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DQ8 Double Transgenic Mice through Pro-inflammatory Cytokine IL-17
Abstracts disease and cause severe complications. We need to develop and use effective immunotherapeutic agents, such as a fusion protein consisting of gelonin and acetylcholine receptor fragment. doi:10.1016/j.clim.2008.03.142
F.31. Bacteroides fragilis Regulates the Induction of FoxP3+CD4+CD25+ Treg Cell Populations in Peripheral Lymph Nodes of Mice with Microflora-depleted Guts Javier Ochoa-Repáraz, Daniel Mielcarz, Lauren Ditrio, Lloyd Kasper. Dartmouth Medical School, Lebanon, NH The role of commensal microorganisms in the immune balance and development of autoimmune diseases such as MS remains unknown. Recent studies demonstrated that peripheral tolerance induced in the gut might be a powerful tool to control experimental autoimmune encephalomyelitis (EAE), suggesting that gut bacteria might be critical to the maintenance of immune homeostasis. Previous works have shown that the polysaccharide A (PSA) of Bacteroides fragilis, one of the most prominent microorganisms of the mammalian gut, is able to induce the activation and proliferation of CD4+ T cells controlling the Th1/Th2 balance into physiological conditions. The effect of B. fragilis and PSA in the regulation of EAE was analyzed. The induction of regulatory FoxP3+CD4+CD25+ Treg cells by B. fragilis expressing PSA and by a PSA-deficient B. fragilis strain was quantified in mice previously treated with anti-microfloral antibiotics. Results show that previous depletion of gut microorganisms provoked a significant reduction of FoxP3 expression in Treg cells of lymph nodes when compared to normal mice. Colonization with B. fragilis producing PSA or not, increased significantly Treg cells percentages and their FoxP3 expression three days post-colonization. Levels remained enhanced two weeks after colonization. By week three, levels were reduced. These increases were particularly significant in animals with depleted microflora. The role of the colonization of guts with Bacteroides and the role of B. fragilis induced-Treg cells in the protection against EAE was tested in C57BL/6 and SJL mice. Our results suggest a role of tolerance induced by commensal components in the control of EAE. doi:10.1016/j.clim.2008.03.143
F.32. HLA-DQ8 (DQB1*0302) Molecule Modulate PLP91-110 Induced EAE in HLA-DR3 (DRB1*0301)/DQ8 Double Transgenic Mice through Pro-inflammatory Cytokine IL-17 Ashutosh Mangalam, Eati Basal, David Luckey, Moses Rodriguez, Chella David. Mayo Clinic, Rochester, MN Multiple sclerosis (MS), a demyelinating disease of the CNS, shows a strong association with certain MHC class II genes such as HLA-DR2, DR3, DR4, DQ6 and DQ8. The role of DR and DQ in MS has been difficult to assess in past due to
S53 heterozygosity in patients populations and linkage disequilibrium between MS associated HLA-DR and -DQ genes. Several studies in MS have raised the question of whether it is a single allele, a combination of two or a complex haplotype that confers an increased risk for disease susceptibility. We have generated double transgenic (Tg) mice expressing both HLA-DQ8 and DR3 (DQ8/DR3.Abo) in order to investigate the role of DQ8 gene on disease susceptible DR3 Tg mice. We have previously shown that PLP91-110 can induce EAE in DR3 (DRB1*0301) but not in DQ8 (DQB1*0302) Tg mice. We observed that the introduction of DQ8 on disease susceptible DR3 Tg mice caused increase in disease incidence as well as in disease severity as compared to DR3.Abo single Tg mice, suggesting a synergistic effect of DQ8 on the development of EAE. We further characterized that the increased susceptibility in DQ8/DR3 Tg mice was due to increased production of pro-inflammatory cytokine IL-17 and GM-CSF by DQ8 specific T cells. Higher IL-17 levels might lead to activation and recruitment of more inflammatory cells inside CNS, while GM-CSF has been known to cause increased antigen presentation inside CNS. These double transgenic mice (HLA-DQ8/DR3) with EAE also showed increased inflammation and demyelination in CNS tissue as compared to single DR3 transgenic mice. Thus our double Tg mouse model provides a novel tool to study gene complementation between different class II genes and suggests that while presence of certain DR gene(s) predispose an individual to MS, polymorphism in DQ gene(s) might play a modulating role. doi:10.1016/j.clim.2008.03.144
F.33. Genetic Variants that Control the Expression of MHC Genes Do Not Affect Susceptibility to Multiple Sclerosis Linda Ottoboni,1 Erica Young,1 Roman Yelensky,2 David Hafler,1 Mark Daly,2 Philip De Jager.1 1Brigham and Women's Hospital, Boston, MA; 2Massachusetts General Hospital, Boston, MA We have screened 4.4 Mb of the major histocompatability complex (MHC) for single nucleotide polymorphisms (SNPs) controlling the RNA expression of genes found within this chromosomal segment in 270 human lymphoblastic cell lines of the HapMap. We have found that 7 genes have at least 2 SNPs with a P-value b 10^-8 for exprerssion of a nearby gene in one of the three HapMap populations (African, East Asian, or European): BTN3A2, CCHCR1, HLA A, HLA DQA1, HLA DRB5, HLA F and ZNRD1. For each of these genes, its expression is controlled by a single major polymorphism, with little residual association once the primary effect is accounted for by logistic regression. None of these seven SNPs have an effect on a second gene within our selected gene set. We have gone on to validate the association of the HLA DQA1 variant, demonstrating association of HLA DQA1 RNA expression at a genome-wide level in another set of human lymphoblastic cell lines as well as association to the level of HLA DQ expression on the cell surface of the HapMap cell lines. Finally, we have assessed these seven alleles for evidence of an effect on susceptibility to multiple sclerosis
