets. Relative amounts of the target gene were calculated using the ⌬ ⌬ Ct formula.

Histology, immunohistochemistry, and atherosclerosis quantifi cation
The extent of atherosclerosis was measured in pinned aortas using the en face technique as previously described ( 17 ). Formalinfi xed, paraffi n-embedded adipose tissues were sectioned and stained with Movat pentachrome histochemical stain using standard protocols. Macrophages in adipose tissue were detected using a rat monoclonal antibody (Mac2; titer 1:2500, Cedarlane Laboratories, Burlington, NC). Adipocyte size was measured using a modifi cation of techniques described previously ( 16 ). Liver sections were stained with Masson's Trichrome stain using standard protocols. Liver cell apoptosis was assessed using the terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay according to the manufacturer's instructions (ApopTag Peroxidase In Situ Apoptosis Detection Kit, Millipore, Billerica, MA) as previously described ( 20 ). Quantifi cation of aortic root lesion area was performed as described previously ( 21 ). Quantifi cation antigen-presenting cells and recognized by V ␣ 14J ␣ 18/ V ␤ 8 on responding T cells ( 10,11 ). Upon activation, NKT cells can rapidly and robustly secrete either proinfl ammatory (Th1) or anti-infl ammatory (Th2) cytokines, which can activate antigen-presenting cells as well as lymphocytes. iNKT cells are therefore uniquely poised as a bridge between the innate and the adaptive immune systems ( 7 ) and can have opposing roles in promoting or resolving infl ammation.
The reported role for iNKT cells in obesity is controversial. However, a reduction in iNKT numbers has been observed in adipose tissue and livers of obese mice and humans (12)(13)(14)(15). Since iNKT-cell numbers are reduced in obesity and since iNKT cells could potentially recognize lipid antigens in expanded adipose tissue of obesity and benefi cially modulate infl ammation, we hypothesized that increasing iNKT-cell numbers would improve metabolic abnormalities and atherosclerosis in a mouse model of diet-induced obesity. We utilized V ␣ 14J ␣ 18 TCR transgenic mice which have an increased complement of iNKT cells ( 16 ). These mice were studied on a LDL receptor-defi cient ( Ldlr Ϫ / Ϫ ) background, since the Ldlr Ϫ / Ϫ mouse is a model of the metabolic syndrome when fed diets rich in fat and refi ned carbohydrates and also allows for evaluation of atherosclerosis ( 17 ). In contrast to our expectation, we show here that increasing iNKT-cell numbers worsens the metabolic complications that accompany obesity in this mouse model.

Animals and diet
V ␣ 14 transgenic ( V ␣ 14tg ) mice from Dr. Albert Bendelac (University of Chicago) ( 16 ) were crossed with Ldlr Ϫ / Ϫ mice as described previously ( 18 ). All animals were in the C57BL/6J background. Littermate Ldlr Ϫ / Ϫ mice were used as controls. Agematched 10-week-old male mice were fed either standard chow or a high-fat, high-sucrose diet with 0.15% cholesterol (HFHSC) (Bioserv F4997, Frenchtown, NJ) for 16 weeks (n = 10 per group). Mice were maintained in a temperature-and light-controlled facility in cages with microisolator fi lter tops. Body weights were measured weekly. Food intake was recorded after 10 weeks of diet and calculated as an average of three sequential days from a known amount of food given. The food was reweighed daily, and the amount of food consumed was calculated. When the animals were euthanized, harvested tissues were snap-frozen in liquid nitrogen and stored at Ϫ 70°C or were fi xed with 10% neutral-buffered formalin and embedded in paraffi n wax. All experimental procedures were undertaken with approval from the Institutional Animal Care and Use Committee of the University of Washington.

Isolation of leukocytes and fl ow cytometry
Mouse adipose tissue, livers, and spleens were collected and weighed after gentle perfusion with PBS. Tissues were minced in fl ow buffer (2% FBS in PBS), and adipose and livers were digested with Collagenase type IV (Sigma, St. Louis, MO) for 30 min at 37°C with shaking. Spleens were processed without collagenase treatment. Adipose stromal vascular cells (SVC), hepatic nonparenchymal cells, or splenocytes thus obtained were passed through a 70 m strainer and centrifuged at 300 g for 5 min. Pellets perigonadal (intra-abdominal) fat-pad weights between the transgenic mice and their controls either in lean (0.4 ± 0.05 g versus 0.5 ± 0.03 g) or obese (2.4 ± 0.1 g versus 2.3 ± 0.1 g) mice. However, body composition analysis revealed increased generalized body fat distribution in the transgenic mice fed the HFHSC diet ( Fig. 2B, C ) compared with obese Ldlr Ϫ / Ϫ mice, suggesting increases in other fat depots. No differences in lean body mass were observed between the obese groups.

