G proteins Gαi1/3 are critical targets for Bordetella pertussis toxin-induced vasoactive amine sensitization.

Pertussis toxin (PTX) is an AB5-type exotoxin produced by the bacterium Bordetella pertussis, the causative agent of whooping cough. In vivo intoxication with PTX elicits a variety of immunologic and inflammatory responses, including vasoactive amine sensitization (VAAS) to histamine (HA), serotonin (5-HT), and bradykinin (BDK). Previously, by using a forward genetic approach, we identified the HA H1 receptor (Hrh1/H1R) as the gene in mice that controls differential susceptibility to B. pertussis PTX-induced HA sensitization (Bphs). Here we show, by using inbred strains of mice, F1 hybrids, and segregating populations, that, unlike Bphs, PTX-induced 5-HT sensitivity (Bpss) and BDK sensitivity (Bpbs) are recessive traits and are separately controlled by multiple loci unlinked to 5-HT and BDK receptors, respectively. Furthermore, we found that PTX sensitizes mice to HA independently of Toll-like receptor 4, a purported receptor for PTX, and that the VAAS properties of PTX are not dependent upon endothelial caveolae or endothelial nitric oxide synthase. Finally, by using mice deficient in individual Gαi/o G-protein subunits, we demonstrate that Gαi1 and Gαi3 are the critical in vivo targets of ADP-ribosylation underlying VAAS elicited by PTX exposure.

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.

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 H(1) receptor (Hrh1/H(1)R). Here, we transgenically overexpressed H(1)R in endothelial cells of Hrh1-KO (H(1)RKO) mice to test the role of endothelial H(1)R directly in Bphs and EAE. Unexpectedly, transgenic H(1)RKO mice expressing endothelial H(1)R under control of the von Willebrand factor promoter (H(1)RKO-vWF(H1R) Tg) were Bphs-resistant. Moreover, H(1)RKO-vWF(H1R) Tg mice exhibited decreased BBB permeability and enhanced protection from EAE compared with H(1)RKO mice. Thus, contrary to prevailing assumptions, our results show that endothelial H(1)R expression reduces BBB permeability, suggesting that endothelial H(1)R 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-H2(q)/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-H2(q)/SgJ (Tyk2(A)) and B10.Q/Ai (Tyk2(G)) mice with the myelin oligodendrocyte glycoprotein peptide 79-96. B10.D1-H2(q)/SgJ mice were resistant to EAE whereas B10.Q/Ai mice were susceptible, and a single copy of the Tyk2(G) allele conferred EAE susceptibility in F(1) hybrids. Furthermore, EAE resistance in B10.D1-H2(q)/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-gamma 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.

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 D(3) (1,25-(OH)(2)D(3)) is a natural inhibitor of autoimmune T cell responses in MS. We found that vitamin D(3) inhibited experimental autoimmune encephalomyelitis (EAE) in intact female mice, but not in ovariectomized females or males. To learn whether 17beta-estradiol (E(2)) is essential for vitamin D(3)-mediated protection, ovariectomized female mice were given E(2) or placebo and evaluated for vitamin D(3)-mediated EAE resistance. Diestrus-level E(2) implants alone provided no benefit, but they restored vitamin D(3)-mediated EAE resistance in the ovariectomized females. Synergy between E(2) and vitamin D(3) occurred through vitamin D(3)-mediated enhancement of E(2) synthesis, as well as E(2)-mediated enhancement of vitamin D receptor expression in the inflamed CNS. In males, E(2) implants did not enable vitamin D(3) to inhibit EAE. The finding that vitamin D(3)-mediated protection in EAE is female-specific and E(2)-dependent suggests that declining vitamin D(3) supplies due to sun avoidance might be contributing to the rapidly increasing female gender bias in MS. Moreover, declining E(2) synthesis and vitamin D(3)-mediated protection with increasing age might be contributing to MS disease progression in older women.

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.Y(B10.S)). EAE was significantly more severe in young male SJL.Y(B10.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.

Lymphocyte reconstitution after DMF discontinuation in clinical trial and real-world patients with MS.

