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Invariant Natural Killer T cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). Here we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal antigen (Ag), IRBP. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with α-C-GalCer, which induces a strong IFN-γ but only a weak IL-4 production by iNKT cells, in contrast to the ligands α-GalCer (both IFN-γ and IL-4) and OCH (primarily IL-4). The protective effect of α-C-Gal-Cer was associated with a reduction of adaptive Ag specific IFN-γ and IL-17 production and was negated by systemic neutralization of IFN-γ. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-γ and a consequent dampening of the Th1 as well as the Th17 effector responses.
Invariant Natural Killer T cells (iNKT) are considered to represent an innate subset of T cells. iNKT cells have a semi-invariant Vα14-Jα18 TCR repertoire specific for lipid antigens that are presented on the MHC class I-like CD1d molecule. GalCer, a synthetic glycolipid derived from marine sponges, is a well known iNKT TCR ligand (1). Recently, natural ligands of iNKT TCR have been described, such as α-glucuronosylceramide and glycosphingolipids from Sphingomonadaceae (2–4), that resemble cell wall constituents of some Gram-negative bacteria, supporting an innate role of iNKT cells in some infectious diseases. Other studies also revealed regulatory and protective properties in various models of autoimmunity such as experimental autoimmune encephalomyelitis (EAE) and type-1 diabetes (reviewed in 1). Most of these studies utilized synthetic ligands for iNKT cell activation and therapeutic regimen.
Upon ligation of their invariant TCR with α–GalCer, iNKT cells rapidly produce large amounts of cytokines such as IFN-γ and IL-4 (5). Analogs of α-GalCer prepared by total synthesis led to the identification of ligands that induce a cytokine pattern that is more biased toward either a Th1-type (IFN-γ) or a Th2-type (IL-4) response. An example of the former is αC-GalCer, while the latter includes the ligand OCH (6, 7). Some studies demonstrated altered biological effects of these α–GalCer analogs on autoimmunity and cancer that could be ascribed to the cytokine profiles they elicit (6, 7).
Experimental autoimmune uveitis (EAU) induced in animals by immunization with retinal antigens in complete Freund’s adjuvant (CFA) is a model for human autoimmune uveitis, a disease that accounts for about 10–15% of severe visual handicap in the US. EAU is induced by immunization with the same retinal antigens that are recognized by uveitis patients and is dependent on CD4+ Th1 and Th17 effector cells (8, 9)}. EAU in mice is induced with the interphotoreceptor retinoid-binding protein (IRBP) or with its pathogenic fragments emulsified in CFA (8).
The relationship between NKT cells and eye-related immune responses is not well understood. Although iNKT cells have been shown to play an important role in the eye-related regulatory phenomenon known as anterior chamber-associated immune deviation (ACAID) (10, 11), the possible role of iNKT cells in regulation of EAU has not been established. In the present study we examine the role of iNKT cells and the effects of the iNKT cell ligands on EAU. Our data show that although natural strain-specific variations or genetically induced lack of iNKT cells do not seem to affect the threshold of susceptibility to EAU, iNKT function seemed to correlate with susceptibility. Importantly, a pharmacological enhancement of these cells using glycolipid iNKT cell ligands was able to inhibit induction of disease. This appeared to be due at least in part to iNKT-produced IFN-γ and a consequent dampening of the adaptive Th1 and Th17 pathogenic effector responses.
B10.RIII, B10.A, C57BL/6, DBA/2, AKR and BALB/c mice (WT and CD1d-KO) were purchased from The Jackson Laboratory (Bar Harbor, ME). All experiments were approved by the NEI Animal Care and Use Committee. Animal care and use conformed to Institutional guidelines and to the ARVO guidelines on the use of animals in ophthalmic and vision research.
