Karen M. Spach

Vitamin D3 Confers Protection from Autoimmune Encephalomyelitis Only in Female Mice

The prevalence of multiple sclerosis (MS) increases significantly with decreasing UV B light exposure, possibly reflecting a protective effect of vitamin D3. Consistent with this theory, previous research has shown a strong protective effect 1,25-dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis (EAE), an MS model. However, it is not known whether the hormone precursor, vitamin D3, has protective effects in EAE. To address this question, B10.PL mice were fed a diet with or without vitamin D3, immunized with myelin basic protein, and studied for signs of EAE and for metabolites and transcripts of the vitamin D3 endocrine system. The intact, vitamin D3-fed female mice had significantly less clinical, histopathological, and immunological signs of EAE than ovariectomized females or intact or castrated males. Correlating with reduced EAE, the intact, vitamin D3-fed female mice had significantly more 1,25-dihydroxyvitamin D3 and fewer CYP24A1 transcripts, encoding the 1,25-dihydroxyvitamin D3-inactivating enzyme, in the spinal cord than the other groups of mice. Thus, there was an unexpected synergy between vitamin D3 and ovarian tissue with regard to EAE inhibition. We hypothesize that an ovarian hormone inhibited CYP24A1 gene expression in the spinal cord, so the locally-produced 1,25-dihydroxyvitamin D3 accumulated and resolved the inflammation before severe EAE developed. If humans have a similar gender difference in vitamin D3 metabolism in the CNS, then sunlight deprivation would increase the MS risk more significantly in women than in men, which may contribute to the unexplained higher MS incidence in women than in men.

IL-10 Signaling Is Essential for 1,25-Dihydroxyvitamin D3-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) results from an aberrant, neuroantigen-specific, T cell-mediated autoimmune response. Because MS prevalence and severity decrease sharply with increasing sunlight exposure, and sunlight supports vitamin D3 synthesis, we proposed that vitamin D3 and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) may protect against MS. In support of this hypothesis, 1,25-(OH)2D3 strongly inhibited experimental autoimmune encephalomyelitis (EAE). This inhibition required lymphocytes other than the encephalitogenic T cells. In this study, we tested the hypothesis that 1,25-(OH)2D3 might inhibit EAE through the action of IL-10-producing regulatory lymphocytes. We report that vitamin D3 and 1,25-(OH)2D3 strongly inhibited myelin oligodendrocyte peptide (MOG35–55)-induced EAE in C57BL/6 mice, but completely failed to inhibit EAE in mice with a disrupted IL-10 or IL-10R gene. Thus, a functional IL-10-IL-10R pathway was essential for 1,25-(OH)2D3 to inhibit EAE. The 1,25-(OH)2D3 also failed to inhibit EAE in reciprocal, mixed bone marrow chimeras constructed by transferring IL-10-deficient bone marrow into irradiated wild-type mice and vice versa. Thus, 1,25-(OH)2D3 may be enhancing an anti-inflammatory loop involving hemopoietic cell-produced IL-10 acting on brain parenchymal cells and vice versa. If this interpretation is correct, and humans have a similar bidirectional IL-10-dependent loop, then an IL-10-IL-10R pathway defect could abrogate the anti-inflammatory and neuro-protective functions of sunlight and vitamin D3. In this way, a genetic IL-10-IL-10R pathway defect could interact with an environmental risk factor, vitamin D3 insufficiency, to increase MS risk and severity.

1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by inhibiting chemokine synthesis and monocyte trafficking.

Multiple sclerosis (MS) is a complex neurodegenerative disease whose pathogenesis involves genetic and environmental risk factors leading to an aberrant, neuroantigen‐specific, CD4+ T cell‐mediated autoimmune response. In support of the hypothesis that vitamin D3 may reduce MS risk and severity, we found that vitamin D3 and 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3) inhibited induction of experimental autoimmune encephalomyelitis (EAE), an MS model. To investigate how 1,25‐(OH)2D3 could carry out anti‐inflammatory functions, we administered 1,25‐(OH)2D3 or a placebo to mice with EAE, and subsequently analyzed clinical disease, chemokines, inducible nitric oxide synthase (iNOS), and recruitment of dye‐labeled monocytes. The 1,25‐(OH)2D3 treatment significantly reduced clinical EAE severity within 3 days. Sharp declines in chemokines, inducible iNOS, and CD11b+ monocyte recruitment into the central nervous system (CNS) preceded this clinical disease abatement in the 1,25‐(OH)2D3‐treated animals. The 1,25‐(OH)2D3 did not directly and rapidly inhibit chemokine synthesis in vivo or in vitro. Rather, the 1,25‐(OH)2D3 rapidly stimulated activated CD4+ T cell apoptosis in the CNS and spleen. Collectively, these results support a model wherein inflammation stimulates a natural anti‐inflammatory feedback loop. The activated inflammatory cells produce 1,25‐(OH)2D3, and this hormone subsequently enhances the apoptotic death of inflammatory CD4+ T cells, removing the driving force for continued inflammation. In this way, the sunlight‐derived hormone could reduce the risk of chronic CNS inflammation and autoimmune‐mediated neurodegenerative disease.

