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Genes controlling immunopathologic diseases of differing etiopathology may also influence susceptibility to autoimmune disease. B10.D1-H2q/SgJ mice with a 2538 G→A missense mutation in tyrosine kinase-2 (Tyk2) are susceptible to Toxoplasma gondii, yet resistant to autoimmune arthritis, unlike the wild-type B10.Q/Ai substrain. To understand if Tyk2 is also important in a second autoimmune model, experimental allergic encephalomyelitis (EAE) was induced in B10.D1-H2q/SgJ (Tyk2A) and B10.Q/Ai (Tyk2G) mice with myelin oligodendrocyte glycoprotein peptide79–96. B10.D1-H2q/SgJ (Tyk2A) mice were resistant to EAE while B10.Q/Ai (Tyk2G) mice were susceptible and a single copy of the Tyk2G allele conferred EAE susceptibility in F1 hybrids. Furthermore, EAE susceptibility in B10.D1-H2q/SgJ (Tyk2A) mice was complemented when pertussis toxin (PTX) was used to mimic the effects of environmental factors derived from infectious agents. Numerous cytokines and chemokines were increased when PTX was included in the immunization protocol. However, only RANTES, interleukin-6, and interferon-γ increased significantly with both genetic compensation and PTX complementation. These data indicate that Tyk2 is a shared autoimmune disease susceptibility gene that can be complemented both genetically and environmentally. Single nucleotide polymorphisms like the one that distinguishes Tyk2 alleles are of considerable significance given the potential role of gene-by-environment interactions in autoimmune disease susceptibility.
Autoimmune diseases have common features and understanding the commonalities underlying them may aid in the design of rational treatments. One approach to understanding these commonalities is to identify genes, termed shared autoimmune disease genes, whose alternately expressed alleles influence susceptibility to multiple autoimmune diseases. Bphs/Hrh1, which controls susceptibility to both experimental allergic encephalomyelitis (EAE) (1) and autoimmune orchitis (2) was the first non-antigen dependent shared autoimmune disease susceptibility gene to be identified and positionally cloned in the mouse (3). Another example of a potential shared autoimmune disease susceptibility gene is Eae3/Idd3/Aod2 (4–6), which controls susceptibility to EAE, autoimmune insulin-dependent type I diabetes mellitus, and day 3 thymectomy induced autoimmune ovarian dysgenesis. In both mouse and humans, genetic clustering of autoimmune disease quantitative trait loci (QTL) supports the hypothesis that susceptibility to autoimmune disease may be controlled by shared genes (7). Moreover, relatives of multiple sclerosis (MS) and celiac disease patients are at increased risk of other autoimmune diseases suggesting a shared genetic susceptibility (8–11). Similarly, multiple autoimmune diseases are often observed within a single patient (12).
Tyrosine kinase 2 (Tyk2/Tyk2) participates in the signaling pathways of multiple cytokines in innate and acquired immunity (13, 14). Tyk2 is a member of the JAK/STAT signaling pathway (15) and contributes to the signaling of IFN-α/β (16), IL-6 (17), IL-10 (18), IL-12 (19), IL-13 (20) and IL-23 (21). Depending on the ligand, cytokine receptor aggregation activates Tyk2 leading to phosphorylation of STAT 1, −3, −4, or −5 (15). The phospho-STAT dimerizes and translocates to the nucleus to promote gene transcription.