BACKGROUND: Delayed-release dimethyl fumarate (DMF) has demonstrated robust efficacy in treating patients with relapsing-remitting multiple sclerosis. Decreases in absolute lymphocyte count (ALC) are a well-known pharmacodynamic effect of DMF treatment, but lymphocyte recovery dynamics are not well characterized after discontinuation of DMF. METHODS: Data sources included the Biogen DMF integrated clinical trial data set, a retrospective US chart abstraction study, and data from MSBase. We assessed rate and time course of lymphocyte reconstitution after DMF discontinuation. RESULTS: The majority of patients who developed lymphopenia while treated with DMF and subsequently discontinued treatment experienced ALC reconstitution. The median time to reach ALC ≥0.8 × 109/L was 2-4 months after discontinuation for patients treated in real-world data sets; the median time to reach ALC ≥0.91 × 109/L was 2 months after discontinuation in DMF clinical trials. Severity of lymphopenia on treatment and decline in ALC within the first 6 months did not affect the ALC reconstitution rate after DMF discontinuation; rather, on-treatment lymphopenia duration influenced the reconstitution rate. In patients with severe, prolonged lymphopenia for ≥3 years, lymphocyte reconstitution to ≥0.91 × 109/L was 12-18 months vs 2-3 months in patients with lymphopenia persisting <6 months. CONCLUSIONS: The majority of patients who discontinued DMF due to lymphopenia experienced ALC reconstitution within 2-4 months following DMF discontinuation. This may help guide clinicians in managing patients who develop lymphopenia during DMF treatment. Prolonged lymphopenia on DMF treatment is associated with slow lymphocyte recovery after DMF discontinuation.

Effect of dimethyl fumarate on lymphocytes in RRMS: Implications for clinical practice.

OBJECTIVE: To assess functional changes in lymphocyte repertoire and subsequent clinical implications during delayed-release dimethyl fumarate (DMF) treatment in patients with multiple sclerosis. METHODS: Using peripheral blood from several clinical trials of DMF, immune cell subsets were quantified using flow cytometry. For some patients, lymphocyte counts were assessed after DMF discontinuation. Incidence of adverse events, including serious and opportunistic infections, was assessed. RESULTS: In DMF-treated patients, absolute lymphocyte counts (ALCs) demonstrated a pattern of decline followed by stabilization, which also was reflected in the global reduction in numbers of circulating functional lymphocyte subsets. The relative frequencies of circulating memory T- and B-cell populations declined and naive cells increased. No increased incidence of serious infection or malignancy was observed for patients treated with DMF, even when stratified by ALC or T-cell subset frequencies. For patients who discontinued DMF due to lymphopenia, ALCs increased after DMF discontinuation; recovery time varied by ALC level at discontinuation. T-cell subsets closely correlated with ALCs in both longitudinal and cross-sectional analyses. CONCLUSIONS: DMF shifted the immunophenotype of circulating lymphocyte subsets. ALCs were closely correlated with CD4+ and CD8+ T-cell counts, indicating that lymphocyte subset monitoring is not required for safety vigilance. No increased risk of serious infection was observed in patients with low T-cell subset counts. Monitoring ALC remains the most effective way of identifying patients at risk of subsequently developing prolonged moderate to severe lymphopenia, a risk factor for progressive multifocal leukoencephalopathy in DMF-treated patients. TRIAL REGISTRATION NUMBERS: EUDRA CT 2015-001973-42, NCT00168701, NCT00420212, NCT00451451, and NCT00835770.

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 approximately 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 >or=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 beta1 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.

Gene expression analysis suggests that 1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by stimulating inflammatory cell apoptosis.

