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Modulation of Aire regulates the expression of tissue-restricted antigens.

Kont V, Laan M, Kisand K, Merits A, Scott HS, Peterson P - Mol. Immunol. (2007)

Bottom Line: Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes.By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern.The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology, Biomedicum, Tartu University, Ravila 19, 50411 Tartu, Estonia.

ABSTRACT
Intrathymic expression of tissue-restricted antigens (TRAs) has been viewed as the key element in the induction of central tolerance and recently, a central role for the autoimmune regulator (Aire) has been suggested in this process. The aim of this study was to establish whether down or up-regulation of Aire leads to alterations in TRA expression and whether this is limited to thymic epithelial cells. This study also characterized whether TRAs follow Aire expression during normal development, and whether thymic microenvironment plays a role in the expression of Aire and TRAs. We did several in vivo and in vitro experiments to manipulate Aire expression and measured expression of four TRAs (Trefoil factor-3, Insulin-2, Major urinary protein-1 and Salivary protein-1) by real-time RT-PCR. Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes. In the thymus, Aire and TRAs were both localized in the medulla and were co-expressed during normal development and involution. In the primary stromal cells as well as thymic epithelial cell line, the adenoviral over-expression of Aire resulted in an increase in TRA expression. By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern. The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity.

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Dose-dependent effect of Aire on TRA expression in C57Bl/6 and Balb/c mice. Four to six weeks old C57Bl/6 or Balb/c mice were genotyped by PCR (A) and whole thymuses from WT, Aire HET and Aire KO mice were analyzed for TRA gene expression by real-time PCR. TRA expression followed the expression of Aire in a dose-dependent manner in C57Bl/6 (B) as well as Balb/c (C) mice. Data are mean with S.E.M. of triplicate measurements of one out of two representative experiments.
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fig1: Dose-dependent effect of Aire on TRA expression in C57Bl/6 and Balb/c mice. Four to six weeks old C57Bl/6 or Balb/c mice were genotyped by PCR (A) and whole thymuses from WT, Aire HET and Aire KO mice were analyzed for TRA gene expression by real-time PCR. TRA expression followed the expression of Aire in a dose-dependent manner in C57Bl/6 (B) as well as Balb/c (C) mice. Data are mean with S.E.M. of triplicate measurements of one out of two representative experiments.

Mentions: Aire deficient mice (C57BL/6J and Balb/c background) were generated at The Walter and Eliza Hall Institute (Melbourne, Australia). The inserted targeting construct containing LacZ gene replaced mouse Aire exon 8. For genotyping, the genomic DNA was extracted using JetQuick Tissue DNA Spin Kit (Genomed), and wild-type (WT) and knockout (KO) alleles were amplified using primers: 1042 5′-cagaagaacgaggat-3′, 1045 5′-cagactgccttggga-3′ or 1043 5′-ctgtcttctgtgaaggcttctagg-3′. As shown in Fig. 1A, primers pair 1042/1043 and 1043/1045 detect WT and KO alleles, respectively. Thymuses from 4- to 6-week-old WT, Aire HET (heterozygote) and Aire KO mice were used. Embryonic (E13.5, E15.5 and E17.5), newborn, neonatal D11 and adult (6 weeks, 6 months and 12 months) mouse tissues were used in developmental dynamics analysis. Mice were maintained at the mouse facility of the Institute of Molecular and Cell Biology, Tartu University. TEC 1C6 cell line (Mizuochi et al., 1992) was kindly provided by G. Holländer (University of Basel, Switzerland). Human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B (Gibco BRL).


Modulation of Aire regulates the expression of tissue-restricted antigens.

Kont V, Laan M, Kisand K, Merits A, Scott HS, Peterson P - Mol. Immunol. (2007)

Dose-dependent effect of Aire on TRA expression in C57Bl/6 and Balb/c mice. Four to six weeks old C57Bl/6 or Balb/c mice were genotyped by PCR (A) and whole thymuses from WT, Aire HET and Aire KO mice were analyzed for TRA gene expression by real-time PCR. TRA expression followed the expression of Aire in a dose-dependent manner in C57Bl/6 (B) as well as Balb/c (C) mice. Data are mean with S.E.M. of triplicate measurements of one out of two representative experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1994210&req=5

fig1: Dose-dependent effect of Aire on TRA expression in C57Bl/6 and Balb/c mice. Four to six weeks old C57Bl/6 or Balb/c mice were genotyped by PCR (A) and whole thymuses from WT, Aire HET and Aire KO mice were analyzed for TRA gene expression by real-time PCR. TRA expression followed the expression of Aire in a dose-dependent manner in C57Bl/6 (B) as well as Balb/c (C) mice. Data are mean with S.E.M. of triplicate measurements of one out of two representative experiments.
Mentions: Aire deficient mice (C57BL/6J and Balb/c background) were generated at The Walter and Eliza Hall Institute (Melbourne, Australia). The inserted targeting construct containing LacZ gene replaced mouse Aire exon 8. For genotyping, the genomic DNA was extracted using JetQuick Tissue DNA Spin Kit (Genomed), and wild-type (WT) and knockout (KO) alleles were amplified using primers: 1042 5′-cagaagaacgaggat-3′, 1045 5′-cagactgccttggga-3′ or 1043 5′-ctgtcttctgtgaaggcttctagg-3′. As shown in Fig. 1A, primers pair 1042/1043 and 1043/1045 detect WT and KO alleles, respectively. Thymuses from 4- to 6-week-old WT, Aire HET (heterozygote) and Aire KO mice were used. Embryonic (E13.5, E15.5 and E17.5), newborn, neonatal D11 and adult (6 weeks, 6 months and 12 months) mouse tissues were used in developmental dynamics analysis. Mice were maintained at the mouse facility of the Institute of Molecular and Cell Biology, Tartu University. TEC 1C6 cell line (Mizuochi et al., 1992) was kindly provided by G. Holländer (University of Basel, Switzerland). Human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B (Gibco BRL).

Bottom Line: Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes.By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern.The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology, Biomedicum, Tartu University, Ravila 19, 50411 Tartu, Estonia.

ABSTRACT
Intrathymic expression of tissue-restricted antigens (TRAs) has been viewed as the key element in the induction of central tolerance and recently, a central role for the autoimmune regulator (Aire) has been suggested in this process. The aim of this study was to establish whether down or up-regulation of Aire leads to alterations in TRA expression and whether this is limited to thymic epithelial cells. This study also characterized whether TRAs follow Aire expression during normal development, and whether thymic microenvironment plays a role in the expression of Aire and TRAs. We did several in vivo and in vitro experiments to manipulate Aire expression and measured expression of four TRAs (Trefoil factor-3, Insulin-2, Major urinary protein-1 and Salivary protein-1) by real-time RT-PCR. Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes. In the thymus, Aire and TRAs were both localized in the medulla and were co-expressed during normal development and involution. In the primary stromal cells as well as thymic epithelial cell line, the adenoviral over-expression of Aire resulted in an increase in TRA expression. By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern. The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity.

Show MeSH
Related in: MedlinePlus