Although Tyk2 activation has been implicated in the signaling of multiple cytokines (22), Tyk2tm1Shmd (Tyk2−/−) mice primarily exhibit defects in responses to IL-12 and type I IFN (14, 23). B10.D1-H2q/SgJ (B10.D1) mice are a substrain of B10.Q mice that have a naturally occurring mutation in Tyk2, designated Tyk2A (24). The 2538 G→A base substitution is predicted to result in a non-conservative amino acid substitution (E775K) within a critical APE motif of the JH2 (pseudokinase) domain of Tyk2. The JH2 domain is required for Tyk2 activation via ligand-activated cytokine receptors (16). Although Tyk2A-specific transcripts are present at normal levels in B10.D1 mice, immunoreactive protein cannot be detected (24). B10.D1 splenocytes exhibit impaired STAT phosphorylation in response to IL-12, IL-23 and IFNα stimulation (24). In addition, neither T cells nor NK cells from B10.D1 mice produce IFNγ when stimulated with IL-12; however, this defect can be overcome by increasing the concentration of IL-12 and the incubation time or by stimulation through an IL-12-independent pathway (25, 26).
Importantly, while B10.Q/Ai mice, which express a wild-type Tyk2G allele, are susceptible to collagen-induced arthritis (CIA) and resistant to Toxoplasma gondii infections, the Tyk2A mutation renders B10.D1 mice resistant to CIA and highly susceptible to Toxoplasma gondii (24–27), demonstrating that Tyk2 is a shared immunopathology gene. Because autoimmune disease susceptibility genes can also be shared (7, 28, 29), we tested the hypothesis that Tyk2 is a shared autoimmune disease gene by assessing susceptibility of B10.D1 and B10.Q/Ai mice to EAE, the principal animal model of MS. We found that Tyk2 is a critical genetic regulator of EAE susceptibility and that the resistant Tyk2A allele can be compensated by one copy of the wild-type Tyk2G allele and by environmental factors such as pertussis toxin (PTX). These results are of particular significance given that TYK2 polymorphisms are associated with increased risk of systemic lupus erythematosus (30, 31) and that TYK2 has recently been identified as a strong MS susceptibility gene in a genome-wide association study (32), and confirmed through independent replication (J. Oksenberg, personal communication).
B10.D1-H2q/SgJ (B10.D1) (Strain #002024) mice bearing the Tyk2rs2538-A (TykA) allele were purchased from The Jackson Laboratory (Bar Harbor, ME) and B10.Q/Ai (Line #4059) mice with the Tyk2rs2538-G (TykG) allele were purchased from Taconic Farms (Tarrytown, NY) through the National Institute of Allergy and Infectious Diseases Animal Supply Contract. Reciprocal F1 hybrid progeny were generated and bred at the University of Vermont. Mice were housed at 25°C with 12:12h light-dark cycles and 40–60% humidity. Naïve, age-matched male and female mice were used throughout. The experimental procedures performed in this study were approved by the Institutional Animal Care and Use Committee of the University of Vermont.
Mice were immunized for the induction of EAE using either the double injection or single injection protocols. For the double injection protocol mice were injected subcutaneously in the posterior right and left flank with a sonicated emulsion of 50 µg myelin oligodendrocyte glycoprotein peptide 79–96 [GKVALRIQNVRFSDEGGY] (MOG79–96) (33) and 200 µg Mycobacterium tuberculosis H37Ra in CFA (Difco Laboratories, Detroit, MI) in 0.1 ml; one week later mice received the same injection on the right and left flank anterior of the original injection site (34). For the single injection protocol with PTX as an auxiliary adjuvant (MOG79–96-CFA + PTX), mice were injected subcutaneously with 100 µg MOG79–96 and 200 µg M. tuberculosis H37Ra in CFA in 0.1 ml on the posterior right and left flank and the scruff of the neck. Immediately afterward, each mouse received 200 ng PTX (List Biological Laboratories, Campbell, CA) in 0.2 ml by intravenous injection (34). EAE was evaluated daily beginning at day 5 as follows: 0, no clinical expression of disease; 1, flaccid tail without hind limb weakness; 2, hind limb weakness; 3, complete hind limb paralysis and floppy tail; 4, hind leg paralysis accompanied by a floppy tail and urinary or fecal incontinence; 5, moribund. Clinical quantitative trait variables were generated as previously described (35). Mice were considered positive for incidence if they showed any clinical signs greater than or equal to one for two or more consecutive days. The severity index is the cumulative disease score per days affected. Histological assessment of EAE neuropathology was done as previously described (35–38). Briefly, brains and spinal cords were dissected from calvarias and vertebral columns, respectively, and fixed by immersion in phosphate buffered (pH 7.2) 10% formalin. Representative areas of the brain and SC, including brainstem, cerebrum, cerebellum, and the cervical, thoracic, and lumbar segments of the SC, were selected for histopathological evaluation. EAE pathology reflects the overall severity of the lesions observed, extent and degree of myelin loss and tissue injury (swollen axon sheathes, swollen axons, and reactive gliosis), severity of the acute inflammatory response (predominantly neutrophils), and the severity of the chronic inflammatory response (lymphocytes/macrophages).
