iNKT cells are a unique subset of T cells that recognize glycolipid antigens and play a role in the regulation of inflammation and immunity. Furthermore, our laboratory9
have shown that activation of iNKT cells is proatherogenic. During conditions of spontaneous hyperlipidemia in apoE−/−
and diet-induced dyslipidema,11
iNKT cell numbers and functions are decreased. However, the etiology behind these changes is not yet known. Modulation of iNKT cell responses is being clinically tested as a possible therapeutic for cancer,25
in humans. In addition, it has been hypothesized that activating iNKT cells during immunization is a superior vaccination strategy.28
Thus, it is imperative to understand how the lipid environment might dampen or skew the iNKT cell response.
In the current study, we sought to examine the mechanisms by which chronic hyperlipidemia might decrease iNKT cell numbers and functions. We observed that apoE−/− mice have decreased numbers of iNKT cells in spleens and livers compared with B6 controls and that iNKT cells present in the spleens of apoE−/− mice have an impaired ability to respond to exogenous stimulation by the glycolipid α-GalCer. This decreased cytokine production seemed to affect IFN-γ responses more than IL-4, suggesting that T helper 1 responses may be more affected than T helper 2.
It is well established that glycolipids play an important role in the development of iNKT cells within the thymus and that normal thymic development of these iNKT cells requires the recognition of natural lipid ligands presented by CD1d. Previous studies have suggested that the endogenous glycosphingolipid isoglobotrihexosylceramide is important for iNKT cell development and self-recognition.29
Because a spontaneous increase in circulating lipids, as seen in apoE−/−
mice, could affect the development of iNKT cells and subsequently lead to decreased peripheral numbers, we analyzed thymic iNKT cells from apoE−/−
and B6 mice by flow cytometry. Our data suggest that decreased iNKT cell numbers in apoE−/−
mice are not the result of defects in thymic development, because numbers and maturation markers of iNKT cells in the thymus remained largely unchanged. Instead, our data support the hypothesis that the iNKT cell defect results from changes in the periphery. This is in stark contrast to studies in mice with deficiencies in the Niemann–Pick type C1 and C2 proteins, where reductions in iNKT cell numbers were observed in the thymus and peripheral lymphoid tissues.30–32
This was also found to be the case in mouse models of other lysosomal lipid storage diseases, such as mice deficient for α- or β-galactosidase or hexosaminidases A and S.33
Therefore, decreased numbers of iNKT cells in the peripheral lymphoid organs of apoE−/−
mice suggest that the endogenous glycolipids necessary to elicit normal selection of iNKT cells in the thymus are not affected by lipid accumulation.
Recent evidence has shown that B cell-mediated activation of iNKT cells is enhanced by apoE and is dependent on the low-density lipoprotein receptor.34
In addition, findings from Van den Elzen et al19
have shown that circulating lipoproteins, such as very low-density lipoproteins, can enhance iNKT cell responses to glycolipid antigen presented by DCs. These experiments elegantly demonstrated that apoE may enhance glycolipid uptake. However, there was no evidence that glycolipid and apoE interact in vivo or that apoE was necessary for normal iNKT cell responses to exogenous lipid antigen. Although the absence of apoE and the effect this might have on antigen uptake is still an important consideration in our system, we have several pieces of evidence suggesting that the absence of apoE alone cannot explain the difference in iNKT cell activation observed in hyperlipidemic mice. First, in our system, DCs from apoE−/−
mice did not appear to be compromised in their ability to present antigen to B6 iNKT cells. Additionally, CD1d loading of GGC and presentation on the cell surface, as measured by L363 antibody staining, was not compromised in apoE−/−
DCs. Therefore, we conclude that although apoE may increase iNKT activation under normolipidemic conditions, the functional defects that we observe in apoE−/−
mice cannot be completely attributed to the lack of apoE.