Increased complement of iNKT cells causes marked metabolic dysregulation in obesity
Hypercholesterolemia and hypertriglyceridemia observed in Ldlr Ϫ / Ϫ mice on the HFHSC were amplifi ed in the transgenic mice. Plasma cholesterol levels were 30% higher in V ␣ 14tg.Ldlr Ϫ / Ϫ mice compared with littermate high-fat-fed controls; similarly, plasma triglyceride levels were 50% higher in V ␣ 14tg.Ldlr Ϫ / Ϫ mice ( Fig. 3A , B ). Differences in plasma cholesterol and triglycerides were observed as early as four weeks of diet feeding (not shown). Lipoprotein profi les showed increased VLDL/IDL and LDL particles in the HFHSC-fed transgenic mice ( Fig. 3C, D ). Fasting hyperglycemia was observed in the transgenic mice on HFHSC feeding; these mice also demonstrated signifi cant fasting hyperinsulinemia ( Fig. 3E, F ). Correspondingly, the insulin resistance index was increased in the transgenic mice, suggesting a greater degree of insulin resistance in states of lipid excess ( Fig. 3G ). Glucose tolerance testing revealed a modest worsening of glucose excursions in the V ␣ 14tg.Ldlr Ϫ / Ϫ compared with Ldlr Ϫ / Ϫ HFHSC-fed control mice with signifi cant hyperinsulinemia ( Fig. 3H, I ). No differences in lipids or insulin sensitivity were observed in the chow-fed animals between the two genotypes.

Adipose tissue infl ammation is worsened in obese mice with excess iNKT cells
Since activated iNKT cells can interact with tissue macrophages ( 12 ), we examined whether the presence of excess iNKT cells infl uenced the macrophage content of adipose tissue. Obese V ␣ 14tg.Ldlr Ϫ / Ϫ mice accumulated more macrophages in perigonadal (intra-abdominal) adipose tissue compared with littermate controls, as demonstrated by increased immunostaining for the macrophage-specifi c protein Mac2 ( Fig. 4A , B ). Adipocyte size was decreased in the obese V ␣ 14tg.Ldlr Ϫ / Ϫ mice ( Fig. 4C ). Analysis of genes involved in inflammation in whole adipose tissue revealed increased expression of a variety of genes in obese mice, although the increase was significantly more pronounced for the chemotactic factor ( Mcp1 ) gene as well as macrophage-specifi c genes, such as Emr1 (F4/80) and Mac2 ( Fig. 4D ). Overall, the accumulated macrophages showed a phenotype of proinfl ammatory M1 activation, as evidenced by increased Tnf ␣ and Cd11c expression with decreased expression of Retnla, an anti-infl ammatory activation M2 marker ( Fig. 4D ). Taken together, these findings indicate an increase in adipose infl ammatory changes in obese mice with excess iNKT cells. of lesion area was performed on digital images of Movat-stained sections using image analysis software (Image Pro Plus software, Media Cybernetics, Bethesda, MD).

Statistical analyses
Data were analyzed using the GraphPad Prism 5 program (Graph-Pad Software Inc., La Jolla, CA) and are represented as means and standard errors. Student t -test was used to detect differences within groups when applicable. One-way ANOVA (ANOVA) was used to compare differences among all groups, and Bonferroni post-hoc testing was used to detect differences among mean values of the groups. P < 0.05 was considered statistically signifi cant.

