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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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ADAM17 and EGF pathway members are expressed on non-TEC thymic stromal cells.Enzymatically digested thymi from B6 mice were FACS-sorted based on TEC and fibroblast markers. (A) Adam17 expression on CD45-Ter119-EpCAM+ TEC and CD45-Ter119-EpCAM- non-TEC stroma with Gapdh serving as the internal control. (B) EGF pathway gene expression levels on the indicated populations were calculated relative to levels on TECs with Gapdh serving as the internal control. Data are from 2 independent experiments with each experiment consisting of n≥3 pooled mice. Data report mean + SD. In these experiments, the cell types were identified as follows: TEC: CD45-Ter119-EpCAM+MTS15-; Fibroblast: CD45-Ter119-EpCAM-MTS15+; Non TEC/Fibroblast: CD45-Ter119-EpCAM-MTS15-. TECs were sorted to an average of 92% purity, fibroblasts to 68.73% purity, and non-TEC/fibroblast to 95.84% purity. Despite lower fibroblast purity, notably, no sorted fibroblasts were detected within the TEC gate upon re-analysis (data not shown).
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pone-0013528-g006: ADAM17 and EGF pathway members are expressed on non-TEC thymic stromal cells.Enzymatically digested thymi from B6 mice were FACS-sorted based on TEC and fibroblast markers. (A) Adam17 expression on CD45-Ter119-EpCAM+ TEC and CD45-Ter119-EpCAM- non-TEC stroma with Gapdh serving as the internal control. (B) EGF pathway gene expression levels on the indicated populations were calculated relative to levels on TECs with Gapdh serving as the internal control. Data are from 2 independent experiments with each experiment consisting of n≥3 pooled mice. Data report mean + SD. In these experiments, the cell types were identified as follows: TEC: CD45-Ter119-EpCAM+MTS15-; Fibroblast: CD45-Ter119-EpCAM-MTS15+; Non TEC/Fibroblast: CD45-Ter119-EpCAM-MTS15-. TECs were sorted to an average of 92% purity, fibroblasts to 68.73% purity, and non-TEC/fibroblast to 95.84% purity. Despite lower fibroblast purity, notably, no sorted fibroblasts were detected within the TEC gate upon re-analysis (data not shown).

Mentions: Since Adam17/Foxn1 thymi do not display the T cell developmental block observed in Adam17 conventional knockout mice [25], it is possible that ADAM17 in another thymic stromal cell type is used during T cell development. To assess this possibility, we examined the non-TEC stroma for presence of ADAM17 and EGF family ligands, which have been shown to be key substrates for ADAM17 in vivo. First, to confirm ADAM17 expression on non-TEC stroma, we FACS sorted CD45−Ter119−EpCAM− cells from normal B6 mice. Quantitative PCR revealed levels of Adam17 expression in these non-TECs, at least as high as that of TECs (Figure 6A). We then proceeded to examine more specific stromal cell populations, including CD45−Ter119−EpCAM−MTS15+ thymic fibroblasts and the CD45−Ter119−EpCAM−MTS15− populations containing thymic endothelial cells and other mesenchyme [35] (Figure 5A). Notably, in thymic fibroblasts, we observed levels of TGFα and HB-EGF that were elevated relative to those of TECs, as well as the presence of the EGF receptor (Figure 6B). This indicates that ADAM17 and EGF pathway substrates shown to be critical in other developmental systems are present on the non-TEC populations in the thymus.


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)

ADAM17 and EGF pathway members are expressed on non-TEC thymic stromal cells.Enzymatically digested thymi from B6 mice were FACS-sorted based on TEC and fibroblast markers. (A) Adam17 expression on CD45-Ter119-EpCAM+ TEC and CD45-Ter119-EpCAM- non-TEC stroma with Gapdh serving as the internal control. (B) EGF pathway gene expression levels on the indicated populations were calculated relative to levels on TECs with Gapdh serving as the internal control. Data are from 2 independent experiments with each experiment consisting of n≥3 pooled mice. Data report mean + SD. In these experiments, the cell types were identified as follows: TEC: CD45-Ter119-EpCAM+MTS15-; Fibroblast: CD45-Ter119-EpCAM-MTS15+; Non TEC/Fibroblast: CD45-Ter119-EpCAM-MTS15-. TECs were sorted to an average of 92% purity, fibroblasts to 68.73% purity, and non-TEC/fibroblast to 95.84% purity. Despite lower fibroblast purity, notably, no sorted fibroblasts were detected within the TEC gate upon re-analysis (data not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958126&req=5

pone-0013528-g006: ADAM17 and EGF pathway members are expressed on non-TEC thymic stromal cells.Enzymatically digested thymi from B6 mice were FACS-sorted based on TEC and fibroblast markers. (A) Adam17 expression on CD45-Ter119-EpCAM+ TEC and CD45-Ter119-EpCAM- non-TEC stroma with Gapdh serving as the internal control. (B) EGF pathway gene expression levels on the indicated populations were calculated relative to levels on TECs with Gapdh serving as the internal control. Data are from 2 independent experiments with each experiment consisting of n≥3 pooled mice. Data report mean + SD. In these experiments, the cell types were identified as follows: TEC: CD45-Ter119-EpCAM+MTS15-; Fibroblast: CD45-Ter119-EpCAM-MTS15+; Non TEC/Fibroblast: CD45-Ter119-EpCAM-MTS15-. TECs were sorted to an average of 92% purity, fibroblasts to 68.73% purity, and non-TEC/fibroblast to 95.84% purity. Despite lower fibroblast purity, notably, no sorted fibroblasts were detected within the TEC gate upon re-analysis (data not shown).
Mentions: Since Adam17/Foxn1 thymi do not display the T cell developmental block observed in Adam17 conventional knockout mice [25], it is possible that ADAM17 in another thymic stromal cell type is used during T cell development. To assess this possibility, we examined the non-TEC stroma for presence of ADAM17 and EGF family ligands, which have been shown to be key substrates for ADAM17 in vivo. First, to confirm ADAM17 expression on non-TEC stroma, we FACS sorted CD45−Ter119−EpCAM− cells from normal B6 mice. Quantitative PCR revealed levels of Adam17 expression in these non-TECs, at least as high as that of TECs (Figure 6A). We then proceeded to examine more specific stromal cell populations, including CD45−Ter119−EpCAM−MTS15+ thymic fibroblasts and the CD45−Ter119−EpCAM−MTS15− populations containing thymic endothelial cells and other mesenchyme [35] (Figure 5A). Notably, in thymic fibroblasts, we observed levels of TGFα and HB-EGF that were elevated relative to those of TECs, as well as the presence of the EGF receptor (Figure 6B). This indicates that ADAM17 and EGF pathway substrates shown to be critical in other developmental systems are present on the non-TEC populations in the thymus.

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