Estrogen Controls Vitamin D3-Mediated Resistance to Experimental Autoimmune Encephalomyelitis by Controlling Vitamin D3 Metabolism and Receptor Expression

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease with a rapidly increasing female gender bias. MS prevalence decreases with increasing sunlight exposure, supporting our hypothesis that the sunlight-dependent hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) is a natural inhibitor of autoimmune T cell responses in MS. We found that vitamin D3 inhibited experimental autoimmune encephalomyelitis (EAE) in intact female mice, but not in ovariectomized females or males. To learn whether 17β-estradiol (E2) is essential for vitamin D3-mediated protection, ovariectomized female mice were given E2 or placebo and evaluated for vitamin D3-mediated EAE resistance. Diestrus-level E2 implants alone provided no benefit, but they restored vitamin D3-mediated EAE resistance in the ovariectomized females. Synergy between E2 and vitamin D3 occurred through vitamin D3-mediated enhancement of E2 synthesis, as well as E2-mediated enhancement of vitamin D receptor expression in the inflamed CNS. In males, E2 implants did not enable vitamin D3 to inhibit EAE. The finding that vitamin D3-mediated protection in EAE is female-specific and E2-dependent suggests that declining vitamin D3 supplies due to sun avoidance might be contributing to the rapidly increasing female gender bias in MS. Moreover, declining E2 synthesis and vitamin D3-mediated protection with increasing age might be contributing to MS disease progression in older women.

Activation of p38 MAPK in CD4 T cells controls IL-17 production and autoimmune encephalomyelitis

Although several transcription factors have been shown to be critical for the induction and maintenance of IL-17 expression by CD4 Th cells, less is known about the role of nontranscriptional mechanisms. Here we show that the p38 MAPK signaling pathway is essential for in vitro and in vivo IL-17 production by regulating IL-17 synthesis in CD4 T cells through the activation of the eukaryotic translation initiation factor 4E/MAPK-interacting kinase (eIF-4E/MNK) pathway. We also show that p38 MAPK activation is required for the development and progression of both chronic and relapsing-remitting forms of experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis. Furthermore, we show that regulation of p38 MAPK activity specifically in T cells is sufficient to modulate EAE severity. Thus, mechanisms other than the regulation of gene expression also contribute to Th17 cell effector functions and, potentially, to the pathogenesis of other Th17 cell-mediated diseases.

Cutting Edge: The Y Chromosome Controls the Age-Dependent Experimental Allergic Encephalomyelitis Sexual Dimorphism in SJL/J Mice

Multiple sclerosis is a sexually dimorphic, demyelinating disease of the CNS, and experimental allergic encephalomyelitis (EAE) is its principal autoimmune model. Young male SJL/J mice are relatively resistant to EAE whereas older males and SJL/J females of any age are susceptible. By comparing a wide age range of proteolipid protein peptide 139–151 immunized mice, we found that female disease severity remains constant with age. In contrast, EAE disease severity increases with age in SJL/J males, with young males having significantly less severe disease and older males having significantly more disease than equivalently aged females. To determine whether the Y chromosome contributes to this sexual dimorphism, EAE was induced in consomic SJL/J mice carrying a B10.S Y chromosome (SJL.YB10.S). EAE was significantly more severe in young male SJL.YB10.S mice compared with young male SJL/J mice. These studies show that a Y chromosome-linked polymorphism controls the age-dependent EAE sexual dimorphism observed in SJL/J mice.

Pertussis toxin induces angiogenesis in brain microvascular endothelial cells.