For ex vivo cytokine and chemokine analysis, spleens and draining lymph nodes were obtained from mice immunized by both methods 10 days earlier for EAE as described above. Single cell suspensions at 1 × 106 cells/ml in RPMI 1640 media (Cellgro Mediatech, Inc., Manassas, VA) plus 5% FBS (HyClone, Logan, UT) were stimulated with 50 µg MOG79–96. Cell culture supernatants were recovered at 72 hours and 23 different cytokine and chemokine levels quantified in duplicate by Bioplex multiplex cytokine assay (Becton Dickinson Bioscience, San Jose, CA), including IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-γ, KC,MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α. To confirm the IL-17 and IFN-γ results, ELISAs were performed as described (34), using primary anti-IL-17A and anti-IFNγ antibodies and their corresponding biotinylated secondary antibodies (BD Biosciences-Pharmingen, San Diego, CA). Other ELISA reagents included horseradish peroxidase–conjugated avidin D (Vector Laboratories, Burlingame, CA), TMB microwell peroxidase substrate and stop solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD), and recombinant IFN-γ and IL-17 (R&D Systems, Inc., Minneapolis, MN) used as standards. IL-6 was confirmed using the mouse IL-6 DuoSet ELISA (R&D Systems, Inc., Minneapolis, MN).
Mice were immunized for EAE induction, and draining lymph nodes and spleens were harvested on day 10. Single cell suspensions were prepared, and 5 × 105 cells/well in RPMI 1640 media (5% FBS) were plated on standard 96-well U-bottom tissue culture plates and stimulated with 0, 2, 10 or 50 µg MOG79–96 for 72 h at 37°C. During the last 18 h of culture, 1µCi of [3H]-thymidine (PerkinElmer, Stelton, CT) was added. Cells were harvested onto glass fiber filters and thymidine uptake was determined by liquid scintillation.
Statistical analyses (two way ANOVA, Kruskal-Wallis test followed by Dunn’s post hoc multiple comparisons, nonlinear regression-based curve fitting (34), and Chi-square test) were performed using GraphPad Prism version 5 (GraphPad Software, San Diego, CA). A p value of < 0.05 was considered significant.