Bovine IRBP was purified as described (12, 13). Complete Freund’s adjuvant (CFA) was purchased from Difco (Detroit, MI) and was supplemented with additional Mycobacterium tuberculosis H37RA to 2.5 mg/ml. Purified derivative of tuberculin (PPD) was purchased from the Statens Seruminstitut (Copenhagen, Denmark). α-GalCer (KRN7000) was provided by the Kirin Brewery Co, Tokyo, Japan (14). α-C-GalCer was synthesized as described previously (6). OCH was synthesized by Drs. Chi-Huey Wong and Douglas Wu of the Scripps Research Institute, La Jolla, CA. Neutralizing anti-IFN-γ antibodies (clone R4-6A2) were obtained from the Biological Resources Branch, NCI, Frederick MD.
Livers were perfused in situ through the hepatic portal vein with RT PBS and minced into small pieces PBS with 2%FCS and 0.02% Sodium Azide (PBS/FCS/Az). The tissue was then pressed though a 200 gauge mesh and cells were suspended in cold (4°C) PBS/FCS/Az. Cells were washed twice (500 g for 7 min). The pelleted cells were resuspended in 37.5% isotonic percoll at room temperature (25ml per liver) and spun at 680g for 12 min. The cell pellet made up of lymphocytes and erythrocytes was collected, washed once in PBS/FCS/Az and erythrocytes were lysed with 2mL red cell lysis buffer (Sigma-Aldrich) for 4min. Cells were then recovered by centrifugation through a layer of fetal bovine serum, resuspended in PBS/FCS/Az, filtered through 100 μm mesh, counted and prepared for immunophenotyping by flow cytometry.
C57BL/6 and BALB/c mice were immunized with 150 μg emulsified in CFA supplemented with Mycobacterium tuberculosis, strain H37RA from Difco (Detroit, MI) to 2.5 mg/ml. Clinical disease was evaluated by fundus examination in a masked fashion and was scored on a scale from 0 (no inflammation) to 4 (complete destruction of the retina) in half-point increments, as described previously (8). Eyes were harvested for histopathology 21 d after immunization. Disease was scored by an ophthalmic pathologist (C.-C. Chan) in a masked fashion as described previously (8).
Unless otherwise noted, 5 μg of either α-GalCer, α-C-GalCer or OCH were added to and emulsified with the uveitogenic Ag preparation (IRBP/CFA 1:1 v/v). The emulsion was injected subcutaneously, divided into 3 doses (both thighs and base of tail). Systemic IFN-γ neutralization was achieved by treatment with monoclonal anti-IFN-γ Ab, 150 ug/mouse injected i.p. on days −2, 0, 2).
Cytokine productionton to α–GalCer analogs in culture was examined on splenocytes obtained from naive mice. Cell suspensions of 2.5 × 106 cells/ml were incubated with 100ng/ml of the stimulant and supernatants were collected after 48 h. Cytokine production in vivo to α–GalCer analogs was measured in sera collected at the indicated time points after intraperitoneal injection of 5 μg of the analog. For determination of Ag specific cytokine production and proliferation, spleens and lymph nodes draining the site of immunization (inguinal and iliac) were collected on day 21 and were pooled within each group. Proliferation to the indicated doses of antigen was assayed by 3H-Thymidine uptake during the last 16 h of a 72 h culture on triplicate cultures of 0.2 ml, as described (15). Cytokine responses were determined using the Pierce Chemical Co. multiplex SearchLight™ Arrays technology (16 and http://www.endogen.com/services).
iNKT cells were enumerated by flow cytometry after exclusion of dead cells by DNA staining with 7-Amino-Actinomycin D (7-AAD). Cells were reacted with PE-labeled CD1d/α–GalCer tetramers, APC-labeled β-TCR and FITC-labeled anti-CD4 antibodies. β-TCR positive cells were gated and the percentage of α-GalCer positive cells (either CD4+ or−) was determined by counting of 1 × 105 viable β-TCR positive cells. Multiplication of percentages by absolute numbers of lymphocytes that were isolated from each organ (beta counter) resulted in the total iNKT numbers shown in figure 1.