Multiple sclerosis (MS) is a debilitating autoimmune disease of the central nervous system (CNS) that develops in genetically susceptible individuals who are exposed to undefined environmental risk factors. Epidemiological, genetic, and biological evidence suggests that insufficient vitamin D may be an MS risk factor. However, little is known about how vitamin D might be protective in MS. We hypothesized that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] might regulate gene expression patterns in a manner that would resolve inflammation. To test this hypothesis, experimental autoimmune encephalomyelitis (EAE) was induced in mice, 1,25-(OH)2D3 or a placebo was administered, and 6 h later, DNA microarray hybridization was performed with spinal cord RNA to analyze the gene expression patterns. At this time, clinical, histopathological, and biological studies showed that the two groups did not differ in EAE disease, but changes in several 1,25-(OH)2D3-responsive genes indicated that the 1,25-(OH)2D3 had reached the CNS. Compared with normal mice, placebo-treated mice with EAE showed increased expression of many immune system genes, confirming the acute inflammation. When 1,25-(OH)2D3 was administered, several genes like glial fibrillary acidic protein and eukaryotic initiation factor 2alpha kinase 4, whose expression increased or decreased with EAE, returned to homeostatic levels. Also, two genes with pro-apoptotic functions, calpain-2 and caspase-8-associated protein, increased significantly. A terminal deoxynucleotidyl transferase-mediated dUTP nicked end labeling study detected increased nuclear fragmentation in the 1,25-(OH)2D3-treated samples, confirming increased apoptosis. Together, these results suggest that sensitization of inflammatory cells to apoptotic signals may be one mechanism by which the 1,25-(OH)2D3 resolved EAE.

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 H(1) (H(1)R) 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 (H(1)R(S)) whereas the L-M-S haplotype is associated with less severe disease (H(1)R(R)). In this study, we show that selective re-expression of the H(1)R(S) allele in T cells fully complements experimental allergic encephalomyelitis susceptibility and the production of disease-associated cytokines while selective re-expression of the H(1)R(R) allele does not. Mechanistically, we show that the two H(1)R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H(1)R(R) allele being retained within the endoplasmic reticulum. Moreover, we show that all three residues (L-M-S) comprising the H(1)R(R) 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.

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.

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 D(3) synthesis, we proposed that vitamin D(3) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) may protect against MS. In support of this hypothesis, 1,25-(OH)(2)D(3) 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)(2)D(3) might inhibit EAE through the action of IL-10-producing regulatory lymphocytes. We report that vitamin D(3) and 1,25-(OH)(2)D(3) strongly inhibited myelin oligodendrocyte peptide (MOG(35-55))-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)(2)D(3) to inhibit EAE. The 1,25-(OH)(2)D(3) 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)(2)D(3) 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 D(3). In this way, a genetic IL-10-IL-10R pathway defect could interact with an environmental risk factor, vitamin D(3) insufficiency, to increase MS risk and severity.

Evidence that the Y chromosome influences autoimmune disease in male and female mice.

Experimental allergic encephalomyelitis (EAE), an autoimmune model of multiple sclerosis, is a complex disease influenced by genetic, intrinsic, and environmental factors. In this study, we questioned whether parent-of-origin effects influence EAE, using reciprocal F2 intercross progeny generated between EAE-susceptible SJL/J (S) and EAE-resistant B10.S/SgMcdJ (B) mice. EAE susceptibility and severity were found to be different in female BS x BS intercross mice as compared with females from the three other birth crosses (BS x SB, SB x SB, and SB x BS), and in fact, both traits in female mice resembled those of their male siblings. This masculinization is associated with transmission of the SJL/J Y chromosome and an increased male-to-female sex ratio. Related studies using progeny of C57BL/6J Y-chromosome substitution strains demonstrate that the Y chromosome again influences EAE in both male and female mice, and that the disease course in females resembles that of their male littermates. Importantly, these data provide experimental evidence supporting the existence of a Y-chromosome polymorphism capable of modifying autoimmune disease susceptibility in both males and females.

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 D(3). Consistent with this theory, previous research has shown a strong protective effect 1,25-dihydroxyvitamin D(3) in experimental autoimmune encephalomyelitis (EAE), an MS model. However, it is not known whether the hormone precursor, vitamin D(3), has protective effects in EAE. To address this question, B10.PL mice were fed a diet with or without vitamin D(3), immunized with myelin basic protein, and studied for signs of EAE and for metabolites and transcripts of the vitamin D(3) endocrine system. The intact, vitamin D(3)-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 D(3)-fed female mice had significantly more 1,25-dihydroxyvitamin D(3) and fewer CYP24A1 transcripts, encoding the 1,25-dihydroxyvitamin D(3)-inactivating enzyme, in the spinal cord than the other groups of mice. Thus, there was an unexpected synergy between vitamin D(3) 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 D(3) accumulated and resolved the inflammation before severe EAE developed. If humans have a similar gender difference in vitamin D(3) 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.