To determine if Tyk2 is a shared autoimmune disease gene, EAE susceptibility was assessed in B10.D1 and B10.Q/Ai mice using the MOG79–96-CFA double-injection protocol. B10.D1 mice were resistant (0/46) to EAE whereas B10.Q/Ai mice were susceptible (31/36; p<0.001) (Fig. 1A and Table I). On day 30, EAE pathology in the brain and spinal cord was assessed using previously defined neuropathologic trait variables (35, 37). The susceptible B10.Q/Ai mice had marked EAE pathology in the spinal cord while B10.D1 had no EAE pathology (Figure 2). Neither strain showed significant EAE pathology in the brain. (B10.D1 × B10.Q/Ai) F1 hybrids (16/18) and the reciprocal (B10.Q/Ai × B10.D1) F1 hybrids (26/31) were both susceptible to MOG79–96-CFA induced EAE compared to B10.D1 mice (p<0.001). The F1 hybrids had significantly greater disease severity for each clinical disease trait compared to B10.D1 (p<0.001), and did not differ significantly from B10.Q/Ai mice (Fig. 1B and Table I). Similarly, lesion severity, monocyte/lymphocyte infiltration, and total EAE pathology score was significantly greater in the reciprocal F1 hybrids compared to B10.D1 mice but did not differ from B10.Q/Ai mice in the spinal cord, p < 0.05 (Figure 2). Again, very little EAE pathology was noted in the brains of reciprocal F1 hybrids. These data indicate that Tyk2 alleles determine EAE susceptibility and that the Tyk2A allele is recessive. Therefore, EAE resistance in B10.D1 mice can be compensated genetically with a single copy of the wild-type Tyk2G allele, and since Tyk2 is also a susceptibility gene in CIA (25), it is by definition a shared autoimmune disease susceptibility gene.
To investigate the mechanism whereby Tyk2 alleles control T cell effector responses, the MOG79–96-specific immune responses of B10.D1, B10.Q/Ai, and F1 hybrid mice were compared ten days after MOG79–96-CFA immunization. Although B10.D1 mice were resistant to MOG79–96-CFA induced EAE, ex vivo proliferative responses did not differ between the strains (Fig. 3G). However, in ex vivo re-stimulation assays, B10.Q/Ai and F1 mice produced significantly more IFNγ, IL-6, and RANTES compared to B10.D1 mice (Fig. 3). Surprisingly, IL-17 was not significantly higher in supernatants from B10.Q/Ai or F1 cells, although a trend toward increased IL-17 production was noted when the results were grouped by strain (Fig. 3C). The three strains did not exhibit significant differences in any of the other cytokines and chemokines assayed (e.g., IL-4 and TNFα, Fig 3, and others not shown). Importantly, IL-12(p40) and IL-12(p70) were not different indicating IL-12 insufficiency is not responsible for EAE resistance in B10.D1 mice.
PTX is an example of an environmental factor derived from an infectious agent that influences susceptibility to EAE and is capable of overriding genetic checkpoints in this autoimmune disease (39). Therefore, we included PTX in the immunization protocol and tested the susceptibility of B10.D1 and B10.Q/Ai mice to EAE. In the MOG79–96-CFA + PTX single injection protocol (34), mice receive the same total amount of MOG79–96 in CFA as the double injection protocol but also receive an i.v. injection of PTX on day 0. In B10.Q/Ai mice injected with MOG79–96-CFA + PTX (Fig. 4B), there was increased incidence and more severe disease compared to B10.Q/Ai mice injected with MOG79–96-CFA alone (Table II). A more dramatic difference was seen in B10.D1 mice, which are much more susceptible (33/37) to EAE induced using PTX (Fig. 4A and Table II) than with MOG79–96-CFA alone. Clearly, EAE susceptibility in B10.D1 (Tyk2A) mice can be complemented by environmental factors derived from infectious agents such as PTX.
The complementation of EAE susceptibility in B10.D1 (Tyk2A) mice following immunization with PTX was not reflected in their antigen-specific ex vivo proliferative responses, which were not significantly different between immunization protocols (Fig. 5B). Inclusion of PTX in the immunization protocol, however, resulted in increased cytokine secretion by cultured cells from both strains, consistent with the increased severity of EAE induced by the PTX protocol. PTX is known to induce many cytokines that are proinflammatory and pathogenic in EAE, but PTX also induces TH2 responses (40, 41). The changes due to inclusion of PTX in the immunization of B10.D1 mice as assayed in cell culture supernatants were consistent with these published reports. For example, cytokines characteristic of TH1 (IFNγ, IL-12, TNFα), TH17 (IL-17, IL-6) and TH2 (IL-4, IL-5, IL-9, and IL-13) responses were elevated compared to those elicited by immunization without PTX (Fig. 5A). In addition, members of the CC chemokine family, MIP-1α and RANTES, were elevated more than fourfold compared to those induced by MOG79–96-CFA alone, suggesting that there is likely to be a distinct mobilization of TH1 cells in the PTX-exposed mice (42, 43). Notably, among the changes in cytokine and chemokine production elicited in B10.D1 mice by the inclusion of PTX in the immunization protocol, IFNγ, IL-6, and RANTES were also significantly elevated by genetic compensation (Fig. 3).