Obesity alters iNKT-cell levels in tissues
We investigated the effects of diet-induced obesity on tissue iNKT-cell numbers in Ldlr Ϫ / Ϫ mice. In line with other studies, Ldlr Ϫ / Ϫ mice fed HFHSC diet for 16 weeks demonstrated a reduced number of iNKT cells in the liver, where iNKT cells represent up to 40% of total resident lymphocytes in mice ( Fig. 1A-E ) ( 7 ). Similarly, as has been demonstrated by others, we observed a decrease in iNKT cells in visceral (perigonadal) adipose tissue of obese Ldlr Ϫ / Ϫ mice. We calculated absolute iNKT-cell numbers per tissue ( Fig. 1C ) and per gram of tissue ( Fig. 1D ). Livers from lean Ldlr Ϫ / Ϫ mice contained ‫ف‬ 2.8 × 10 6 iNKT cells per gram compared with 0.9 × 10 5

V ␣ 14tg.Ldlr
Ϫ / Ϫ mice have an increased complement of iNKT cells and develop excess weight gain with high-fat, high-sucrose diet feeding To evaluate the role of iNKT cells in obesity-associated metabolic derangements, we utilized a gain-of-function approach. As anticipated, V ␣ 14tg mice have a selective increase in iNKT-cell numbers ( 16 ) in the spleen, liver, and adipose tissue ( Fig. 1F-J ). The HFHSC diet did not alter the levels of iNKT cells in spleen, liver, or adipose tissues in the V ␣ 14tg mice (not shown).
We next evaluated the hypothesis that excess iNKT cells may benefi cially impact obesity and related metabolic consequences. We previously showed that Ldlr Ϫ / Ϫ mice fed a HFHSC diet for 24 weeks developed obesity, hyperinsulinemia, and hyperlipidemia as well as significant atherosclerosis ( 17 ). V ␣ 14tg.Ldlr Ϫ / Ϫ and littermate Ldlr Ϫ / Ϫ mice were placed on standard chow or HFHSC for 16 weeks. These mice were fertile and appeared healthy. No differences in body weight or adiposity were observed between chow-fed V ␣ 14tg.Ldlr Ϫ / Ϫ and control Ldlr Ϫ / Ϫ mice; however, when challenged with the HFHSC diet, weight gain was signifi cantly higher in V ␣ 14tg.Ldlr Ϫ / Ϫ mice compared with control Ldlr Ϫ / Ϫ mice ( P < 0.001, Fig. 2A ). Caloric intake was equivalent between the HFHSC diet groups (not shown). There were no differences in the mice ( Fig. 5C ), suggesting increased hepatocyte damage, although we did not observe an increase in TUNEL-positive cells ( Fig. 5D ). Plasma circulating SAA levels, an infl ammatory marker derived from the liver, were increased in both groups of obese mice ( Fig. 5E ). Additionally, gene expression analysis did not reveal evidence of increased hepatic infl ammation in transgenic mice ( Fig. 5E ).