Pertussis toxin (PTX) is an ancillary adjuvant used to elicit experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis. One mechanism whereby PTX potentiates EAE is to increase blood–brain barrier (BBB) permeability. To elucidate further the mechanism of action of PTX on the BBB, we investigated the genomic and proteomic responses of isolated mouse brain endothelial cells (MBEC) following intoxication. Among ∼14,000 mouse genes tracked by cDNA microarray, 34 showed altered expression in response to PTX. More than one‐third of these genes have roles in angiogenesis. Accordingly, we show that intoxication of MBEC induces tube formation in vitro and angiogenesis in vivo. The global effect of PTX on signaling protein levels and phosphorylation in MBEC was investigated by using Kinex antibody microarrays. In total, 113 of 372 pan‐specific and 58 of 258 phospho‐site‐specific antibodies revealed changes ≥25% following intoxication. Increased STAT1 Tyr‐701 and Ser‐727 phosphorylation; reduced phosphorylation of the activating phospho‐sites in Erk1, Erk2, and MAPKAPK2; and decreased phosphorylation of arrestin β1 Ser‐412 and Hsp27 Ser‐82 were confirmed by Kinetworks multi‐immunoblotting. The importance of signal transduction pathways on PTX‐induced MBEC tube formation was evaluated pharmacologically. Inhibition of phospholipase C, MEK1, and p38 MAP kinase had little effect, whereas inhibition of cAMP‐dependent protein kinase, protein kinase C, and phosphatidylinositol 3‐kinase partially blocked tube formation. Taken together, these findings are consistent with the concept that PTX may lead to increased BBB permeability by altering endothelial plasticity and angiogenesis.

Endothelial histamine H1 receptor signaling reduces blood–brain barrier permeability and susceptibility to autoimmune encephalomyelitis

Disruption of the blood–brain barrier (BBB) underlies the development of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Environmental factors, such as Bordetella pertussis, are thought to sensitize central endothelium to biogenic amines like histamine, thereby leading to increased BBB permeability. B. pertussis-induced histamine sensitization (Bphs) is a monogenic intermediate phenotype of EAE controlled by histamine H1 receptor (Hrh1/H1R). Here, we transgenically overexpressed H1R in endothelial cells of Hrh1-KO (H1RKO) mice to test the role of endothelial H1R directly in Bphs and EAE. Unexpectedly, transgenic H1RKO mice expressing endothelial H1R under control of the von Willebrand factor promoter (H1RKO-vWFH1R Tg) were Bphs-resistant. Moreover, H1RKO-vWFH1R Tg mice exhibited decreased BBB permeability and enhanced protection from EAE compared with H1RKO mice. Thus, contrary to prevailing assumptions, our results show that endothelial H1R expression reduces BBB permeability, suggesting that endothelial H1R signaling may be important in the maintenance of cerebrovascular integrity.

A Single Nucleotide Polymorphism in Tyk2 Controls Susceptibility to Experimental Allergic Encephalomyelitis

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 the tyrosine kinase-2 gene (Tyk2) are susceptible to Toxoplasma gondii yet resistant to autoimmune arthritis, unlike the wild-type B10.Q/Ai substrain. To understand whether 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 the myelin oligodendrocyte glycoprotein peptide 79–96. B10.D1-H2q/SgJ mice were resistant to EAE whereas B10.Q/Ai mice were susceptible, and a single copy of the Tyk2G allele conferred EAE susceptibility in F1 hybrids. Furthermore, EAE resistance in B10.D1-H2q/SgJ mice was overridden 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, IL-6, and IFN-γ increased significantly with both genetic compensation and PTX treatment. These data indicate that Tyk2 is a shared autoimmune disease susceptibility gene whose genetic contribution to disease susceptibility can be modified by environmental factors. 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 Disease-Associated Histamine Receptor H1 Alleles Exhibit Differential Protein Trafficking and Cell Surface Expression

Structural polymorphisms (L263P, M313V, and S331P) in the third intracellular loop of the murine histamine receptor H1 (H1R) are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis and experimental allergic orchitis. The P-V-P haplotype is associated with increased disease susceptibility (H1RS) whereas the L-M-S haplotype is associated with less severe disease (H1RR). In this study, we show that selective re-expression of the H1RS allele in T cells fully complements experimental allergic encephalomyelitis susceptibility and the production of disease-associated cytokines while selective re-expression of the H1RR allele does not. Mechanistically, we show that the two H1R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H1RR allele being retained within the endoplasmic reticulum. Moreover, we show that all three residues (L-M-S) comprising the H1RR haplotype are required for altered expression. These data are the first to demonstrate that structural polymorphisms influencing cell surface expression of a G protein-coupled receptor in T cells regulates immune functions and autoimmune disease susceptibility.