We examined in this study the effects of NKT cell activation on mercury-induced autoimmunity. The two synthetic NKT cell ligands, PBS 57 and 4-deoxy α-GalCer, differentially modulated mercury-induced autoimmunity. When tested in a model of tolerance induction, NKT cell activation prevented tolerance establishment, but was by itself insufficient to break established tolerance. To disrupt tolerance, NKT cells required an additional signal, which was in this case provided by a TLR9 agonist. In addition, we observed that different NKT ligands can either synergize with or antagonize TLR9-induced breakage of tolerance. Lastly, bacteria-bearing NKT cell ligands have an exacerbating effect on our model of chemically induced autoimmunity.
The outcome of NKT cell activation in autoimmunity depends on a variety of factors, including the mouse strain studied (31
), the timing of administration of NKT cell-activating ligand (33
), and the NKT cell-activating ligand itself (34
). Our results emphasize the importance of the mouse strain and the activating ligand. In the C57BL/6.SJL strain, both NKT cell ligands potentiated development of autoimmunity. However, in the A.SW strain of mice, NKT cell activation has profoundly different results depending on the glycolipid used to stimulate them. The 4-deoxy α-GalCer consistently exacerbated mercury-induced autoantibody production, whereas PBS 57 either had no effect on or down-regulated autoantibody levels. Thus, NKT cells are able to modulate heavy metal-induced autoimmunity differently depending on the ligand that activates them. These results are surprising because both ligands are derivatives of the widely used NKT cell ligand α-GalCer, with earlier studies indicating that their immunostimulatory properties are comparable to the parental ligand (16
). We expanded our study by comparing the two ligands in a model of tolerance induction. Because microbial infections can expose the immune system simultaneously to both NKT cell ligands and TLR ligands, we examined interplay between two NKT cell ligands and a TLR9 ligand, CpG 1826, in the breakage of established tolerance. The 4-deoxy α-GalCer-induced NKT cell activation strongly synergized with TLR9 stimulation to break established tolerance. This result is in agreement with a recent study wherein coexposure to a NKT cell ligand, α-GalCer, and various TLR ligands resulted in enhancement of dendritic cell (DC) maturation with increased T cell and B cell responses, suggesting that synergy between NKT ligands and different TLR ligands is a generalized phenomenon (36
). Surprisingly, our results showed that NKT cell activation can also antagonize TLR signals. NKT cell activation by PBS 57 antagonized the effects of TLR9 stimulation, reducing TLR9-induced autoantibody production.
Because the two NKT ligands had opposite effects on autoantibody production when administered with mercury, we examined the in vivo effects of dual administration of NKT ligands and mercury. In mercury-exposed animals, PBS-57-induced NKT cell activation increased the frequency of Tregs
, whereas 4-deoxy α-GalCer-induced NKT cell activation had the opposite effect. Moreover, in the tolerance model, PBS 57, but not 4-deoxy α-GalCer, restored the balance between T regs
, which had been disrupted by TLR9 stimulation. The importance of this balance has been highlighted by a recent study that demonstrated that a decrease in the ratio of Tregs
correlated with progression of disease in NOD mice (37
). Similarly, our study demonstrates that a treatment that disrupts tolerance decreases this ratio and suggests that this balance can be modulated by interplay between NKT cell agonists and a TLR9 ligand.
PBS 57 increased Th2 skewing in mercury-treated mice, whereas 4-deoxy α-GalCer increased Th1 skewing in tolerized mice challenged with CpG 1826 and mercury. Because autoantibody production in the mercury model is dependent on IFN-γ, but not IL-4 (8
), these results suggest an explanation as to why PBS 57 negatively regulates mercury-induced autoantibody production and why 4-deoxy α-GalCer and PBS 57 have opposing effects on CpG 1826-induced autoantibody production in tolerized mice. Strikingly, because mercury increased Th2 skewing in PBS 57-treated, but not 4-deoxy α-GalCer-treated animals, these results also suggest that mercury (a common environmental pollutant) can modify the outcome of NKT cell activation by certain agonists.