All experiments were performed at least twice and results were highly reproducible. Figures show data from representative or from pooled experiments, as specified. EAU severity is represented by fundoscopy scores determined on day 19–21. All fundoscopy scores were confirmed by histopathology. Where appropriate, statistical analysis of EAU severity was performed using the Snedecor and Cochran z test for linear trend in proportions (17). This is a nonparametric, frequency-based test that takes into account both disease severity and incidence. Probability values of < 0.05 were considered to be significant. Values determined to be significantly different from controls are marked with an asterisk in the figures.
EAU is a disease model where susceptibility varies considerably among different mouse strains. The strain with the highest known susceptibility is B10.RIII. B10.A is another susceptible strain that can develop high disease scores, but unlike the B10.RIII strain it requires administration of pertussis toxin at the time of immunization in order to develop EAU, as do all other susceptible strains. C57BL/6 and DBA/2 mice have mild to moderate susceptibility and AKR as well as BALB/c mice are resistant to disease. We asked the question whether susceptibility to disease in a series of EAU-characterized mouse strains correlated with their numbers of iNKT cells. Figure 1a shows a schematic representation of typical EAU scores for six mouse strains, summarizing previously reported findings (18–20). Representative pictures of severe mild and no disease are shown in the inset.
Most of the iNKT cells in the body are concentrated in the thymus, liver and spleen. We enumerated invariant TCR-bearing NKT cells in these 3 organs in the different mouse strains using flow cytometry, by binding of PE-labeled CD1d/α–GalCer-Tetramers. Although there were strain-specific differences in iNKT cell numbers and variations in their content in the different organs, there was no correlation with strain-specific differences in disease susceptibility (Fig. 1b and 1c). In addition, a low iNKT number in one organ (e.g. thymus B10.A, spleen AKR) tended to be counterbalanced by a higher number in another organ (e.g. liver of B10.A and AKR) in some strains. Consequently, the total number of iNKT cells was often similar between strains with different EAU susceptibilities (Fig. 1b).
We next examined the percent of CD4+ iNKT cells out of the total iNKT cells, as this subset plays a role in the eye-specific regulatory phenomenon known as ACAID (11, 21). CD4+ iNKT cells were enumerated by double staining for CD4 and for the invariant TCR using CD1d/α–GalCer tetramers. The data revealed that the differences in CD4+ iNKT cells between the strains were even less pronounced than total iNKT numbers and did not correlate with disease susceptibility (Fig. 1d).
Lastly, we selected 3 strains that were either resistant, moderately susceptible or highly susceptible to EAU (Balb/c, C57Bl/6 and B10RIII, respectively) and tested their iNKT cells in vitro using two different ligands: α-GalCer and OCH, each known to elicit differing cytokine profiles (4, 22). Single cell suspensions were made from whole spleens and were stimulated with the indicated ligands for 48 hours. Analysis of culture supernatants revealed that iNKT cells in resistant Balb/c mice produce significantly higher levels of IFN-γ, IL-4 and IL-2, irrespective of the ligand used for stimulation (Figure 2). Interestingly, the opposite was the case with IL-17, with the highest levels detected in supernatant of B10RIII splenocytes. iNKT cells have recently been identified as a source of innate IL-17 (23), although the significance of this response for autoimmune disease is yet to be defined. No inter-strain differences were found in other cytokines examined such as IL-10, IL-13 and IL-5 (not shown).
If iNKT cells had a role in raising the threshold of susceptibility to EAU, we would expect that iNKT deficiency would result in more severe disease. However, mice deficient in CD1d (lacking CD1d-dependent NKT cells) (24) did not show enhanced EAU susceptibility compared to their WT counterparts (Fig. 3). The time of onset as well as the course of disease as determined by periodic fundus examinations were also not affected (data not shown). Mice deficient in CD1d on the resistant BALB/c background remained resistant (Fig. 3). These data are consistent with observations made by others in the EAE model (25–27).