It has been shown previously in atherosclerosis studies that lipids, such as the glycosphingolipid β-glucosylceramide35
and the disialoganglioside GD3,36
accumulate in the serum of apoE−/−
mice as well as in humans. In addition, natural activation of iNKT cells during microbial infection is often dependent on both IL-12 and presentation of endogenous glycolipid antigen by DCs.37
Thus, a possible explanation for the iNKT cell hyporesponsiveness that we observe in apoE−/−
mice is that these cells are chronically activated by increased levels of endogenous glycolipid. Although recent data from VanderLaan et al12
suggest that endogenous iNKT antigen was not present in the serum of apoE−/−
mice fed a high-fat diet, it is possible that presence of an endogenous ligand in tissue, such as in the liver, spleen, or lymph node, and not serum is responsible for the changes in iNKT cells that we observed. Alternatively, given that glycolipid antigens are processed and loaded onto CD1d within the APC after uptake, lysosomal accumulation of lipids during hyperlipidemia may play a role in the differences that we observe. Supporting this possibility, a recent study from Bai et al38
shows that lipid exchange within the lysosome is regulated by lipid structure as well as acidic lysosomal pH. However, our results indicate that DCs from apoE−/−
mice retain their ability to activate iNKT cells, suggesting that the iNKT cell hyporesponsiveness in apoE−/−
mice is due to chronic activation rather than defective antigen presentation.
Previous studies have shown that repeated activation of conventional T cells, such as in chronic viral infection, results in T cell exhaustion.39
Similarly, repeated activation of iNKT cells by α-GalCer also results in a functionally unresponsive anergic phenotype.15
Our data show that apoE−/−
iNKT cells display a phenotype similar to those rendered anergic due to repeated activation. This evidence supports our hypothesis that spontaneous dyslipidemia, as observed in apoE−/−
mice, leads to chronic activation of iNKT cells. Several recent reports have shown that the inhibitory receptor PD-1 is upregulated on exhausted T cells due to chronic activation.40,41
In addition, PD-1 has been implicated in the induction and maintenance of iNKT cell anergy.23
In a recent study by Parekh et al,24
it was shown that blocking PD-1/PD-L interactions could prevent iNKT cell anergy but could not overcome anergy once established. Our study illustrates that hyperlipidemic apoE−/−
mice have spontaneously increased PD-1 expression on iNKT cells, although blocking PD-1 in vitro was not able to restore responsiveness to α-GalCer stimulation. Consistent with our data, recent clinical findings in HIV-infected humans indicated that these patients have increased PD-1 expression on iNKT cells, and PD-1 blockade did not restore iNKT cell function.36
Taken together, these data suggest that in our system, decreased functionality of chronically activated iNKT cells is not dependent on PD-1 expression and may be irreversible once established.
It is known that iNKT cells become undetectable with tetramer soon after in vivo stimulation with α-GalCer due to surface TCR downregulation.18
Our flow cytometry analyses demonstrate that there is a significant increase in the intracellular TCRβ in the apoE−/−
mice as compared with B6 mice. Importantly, these studies were performed ex vivo without exogenous stimulation. In addition, we have shown that hyperlipidemic apoE−/−
mice have modest but significant increases in myristic acid (14:0) and palmitic acid (16:0) (supplemental Figure II
). Although the physiological relevance of this increase is not tested in the current study, the serum concentrations that we observe are in excess of fatty acid concentrations that have been shown to activate APCs through toll-like receptor activation.42
Therefore, it is possible that saturated fatty acid signaling in DCs via toll-like receptor 4 leads to the production of IL-12, thus activating iNKT cells by the alternative or indirect pathway.43
Collectively, these data support the hypothesis that the hyperlipidemic environment of the apoE−/−
mice spontaneously activates the iNKT cells, rendering them unresponsive to further stimulation. Further studies to test this hypothesis are warranted.
In summary, our results indicate that an increase in circulating lipids leads to decreased iNKT cell functionality and that this dyslipidemia-associated decrease is iNKT cell intrinsic. The functional characteristics of iNKT cells are important to understand not only in chronic conditions of dyslipidemia, such as atherosclerosis, but also in diseases for which therapeutic approaches involving manipulation of iNKT cells are being considered. In addition, given that similar decreases in iNKT cell numbers and functions are observed in wild-type mice fed a high-fat diet,11
caution may be warranted in immunologic studies involving the use of lipid-laden diets and therapies that give rise to dyslipidemia.