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ADAM17 deletion in thymic epithelial cells alters aire expression without affecting T cell developmental progression.

Gravano DM, McLelland BT, Horiuchi K, Manilay JO - PLoS ONE (2010)

Bottom Line: However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult.Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo.The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development remain to be identified.

View Article: PubMed Central - PubMed

Affiliation: School of Natural Sciences, University of California at Merced, Merced, California, United States of America.

ABSTRACT

Background: Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs) are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach.

Methodology/principal findings: We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted.

Conclusions/significance: In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development remain to be identified.

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Aire mRNA expression is reduced in Adam17/Foxn1 EpCAM+ TECs.FACS-sorted CD45-Ter119-EpCAM+ TECs were pooled from 8-week old Control and Adam17/Foxn1 mice (n≥3 for each pool). qRT-PCR for Aire (A) and TRA (B) expression was determined from 3 independent experiments. Rpl7 was used as the internal control and expression levels were determined relative to mean expression in Control mice. Data represent mean + SD; p<0.05 (*), p<0.01 (**), p<0.001 (***). Control: fl/+ or fl/fl; Adam17/Foxn1: Cre fl/fl.
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pone-0013528-g003: Aire mRNA expression is reduced in Adam17/Foxn1 EpCAM+ TECs.FACS-sorted CD45-Ter119-EpCAM+ TECs were pooled from 8-week old Control and Adam17/Foxn1 mice (n≥3 for each pool). qRT-PCR for Aire (A) and TRA (B) expression was determined from 3 independent experiments. Rpl7 was used as the internal control and expression levels were determined relative to mean expression in Control mice. Data represent mean + SD; p<0.05 (*), p<0.01 (**), p<0.001 (***). Control: fl/+ or fl/fl; Adam17/Foxn1: Cre fl/fl.

Mentions: We next examined whether deletion of ADAM17 in TECs affected their development and expression of TEC-specific genes in a cell-autonomous fashion. Analysis of gene expression in FACS-sorted EPCAM+ TECs by qRT-PCR yielded the unexpected finding that the autoimmune regulator transcription factor, Aire, was diminished in Adam17/Foxn1 mice (Figure 3A). Aire mutations have been implicated in the human disease APECED, which may result from defective medullary TEC development, and/or lack of expression of tissue-specific antigens (TRAs) that result in alterations in negative selection of thymocytes (reviewed in [29]). Aire mRNA expression in TECs in Adam17/Foxn1 mice was less than 10% of control mice. TRAs whose expression is dependent or independent of Aire have been previously described [30]. The Aire-dependent TRA, Casein α (Csnα), was significantly reduced in Adam17/Foxn1 mice relative to controls (Figure 3B). However, other Aire-dependent TRAs, Casein γ (Csnγ), Salivary protein 1 (Spt1), and Insulin 2 (Ins2), were not reduced. As expected, Aire-independent TRAs, Casein β (Csnβ), Casein κ (Csnκ), and Glutamic acid decarboxylase-67 (Gad67), were expressed similarly between Adam17/Foxn1 and control mice (Figure 3B).


ADAM17 deletion in thymic epithelial cells alters aire expression without affecting T cell developmental progression.

Gravano DM, McLelland BT, Horiuchi K, Manilay JO - PLoS ONE (2010)

Aire mRNA expression is reduced in Adam17/Foxn1 EpCAM+ TECs.FACS-sorted CD45-Ter119-EpCAM+ TECs were pooled from 8-week old Control and Adam17/Foxn1 mice (n≥3 for each pool). qRT-PCR for Aire (A) and TRA (B) expression was determined from 3 independent experiments. Rpl7 was used as the internal control and expression levels were determined relative to mean expression in Control mice. Data represent mean + SD; p<0.05 (*), p<0.01 (**), p<0.001 (***). Control: fl/+ or fl/fl; Adam17/Foxn1: Cre fl/fl.
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Related In: Results  -  Collection

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pone-0013528-g003: Aire mRNA expression is reduced in Adam17/Foxn1 EpCAM+ TECs.FACS-sorted CD45-Ter119-EpCAM+ TECs were pooled from 8-week old Control and Adam17/Foxn1 mice (n≥3 for each pool). qRT-PCR for Aire (A) and TRA (B) expression was determined from 3 independent experiments. Rpl7 was used as the internal control and expression levels were determined relative to mean expression in Control mice. Data represent mean + SD; p<0.05 (*), p<0.01 (**), p<0.001 (***). Control: fl/+ or fl/fl; Adam17/Foxn1: Cre fl/fl.
Mentions: We next examined whether deletion of ADAM17 in TECs affected their development and expression of TEC-specific genes in a cell-autonomous fashion. Analysis of gene expression in FACS-sorted EPCAM+ TECs by qRT-PCR yielded the unexpected finding that the autoimmune regulator transcription factor, Aire, was diminished in Adam17/Foxn1 mice (Figure 3A). Aire mutations have been implicated in the human disease APECED, which may result from defective medullary TEC development, and/or lack of expression of tissue-specific antigens (TRAs) that result in alterations in negative selection of thymocytes (reviewed in [29]). Aire mRNA expression in TECs in Adam17/Foxn1 mice was less than 10% of control mice. TRAs whose expression is dependent or independent of Aire have been previously described [30]. The Aire-dependent TRA, Casein α (Csnα), was significantly reduced in Adam17/Foxn1 mice relative to controls (Figure 3B). However, other Aire-dependent TRAs, Casein γ (Csnγ), Salivary protein 1 (Spt1), and Insulin 2 (Ins2), were not reduced. As expected, Aire-independent TRAs, Casein β (Csnβ), Casein κ (Csnκ), and Glutamic acid decarboxylase-67 (Gad67), were expressed similarly between Adam17/Foxn1 and control mice (Figure 3B).

Bottom Line: However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult.Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo.The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development remain to be identified.

View Article: PubMed Central - PubMed

Affiliation: School of Natural Sciences, University of California at Merced, Merced, California, United States of America.

ABSTRACT

Background: Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs) are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach.

Methodology/principal findings: We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted.

Conclusions/significance: In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development remain to be identified.

Show MeSH
Related in: MedlinePlus