We next examined whether functional triggering of iNKT cells using invariant TCR ligands can affect EAU. Five micrograms of α-GalCer incorporated into the IRBP/CFA emulsion ameliorated EAU severity and incidence in C57Bl/6 mice (Fig. 4a). To examine the effect of α–GalCer analogs, mice were treated with α-C-Gal-Cer and OCH. The data showed that OCH protected no better than α–GalCer, whereas α-C-GalCer was the most effective (Fig. 4b).
This pattern of protection was unexpected because OCH deviates the iNKT response to TCR ligation towards IL-4 production (whereas α–C-Gal-Cer skews towards IFN-γ) and has moreover been shown in experimental models of arthritis, diabetes in the NOD mouse and encephalomyelitis (EAE) to be more protective than α–GalCer in its original form through an IL-4 dependent mechanism (reviewed in 7, 22). We therefore examined whether our α–GalCer, α-C-Gal-Cer and OCH preparations had the expected effect on iNKT cytokine production. Data obtained by testing IL-4 and IFN-γ in serum of mice injected with the three α–GalCer analogs confirmed that indeed the three analogs elicited cytokine profiles that were in keeping with what has been reported by others (Fig 5). These data suggested that an IFN-γ-dominated cytokine profile elicited by α-C-Gal-Cer is more efficient in protecting from EAU than a deviation towards an IL-4 dominated profile in this model.
As we have previously observed that systemically produced IFN-γ can have a protective role in EAU (19, 28, 29), we decided to examine whether the enhanced protective effect of α-C-Gal-Cer compared to the other analogs was due to its ability to induce enhanced production of IFN-γ by iNKT cells. We therefore injected mice immunized for EAU in the presence of α-C-Gal-Cer with neutralizing Abs to IFN-γ at the time of immunization, when innate production of IFN-γ induced by α-C-Gal-Cer would be occurring (according to the timeline established in Fig 5). EAU and adaptive IFN-γ and IL-17, representing pathogenic effector Th1 and Th17 cell responses were examined. Mice protected from disease with α-C-Gal-Cer had strongly reduced adaptive IFN-γ and IL-17 responses (Fig. 6). Neutralization of IFN-γ at the time of immunization restored EAU scores in αC-Gal-Cer treated mice, but had no effect on disease progression in the control group, demonstrating a specific requirement for IFNγ in the protection. In addition, subsequent Ag-specific production of IFN-γ and IL–17 was restored (Fig. 6b and 6c), supporting the notion that innate production of IFN-γ elicited by α-C-Gal-Cer had a role in the protection and inhibition of adaptive responses induced by this analog.
In the present study we demonstrate that iNKT cells can have a role in EAU regulation. Their role appears to be not in setting the threshold of susceptibility to EAU, as do the natural CD4+CD25+ regulatory cells whose function in deterring development of ocular autoimmunity we have characterized in recent studies (30, 31). Rather, they can inhibit developing disease following a pharmacological enhancement of their activity at or around the time of priming. In chronic autoimmunity, priming of new effector T cells is believed to be occurring on a continuous basis. Since endogenous ligands for iNKT cells exist in the body and can trigger iNKT activity (4, 5), it is conceivable that iNKT cells can participate in modulating the course of ocular autoimmune disease. Thus, there appears to be a “division of labor” between the natural CD4+CD25+ regulatory T cells and iNKT cells, with the former setting the threshold of susceptibility, and the latter possibly regulating the autoimmune response after that threshold has been passed.
The groups of Streilein and Stein-Streilein demonstrated that iNKT cells have a central role in ACAID, a prototypic regulatory phenomenon elicited by injection of Ag into the anterior chamber of the eye and its transport by eye-derived APC to the spleen (10, 11, 21). iNKT cells are recruited into the spleen via a mechanism involving MIP-2 and participate in priming the adaptive T regulatory cells typically associated with ACAID. Although prior elicitation of ACAID to IRBP can inhibit a subsequent episode of EAU (32), it is unlikely that the protection from EAU by iNKT that we observe here bears a relationship to their role in ACAID. In ACAID the eliciting Ag originates from the eye and the eye has to be perturbed (injected with Ag) in order for this phenomenon to be observed, and iNKT activation, if any, occurs without additional manipulation. In contrast, in our study, pharmacological activation of iNKT cells is needed and is applied when the eye is still intact. Thus, it is conceivable that iNKT cells may regulate ocular immune responses at more than one level.