A naturally-occurring single nucleotide polymorphism (SNP) within the pseudokinase domain of Tyk2 influences the immunopathologic outcomes of CIA (25) and Toxoplasma gondii infection in B10.Q/Ai and B10.D1 (26), making Tyk2 a shared immunopathology gene. The data presented here showing that Tyk2 alleles control EAE susceptibility demonstrate that Tyk2 is also a shared autoimmune disease susceptibility gene. Specifically, B10.D1 mice which possess the Tyk2A allele are completely resistant to MOG79–96-CFA-induced EAE while B10.Q/Ai substrain mice expressing the Tyk2G allele are susceptible. Moreover, a single copy of the Tyk2G allele fully confers EAE susceptibility in B10.D1 F1 hybrids clearly establishing that Tyk2 is important in controlling autoimmune disease susceptibility. When PTX was included in the immunization regime to mimic the effects of environmental agents derived from infectious agents, EAE susceptibility was also complemented thus emphasizing the contextual role that gene-by-environment interactions play in determining susceptibility to autoimmune diseases.
It is not surprising that Tyk2 is a player in mouse autoimmunity and inflammation, due to its importance in supporting IL-12-induced IFN-γ responses (14, 15, 44), and its ability to down-regulate TH2-mediated antibody production, especially IgE (45). There are differences between the requirements for Tyk2 in mouse as compared to human immune responses. For example, human TYK2-deficient cell lines are completely unresponsive to type 1 IFN, and IL-6 and IL-10 signaling is severely impaired (13), whereas these phenotypes are leaky in Tyk2−/− mice, and high concentrations of IFNα can overcome the mouse Tyk2 deficiency MHC class I expression (23). However, evidence exists that Tyk2 shares some functions between these two species. There is a report of one patient with a mutation in TYK2, who had a remarkably similar immune response to that seen in B10.D1 mice, with hyper-IgE syndrome, increased susceptibility to multiple microbial pathogens, an impaired STAT-4 phosphorylation pattern, and undetectable IFNγ production in response to IL-12 (13). This supports the hypothesis that human TYK2 is also a shared immunopathology gene. Supporting the hypothesis that TYK2 is a shared human autoimmune disease susceptibility gene, it has recently been identified as a strong MS susceptibility gene in a genome-wide association study (32), and confirmed through independent replication (J. Oksenberg, personal communication) and TYK2 polymorphisms have also been associated with increased risk of systemic lupus erythematosus (30, 31).
However, the manner by which Tyk2 complements autoimmunity is not known. T cell proliferation induced by IL-12 is not Tyk2 dependent (23) and likewise, no differences in antigen-specific proliferation were observed between B10.D1 and wild type B10.Q/Ai mice following immunization with MOG79–96-CFA in this study. B10.D1 mice make IL-12 in normal abundance (26) and both IL-12 and TNFα levels were similar between B10.D1 and B10.Q/Ai and F1 hybrid mice after induction of EAE without PTX. Therefore, differences in proliferation and/or levels of these cytokines are not likely an important mechanism by which B10.D1 mice are resistant to EAE. The Tyk2A mutation impairs signaling through both IL-12R and IL-23R pathways (24). It is more likely therefore that their EAE resistance is due to this signaling defect, leading to an inability to up-regulate encephalitogenic levels of IFNγ (via IL-12R, reviewed in (44)) and IL-17 (via IL-23R, reviewed in (44)) or to activate T cells that make these cytokines. We observed differences between B10.D1 and B10.Q/Ai mice for both of these effector molecules, although the difference in IL-17 was only a trend. In addition, B10.D1 (Tyk2A) mice had significantly impaired ability to produce pro-inflammatory molecules such as IL-6 and RANTES. IL-17 and IFNγ have well-documented roles in EAE (46–49), and the development of EAE is blocked by antibodies or antagonists of IL-6 and RANTES (50, 51). It is important to note that the effects of the Tyk2A allele may not be exclusively in T cells as Tyk2 is also required in dendritic cells for IL-12, IL-23 and IFNγ production (52).