Aortic atherosclerosis and infl ammation is worsened in obese
Mice on the Ldlr Ϫ / Ϫ background are particularly susceptible to atherosclerosis when placed on high-fat diets. iNKT cells have been shown to enhance atherosclerotic lesion development ( 22 ). To assess atherosclerosis in our mouse model of excess iNKT cells, we fi rst measured aortic atherosclerotic lesion area using the en face technique.
Obese V ␣ 14tg.Ldlr Ϫ / Ϫ mice demonstrate increased hepatic steatosis but not infl ammation Changes in the liver often accompany those seen in adipose tissue with diet-induced obesity. Since lipid-sensing iNKTs are highly enriched in the hepatic lymphocyte pool ( 7 ), we evaluated whether excess iNKT cells also worsens hepatic steatosis and infl ammation. As with the adipose tissue, liver weights were increased in obese over lean animals. In this case, however, the transgenic mice had signifi cantly larger livers when fed either chow (1.1 ± 0.06 g versus 1.35 ± 0.07 g, P < 0.001) or the HFHSC diet (1.9 ± 0.2 versus 3.0 ± 0.2 g, P < 0.0001). Increased hepatic steatosis was observed in the obese V ␣ 14tg.Ldlr Ϫ / Ϫ mice ( Fig. 5A ). Correspondingly, hepatic triglyceride content was significantly greater in the transgenic mice on HFHSC diet ( Fig. 5B ). Plasma ALT levels were also increased in these this is the fi rst reported study in which this transgenic mouse has been employed for the study of obesity and its associated pathophysiology. Contrasted with control Ldlr Ϫ / Ϫ mice, the transgenic Ldlr Ϫ / Ϫ mice exhibited increased weight gain, hyperlipidemia, adiposity and infl ammation, glucose intolerance, and insulin resistance, as well as increased aortic atherosclerosis. In obesity, adipocyte expansion in adipose tissue is accompanied by infi ltration of macrophages, as well as an altered balance of T cells, including alterations in iNKT cells, CD8+ T cells, and CD4+ regulatory T cells. Changes in adipose tissue mass and infl ammation are associated with the release of cytokines and adipokines and with metabolic dysfunction, in which glucose intolerance, insulin resistance, and increased hepatic lipid content are most extensively studied. The molecular mediators of these changes are not always clear.
There has been much recent investigation of the potential role of NKT cells in obesity using a variety of approaches. These include the examination of adipose tissue for the presence of NKT cells in lean and obese mice; stimulation of iNKT cells with its characteristic exogenous agonist ␣ -galactosylceramide; and the response to diet-induced obesity of mice lacking iNKT cells only ( J ␣ 18 Ϫ / Ϫ mice) or lacking both iNKT and vNKT cells ( CD1d Ϫ / Ϫ mice). The outcome of these studies has not been consistent in all cases. Some of these studies suggest that iNKT cells reduce the development of obesity and its metabolic accompaniments ( 12,13,15 ). On the other hand, one study showed that, in the absence of iNKT cells, there was a reduction in Chow-fed mice in both groups did not show any evidence of atherosclerosis. As anticipated, signifi cant atherosclerosis was observed in the Ldlr Ϫ / Ϫ mice on the HFHSC diet, and it was substantially amplifi ed in the obese V ␣ 14tg.Ldlr Ϫ / Ϫ mice ( P < 0.002, Fig. 6A ). A similar trend was also observed in the aortic root lesion area, although it did not reach statistical signifi cance (58,645 ± 10,999 µm 2 versus 87,712 ± 13,274 µm 2 ). In line with these observations, aortic expression of Il6 and Vcam1 genes was signifi cantly increased in the obese transgenic mice compared with the obese Ldlr Ϫ / Ϫ mice, which in turn were much higher than in the aortae of the lean animals ( Fig. 6B ). Aortic lesion area correlated with plasma cholesterol ( r = 0.58, P < 0.05) and triglyceride levels ( r = 0.70, P < 0.01) in the obese animals fed highfat diets.