F.31. Bacteroides fragilis Regulates the Induction of FoxP3+CD4+CD25+ Treg Cell Populations in Peripheral Lymph Nodes of Mice with Microflora-depleted Guts Javier Ochoa-Repáraz, Daniel Mielcarz, Lauren Ditrio, Lloyd Kasper. Dartmouth Medical School, Lebanon, NH The role of commensal microorganisms in the immune balance and development of autoimmune diseases such as MS remains unknown. Recent studies demonstrated that peripheral tolerance induced in the gut might be a powerful tool to control experimental autoimmune encephalomyelitis (EAE), suggesting that gut bacteria might be critical to the maintenance of immune homeostasis. Previous works have shown that the polysaccharide A (PSA) of Bacteroides fragilis, one of the most prominent microorganisms of the mammalian gut, is able to induce the activation and proliferation of CD4+ T cells controlling the Th1/Th2 balance into physiological conditions. The effect of B. fragilis and PSA in the regulation of EAE was analyzed. The induction of regulatory FoxP3+CD4+CD25+ Treg cells by B. fragilis expressing PSA and by a PSA-deficient B. fragilis strain was quantified in mice previously treated with anti-microfloral antibiotics. Results show that previous depletion of gut microorganisms provoked a significant reduction of FoxP3 expression in Treg cells of lymph nodes when compared to normal mice. Colonization with B. fragilis producing PSA or not, increased significantly Treg cells percentages and their FoxP3 expression three days post-colonization. Levels remained enhanced two weeks after colonization. By week three, levels were reduced. These increases were particularly significant in animals with depleted microflora. The role of the colonization of guts with Bacteroides and the role of B. fragilis induced-Treg cells in the protection against EAE was tested in C57BL/6 and SJL mice. Our results suggest a role of tolerance induced by commensal components in the control of EAE. doi:10.1016/j.clim.2008.03.143
F.32. HLA-DQ8 (DQB1*0302) Molecule Modulate PLP91-110 Induced EAE in HLA-DR3 (DRB1*0301)/DQ8 Double Transgenic Mice through Pro-inflammatory Cytokine IL-17 Ashutosh Mangalam, Eati Basal, David Luckey, Moses Rodriguez, Chella David. Mayo Clinic, Rochester, MN Multiple sclerosis (MS), a demyelinating disease of the CNS, shows a strong association with certain MHC class II genes such as HLA-DR2, DR3, DR4, DQ6 and DQ8. The role of DR and DQ in MS has been difficult to assess in past due to
S53 heterozygosity in patients populations and linkage disequilibrium between MS associated HLA-DR and -DQ genes. Several studies in MS have raised the question of whether it is a single allele, a combination of two or a complex haplotype that confers an increased risk for disease susceptibility. We have generated double transgenic (Tg) mice expressing both HLA-DQ8 and DR3 (DQ8/DR3.Abo) in order to investigate the role of DQ8 gene on disease susceptible DR3 Tg mice. We have previously shown that PLP91-110 can induce EAE in DR3 (DRB1*0301) but not in DQ8 (DQB1*0302) Tg mice. We observed that the introduction of DQ8 on disease susceptible DR3 Tg mice caused increase in disease incidence as well as in disease severity as compared to DR3.Abo single Tg mice, suggesting a synergistic effect of DQ8 on the development of EAE. We further characterized that the increased susceptibility in DQ8/DR3 Tg mice was due to increased production of pro-inflammatory cytokine IL-17 and GM-CSF by DQ8 specific T cells. Higher IL-17 levels might lead to activation and recruitment of more inflammatory cells inside CNS, while GM-CSF has been known to cause increased antigen presentation inside CNS. These double transgenic mice (HLA-DQ8/DR3) with EAE also showed increased inflammation and demyelination in CNS tissue as compared to single DR3 transgenic mice. Thus our double Tg mouse model provides a novel tool to study gene complementation between different class II genes and suggests that while presence of certain DR gene(s) predispose an individual to MS, polymorphism in DQ gene(s) might play a modulating role. doi:10.1016/j.clim.2008.03.144
F.33. Genetic Variants that Control the Expression of MHC Genes Do Not Affect Susceptibility to Multiple Sclerosis Linda Ottoboni,1 Erica Young,1 Roman Yelensky,2 David Hafler,1 Mark Daly,2 Philip De Jager.1 1Brigham and Women's Hospital, Boston, MA; 2Massachusetts General Hospital, Boston, MA We have screened 4.4 Mb of the major histocompatability complex (MHC) for single nucleotide polymorphisms (SNPs) controlling the RNA expression of genes found within this chromosomal segment in 270 human lymphoblastic cell lines of the HapMap. We have found that 7 genes have at least 2 SNPs with a P-value b 10^-8 for exprerssion of a nearby gene in one of the three HapMap populations (African, East Asian, or European): BTN3A2, CCHCR1, HLA A, HLA DQA1, HLA DRB5, HLA F and ZNRD1. For each of these genes, its expression is controlled by a single major polymorphism, with little residual association once the primary effect is accounted for by logistic regression. None of these seven SNPs have an effect on a second gene within our selected gene set. We have gone on to validate the association of the HLA DQA1 variant, demonstrating association of HLA DQA1 RNA expression at a genome-wide level in another set of human lymphoblastic cell lines as well as association to the level of HLA DQ expression on the cell surface of the HapMap cell lines. Finally, we have assessed these seven alleles for evidence of an effect on susceptibility to multiple sclerosis