In the present study, PTX was included in the EAE induction protocol to reveal gene-by-environment interactions, and especially the effects of an environmental factor derived from an infectious agent. B10.D1 mice were susceptible to EAE only when it was induced with MOG79–96-CFA + PTX, and they produced significantly higher levels of multiple TH1-, TH2-, and TH17-type cytokines and chemokines compared to the MOG79–96-CFA-inoculated mice (Fig. 5A). Of interest, both GM-CSF and IL-5 showed large increases with PTX treatment. Multiple sclerosis patients in the active phase of disease have elevated levels of GM-CSF compared to patients in remission (53) and mice lacking GM-CSF are resistant to MOG35–55-induced EAE (54). Taken together, these reports and the present study suggest that GM-CSF is critically important in EAE and MS pathogenesis. A small study of MS patients and controls found patients with highly proliferating MBP-specific T cells produced higher levels of IL-5 and IL-17 and this correlated with number of MRI-identified active plaques (55). However IL-5 has also been shown to increase in MS patients treated with IFN-beta (56) or glatiramer acetate (57).
Although it is not known how PTX restores EAE susceptibility in B10.D1 mice, several possibilities exist. PTX may act intrinsically in T cells because they express PTX-sensitive Gi/o proteins. Inhibitory Gi/o proteins are inactivated by PTX-mediated ADP-ribosylation (58). Following direct TCR stimulation, T cells from Gαi2−/− mice produce more IL-2, IL-4 and IFNγ than wild type mice (59). Thus, the PTX-mediated abrogation of Gi coupled inhibitory signals in T cells would increase the magnitude of the cytokine responses. This may be one mechanism by which PTX complements EAE susceptibility in B10.D1 mice. Accordingly, PTX also increases the expression of the co-stimulatory molecule CD28 on T cells (41) which would therefore potentiate the immune response.
PTX could also increase T cell cytokine responses and complement EAE susceptibility through its effects on APCs. PTX increases secretion of proinflammatory cytokines such as IL-1β (41), IL-6 (60), IL-12 (61) and TNFα (62). Increased IL-6 production by PTX-treated APCs promotes the generation of IL-17 producing T cells (60). Indeed, IL-6 and IL-17 were up-regulated by PTX in both B10.D1 (Tyk2A) and B10.Q/Ai (Tyk2G) mice. PTX also enhances the ability of the APC to activate T cells by inducing dendritic cell maturation (62) and up-regulating expression of the co-stimulatory molecules CD80 and CD86 in the spleen and spinal cord (40, 41, 63). PTX-induced cytokine production and T cell clonal expansion are thought to occur primarily through the CD28:CD80/86 costimulatory pathway (64). The PTX-induced changes in EAE susceptibility of B10.D1 mice cannot be attributed solely to effects on either T cells or APCs based on these experiments. Although it is tantalizing to speculate that PTX increases co-stimulatory molecules on APCs which then increases both APC maturation and T cell differentiation and clonal expansion as evidence suggests (40, 63, 65), leading to the cytokine differences we observed, careful bone marrow chimera experiments must be performed to conclude this. These experiments are underway to attempt to determine if the effect of PTX is directly on APC or T cells through APCs.