DISCUSSION
We previously showed that Ldlr Ϫ / Ϫ mice develop many features of the metabolic syndrome when fed diets rich in saturated fat and refi ned carbohydrates, effects that can be exacerbated by the addition of moderate amounts of dietary cholesterol ( 17 ). We utilized this model to evaluate the effects of iNKT-cell excess on weight gain and glucose homeostasis, as well as to evaluate atherosclerosis. Here we show that obesity and features of the metabolic syndrome are aggravated by the presence of increased numbers of invariant NKT cells, as achieved by crossing the V ␣ 14 TCR transgenic mouse with the Ldlr Ϫ / Ϫ mouse. To our knowledge, the transgenic animals exhibited no differences in body weight, hypercholesterolemia, or atherosclerosis over the Ldlr Ϫ / Ϫ controls. Overall, our results indicate that the overabundance of iNKT cells interacts critically with the diet and the presence of obesity in relation to the observed pathophysiological consequences. The evolution of obesity with time involves a very dynamic network of cellular changes. Like the liver, white adipose tissue is highly enriched in NKT cells compared with the spleen and other lymphoid tissues. With the feeding of high-fat diets, there is a decline in the NKT-cell complement in two tissues: adipose and liver ( 13,25 ). This was more pronounced in one study ( 13 ) than another ( 25 ), in which the reduction was only seen after 20 weeks of high-fat feeding. The restoration of chow to high-fat-fed animals rapidly increased adipose tissue iNKT cells. In our hands, the HFHSC diet also caused a reduction in iNKTcell numbers in liver as well as adipose tissue. However, our data suggests that increasing the numbers of iNKT cells via enforced expression of a transgenic TCR, which drives positive thymic selection, may overwhelm the regulatory effects of obesity on iNKT. Nonetheless, in our experiments, it is clear that the exacerbation of obesity and metabolic syndrome was a result of the HFHSC diet acting through the NKT cells rather than an intrinsic effect of increased iNKT numbers, because there was no difference obesity on a high-fat diet ( 23 ). A recent study evaluating the effect of iNKT-cell defi ciency in lean mice suggests that adipose tissue NKT cells may protect against the development of insulin resistance ( 15 ). Others have shown that activation of iNKT cells with ␣ -galactosylceramide exacerbates adipose tissue infl ammation, insulin resistance, and hepatic steatosis in obese mice ( 24,25 ). Ours is the fi rst example of the use of a transgenic model overrepresenting iNKT cells. Each of the above-cited studies were performed with Ldlr +/+ mice, whereas ours employed Ldlr Ϫ / Ϫ mice. Reasons for discordant results between laboratories include variations in experimental protocol, such as diet composition, level of enrichment in dietary fat, and duration of dietary therapy, and experimental location, which could infl uence the gut microbiota, which are known to be infl uenced by obesity ( 26 ). Some investigators have employed the commonly used Western-type diet containing 21% fat (40% kcals from fat), others have used a slightly higher dietary fat (45% kcals) ( 15 ), while most have employed diets in which 60% kcals are from fat ( 12,13,25,27,28 ). None of these studies used diets supplemented with cholesterol. We used a high-fat, high-sucrose diet (HFHSC), providing 60% calories as fat derived mostly from lard, supplemented with dietary cholesterol (0.15% in the HFHSC diet). In a separate study (data not shown), we found that with feeding the Westerntype diet (40% calories from milk fat and 0.2% cholesterol), adipose tissue with the three-day feeding but did not report on this with prolonged exposure to the high fat. Our Ldlr Ϫ / Ϫ mice also exhibited increased adipose SAA3 expression when fed the HFHSC diet for 16 weeks, but the effect of HFHSC on SAA3 was attenuated in V ␣ 14tg.Ldlr Ϫ / Ϫ mice. The results with the Rag Ϫ / Ϫ mice suggest that some of the metabolic effects of obesity may derive partly from the expansion of the adipocytes rather than from the participation of the adaptive immune system, including NKT cells, in the adipose tissue infl ammation.
The macrophages that accumulated in mice with NKTcell enrichment in our experiments have features of proinfl ammatory, M1 macrophages that may contribute to the metabolic dysfunction observed. In experiments with short-term, high-fat feeding (four days), the macrophages of the adipose tissue were predominantly of the M2 subtype between chow-fed V ␣ 14tg.Ldlr Ϫ / Ϫ mice and their control Ldlr Ϫ / Ϫ counterparts.
The relative contributions of the increased adipocyte number/size and of adipose infl ammation to metabolic dysfunction are not clear. Both increase with the duration of the high-fat feeding. In one study, four days of a high-fat diet was suffi cient to induce an increment in the size of adipocytes and enrichment of iNKT cells in the adipose tissue, resulting in impaired glucose tolerance and insulin sensitivity, though an increase in plasma insulin was only seen at eight weeks of high-fat feeding ( 27 ). In another study, three days of high-fat feeding resulted in increased adipocyte size and glucose intolerance ( 29 ). High-fat feeding in Rag Ϫ / Ϫ mice lacking all T cells, pro-and anti-infl ammatory, led to elevated fat mass and a small degree of insulin resistance ( 5 ). These authors noted an increase in SAA3 in