Studies in the models of experimental arthritis, NOD diabetes and EAE (reviewed in 22) had indicated that activation of iNKT by OCH is more effective than by α–GalCer, which was attributed to its induction of IL-4 and Th2 skewing. We were therefore surprised to find that OCH was not more effective than α–GalCer in protecting from EAU, and that the most efficient protection followed administration of α-C-Gal-Cer, which induces an IFN-γ dominated iNKT cytokine response. Thus, effectiveness of protection paralleled the innate IFN-γ inducing ability of the invariant TCR ligand. The protection was accompanied by reduction in the IRBP-specific adaptive Th1 and Th17 pathogenic effector responses, as judged by production of their respective hallmark cytokines IFN-γ and IL-17 to in vitro recall with IRBP. The functional role of IFN-γ in the protective and regulatory effects of iNKT are strongly supported by direct evidence showing that neutralization of innate IFN-γ reversed the protective effect of α-C-Gal-Cer and restored the subsequent proinflammatory cytokine production of the adaptive response. This is not to say that the mechanism of protection is the same for all the three analogs. Our data do not negate the possibility that protection from EAU by OCH and by α–GalCer could involve IL-4, as was previously demonstrated in several other autoimmune disease models (22).
Our data are in line with some previous reports, which revealed that protection from autoimmune disease by iNKT may not always involve IL-4 and Th2 skewing. Studies by Lehuen and her colleagues demonstrated that even in the absence of IL-4 iNKT cells can control EAE (33) and experimental type 1 diabetes (T1D) (34). This was associated with a decrease in Th1-associated pathogenic autoimmune responses without inducing Th2 responses, and was due at least in part to induction of anergy in the autoreactive T cells (35). Such a mechanism could also be involved in the prevention of EAU observed here. Although these studies did not directly implicate IFN-γ in these effects, participation of IFN-γ (rather than IL-4) in protection from EAE was suggested by Furlan et al. (27).
It should be noted that high systemic levels of IFN-γ early or late in the disease can be protective, but likely by different mechanisms. Initial production of IFN-γ would be mostly from NKT and NK cells, whereas later in disease Ag specific Th1 cells are a major source of IFN-γ. We previously showed that early upregulation of IFN-γ by injections of IL-12 inhibits development of EAU and associated immunological responses by aborting priming, through a process that involves induction of nitric oxide and apoptosis (29). The innate IFN-γ produced by α-C-Gal-Cer-triggered NKT cells may well work in a similar fashion. On the other hand, protective effects of IFN-γ later in the disease appear to be due to its role in elimination of spent effector cells by activation-induced cell death (36, 37). Thus, neutralization of systemic IFN-γ at that stage also enhances disease (19), although at that point it is not possible to distinguish between effects of IFN-γ produced by Ag specific T cells and iNKT cells.
In summary, we have demonstrated that iNKT cells can actively participate in regulating the autoimmune response to immunologically privileged retinal Ags. This apparently occurs at a different level than their role in induction of ACAID. The mechanism involves the induction of innate IFN-γ production through ligation of the invariant TCR and results in inhibited development of adaptive Th1 and Th17 responses that represent pathogenic effector mechanisms in uveitis.
The authors thank Dr. S. Yamano of the Kirin Brewery, Tokyo, Japan for providing α–GalCer (KRN7000) and Drs. Chi-Huey Wong and Douglas Wu of the Scripps Research Institute, La Jolla, CA, for synthesizing the OCH used in this study.
1Funding sources: NIH Intramural funding;