In B10.D1 (Tyk2A) mice, IFNγ and IL-17 levels were both elevated, indicating that PTX must enhance their production via Tyk2-independent pathways, such as IL-18R-mediated IFNγ production (26). The Tyk2−/− mouse has decreased expression of Il-18R (66), so if PTX modulated production of IFN-γ via IL-18, it would also have to up-regulate the IL-18R for this to occur. The role of IL-18 is paradoxical, despite its apparently excellent candidacy. IL-18Rα-deficient mice are protected from EAE, in contrast to IL-18-deficient mice, which are susceptible (67). Further complicating matters, an alternative unidentified ligand for IL-18R on APCs responsible for Th17 pathogenicity has been postulated (68). Within the type I IFN pathway, alternate routes of STAT4 phosphorylation have also been noted (69). Additionally, PTX intoxication may produce sufficient IL-12 stimulation as to cause receptor aggregation leading to phosphorylation of Jak2 that can stand in for Tyk2. We observed high levels of active IL-12 after PTX inoculation (Fig. 5A), and it has been shown that greater exposure to IL-12 in vitro can overcome the effects of the Tyk2A allele as measured by STAT4 phosphorylation (25). Thus, IL-12 alone could explain the results we have observed. Because the issue of direct and indirect effects on the pathogenic T cells is further complicated by the independent roles of IL-12, IL-18, and an unknown IL-18R-ligand, it is unclear which pathway PTX uses to modulate IFN-γ and IL-17 in the B10.D1 mice.
The ability of environmental factors derived from infectious agents to alter autoimmune disease susceptibility controlled by a SNP highlights the contextual importance of gene-by-environment interactions in determining autoimmune disease susceptibility. There is increasing evidence that human autoimmune disease results from a complex interaction of environmental effects in genetically susceptible individuals. The 75% discordance rate for MS in monozygotic twins (70) suggests that environmental factors are important in MS. In particular, low ultraviolet light exposure (71–74) and the resulting low serum 25-(OH)2D3 levels correlate with increased MS risk (75–78). It is therefore interesting in this regard, that in vivo treatment of mice with 1,25-dihydroxyvitamin D3 ameliorates EAE (79), and treatment of activated T cells in vitro with 1,25-dihydroxyvitamin D3 inhibits IL-12-induced tyrosine phosphorylation of Tyk2, thereby reducing T cell responses to antigen (80). Another important finding is that the sexual dimorphism observed in MS is increasing in the last 50 years suggesting that emergent factors such as environmental estrogens could selectively promote MS in women (81). The phytoestrogen quercetin, known to reduce signs of EAE in mice, is also capable of blocking IL-12 induced phosphorylation of Tyk2 (82). In preliminary work we note that EAE susceptibility in B10.D1 (Tyk2A) and B10.Q/Ai (Tyk2G) mice is in fact sexually dimorphic (Blankenhorn, Spach, and Teuscher, in preparation, 2009). In the context of the present report, therefore, the mutant Tyk2A allele is a good candidate for an environmentally sensitive genetic modifier of demyelinating diseases, responding to a wide variety of environmental factors including 1,25-dihydroxyvitamin D3, estrogenic compounds, and toxins produced by microorganisms, such as PTX.
In summary, we demonstrate that Tyk2 is both a shared immunopathology gene and also a shared autoimmune disease susceptibility gene in mice. We further demonstrate that it can be complemented both genetically and environmentally. As such, this model provides a unique opportunity to identify additional environmental factors impacting a core genetic network underlying susceptibility to autoimmune disease.
We wish to thank Nathan Zalik for animal husbandry and Dr. Mercedes Rincon, Will Dixon and Laure Case for critically reading the manuscript.
This work was supported by the National Institutes of Health grants NS36526, AI41747, AI058052, NS061014, NS060901, and AI45666 and by National Multiple Sclerosis Society grant RG3575.
The authors have no conflicting financial interests.