We used mice on the Ldlr
Ϫ / Ϫ background for our studies to evaluate atherosclerosis. A role for the LDL receptor in lipid antigen presentation has been suggested. In a series of elegant studies, apoE was shown to be essential for exogenous lipid antigen uptake and presentation by antigenpresenting cells ( 33 ). The same authors also demonstrated a diminished IFN ␥ response in Ldlr Ϫ / Ϫ mice treated with the lipid antigen galactosyl galactosylceramide, but this response was nonetheless ‫ف‬ 8-fold higher than in control mice. This fi nding suggests that the LDL receptor is not required for exogenous lipid antigen uptake. In corroboration, recently it was shown that the absence of the LDL receptor does not affect lipid antigen uptake; Ldlr Ϫ / Ϫ mice also demonstrated appropriate IFN ␥ responses to ␣ -galactosylceramide in vivo ( 34 ). For some natural antigens, these authors implicated scavenger receptors as an important recognition receptor. Taken together, these data point to the presence of other less studied pathways by which lipids can be presented to iNKT cells and that the LDL receptor plays a role in but is not necessary for lipid antigen uptake and presentation. Evidence that iNKT cells mediate an increase in atherosclerosis has been demonstrated previously. Absence of CD1d and thus NKT cells resulted in decreased atherosclerosis in Ldlr Ϫ / Ϫ and ApoE Ϫ / Ϫ mice fed a high fat-diet (35)(36)(37). Adoptive transfer of splenocytes from V ␣ 14tg to Rag Ϫ / Ϫ mice led to an increase in atherosclerotic lesions ( 18 ). With the HFHSC diet used in our study, we noted a marked enhancement of aortic atherosclerosis with NKTcell enrichment, in contrast to what we observed in the same strains fed the Western-type diet (data not shown). This again indicates a strong interaction between the former diet and NKT-cell enrichment in promoting atherosclerosis likely mediated in part by the enhanced hyperlipidemia, including increase in VLDL/IDL particles in the transgenic mice. We previously noted that atherosclerosis is well correlated with VLDL cholesterol levels ( 38 ). While hyperlipidemia developed in Ldlr Ϫ / Ϫ mice fed HFHSC diets, hypercholesterolemia and hypertriglyceridemia were worsened in the obese transgenic mice. Another contributor to the increased atherosclerosis could be related to the presence of increased LDL particles, mediated by IL-4/Stat 6 activation ( 12 ). A study of the time-dependent evolution of macrophage subtypes in the adipose tissue has not been undertaken and would be of interest, since these cells may modify their expression patterns with the duration of fat feeding. Also included in the variety of cellular players in obesity are regulatory T cells, levels of which are decreased in obese adipose tissue, thus contributing to insulin resistance ( 30 ). Recent reports suggest a reciprocal cross-talk between iNKT cells and regulatory T cells, which can suppress cytokine production and cytotoxic activity of iNKT cells ( 31,32 ). Thus, it is conceivable that the increase in iNKT-cell numbers accompanied by a likely decrease in regulatory T cells could account for the accelerated proinfl ammatory response that we observed in our obese transgenic mice. One approach to the activation of iNKT cells involves the administration of the Cd1d agonist ␣ -galactosylceramide. One might expect that this would yield results similar to those observed with NKT-cell enrichment. However, as with other studies of the role of NKT cells in obesity, the results of agonist administration are not fully consistent. The response to the agonist is not seen in J ␣ 18 Ϫ / Ϫ mice defi cient in iNKT cells. Wu and colleagues noted that while weekly injections of agonist for eight weeks into obese mice did not increase body weight, worsening of insulin sensitivity and increased liver fat was observed ( 25 ). The same agonist given to obese mice in another study ( 29 ) reduced body weight and fat, with a decrease in adipocyte size, an improvement in glucose tolerance, and a reduction in plasma and liver triglycerides. Similar results were reported by Ji and colleagues ( 12 ). Ohmura and colleagues found that ␣ -galactosylceramide administration resulted in a major accumulation of iNKT cells in adipose tissue, moderate glucose intolerance, and increased mRNA for Ifng, Tnfa , and Mcp-1 ( 24 ). Their results are similar to ours reported here with iNKT-cell enrichment. The activation of iNKT cells by endogenous agonists presumably accounts for the activity of these cells in diet-induced obesity. The nature of these agonists is not known, although a dietary origin must be considered, given the variability of results with diets containing various lipids and fatty acids. which have been demonstrated to stimulate the V ␣ 14J ␣ 18 TCR ( 18 ).
In conclusion, the role of iNKT cells in diet-induced obesity and its sequelae remains inconclusive. Results vary across a large spectrum, presumably depending upon the level and nature of the fat in the diet, the duration of high-fat feeding, the possible differences in gut microbiota, and other unrecognized factors. The resolution of the differences noted in this discussion will require careful further experimentation, in which the composition of the diet and its duration of feeding are carefully standardized, preferably in the same laboratory microenvironment. The activity of iNKT cells at various stages in the evolution of the obesity also needs to be considered. However, our fi ndings are that the enrichment of tissues, including the adipose with iNKT cells as a result of transgenesis results in increased weight gain, adiposity, adipose tissue infl ammation, liver steatosis, hyperlipidemia, hyperglycemia, glucose intolerance, insulin resistance, as well as a notable increment in aortic atherosclerosis, compared with control Ldlr Ϫ / Ϫ mice fed the same diet. Rather than knocking out a population that may have opposing physiological effects, our approach was to use a system that employs the expansion of iNKT numbers, while allowing them to respond naturally to endogenous physiological cues. Therefore, our data represent an augmentation of the natural role of iNKT cells and suggest that they act to promote diet-induced obesity and its related comorbidities when fed a diet rich in saturated fat and refi ned carbohydrates.