We also examined effects of the NKT cell activation in a model of tolerance induction. Current models of tolerance induction hypothesize that Ag presentation in the steady state by DCs induces peripheral tolerance (38
). The nature of these tolerogenic DCs is, however, unclear. Certain studies suggest that tolerogenic DCs are phenotypically immature, whereas maturation imparts the ability to be immunogenic (35
). However, in certain cases, phenotypically mature DCs can be tolerogenic (42
). Menges et al. (43
) showed that DCs matured with repeated injections of TNF-α induce Ag-specific protection of mice against expermental autoimmune encephalomyelitis. Activation of NKT cells induces phenotypic maturation of DCs, causing up-regulation of CD40, CD80, and CD86 costimulatory molecules and MHC class II Agpresenting molecules (46
). This study (45
) and others (38
) suggest that NKT cell activation could produce immunogenic DCs. In contrast, studies that examined the role of NKT cells in two models of tolerance have shown that this cell population is necessary for tolerance establishment (47
). In a model of tolerance establishment following oral administration of the contact allergen nickel, CD4+
NKT cells producing IL-4 and IL-10, but not IFN-γ, were required for the induction of tolerogenic APCs (47
). Somewhat in contrast to these findings, we demonstrate that NKT cell activation was sufficient to prevent establishment of tolerance to mercury. In our experiment, NKT cell activation by PBS 57, but not 4-deoxy α-GalCer, prevented tolerance establishment. PBS 57 is also a stronger NKT cell agonist than 4-deoxy α-GalCer. It is a more potent inducer of several cytokines, including IL-17 (our unpublished observations). A recent study has demonstrated that IL-17 can block establishment of oral tolerance (49
). Further studies are required to elucidate the mechanisms involved in PBS 57-induced prevention of tolerance.
A principal evolutionary role for NKT cells has been identified as antimicrobial defense (30
); this cell type is required for clearance of a large number of microbes (18
). Bacterial, viral, and parasitic infections have been implicated in the development and exacerbation of autoimmune diseases (6
). A number of other studies have shown that exposure to chemicals (drugs or heavy metals) can also trigger or exacerbate autoimmune disease (19
). However, the effects of infections and chemicals on autoimmune disease have for the most part been studied separately, whereas human patients are likely to be exposed to both factors. Hence, in this study, we tested the effect of a commonly dispersed chemical and an infectious agent on autoimmunity. NKT cell ligand-bearing bacteria of the Sphingomonas
strain are abundant soil microbes, and have been detected in the stools of 25% of healthy individuals (51
). Although contact levels vary among individuals, mercury exposure is virtually universal because of its natural release in the environment, abundance as a pollutant, and presence in dental amalgams, cosmetics, preservatives, fumigants, and vaccine preparations (7
). As previously demonstrated, normally maintained immune tolerance is broken by exposure to mercury. Administration of both mercury and bacteria induced pronounced anti-nucleolar reactivity and exacerbated the heavy metal-induced autoimmunity. Unlike many bacterial species that primarily activate innate immunity via TLR signaling, S. capsulata
bears triggers for both NKT cells and TLRs. These dual stimuli may have contributed to exacerbation of autoimmune manifestations.
Certain synthetic NKT ligands, such as OCH or the C-glycoside analog of α-GalCer, induce strongly different effects, owing to structural differences that result in highly polarized cytokine production. In contrast, PBS 57 and 4-deoxy α-GalCer have both been described as similar to α-GalCer. However, our study demonstrates that these NKT agonists, in combination with immunomodulator mercury, yield strikingly different effects. There could be several explanations for this. PBS 57 is more soluble due to the amide and the double bond in the acyl chain. Differential solubility can affect loading of lipids onto CD1d, which in turn can modulate uptake and presentation by CD1d-expressing APCs (53
). Additionally, decrease in acyl chain length (54
) and increased unsaturation (55
) increase Th2 cytokine production. The latter findings may represent explanations as to why more IL-4 is detected with PBS 57. Mercury is an immunomodulatory agent that increases skewing toward Th2 (8
). It is possible that the combination of the two agents results in increased Th2 skewing, and this could be, in part, contributing to the regulatory effects of PBS 57 in mercury-administered A.SW animals. Our study demonstrates that an environmental agent with immunomodulatory capacity can strongly influence the effect of NKT cell activation. This observation serves as a cautionary note when considering NKT cell activators as therapeutics in patients who may be simultaneously exposed to various environmental agents.