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RNA microarray of sorted dendritic epidermal T cells of aryl hydrocarbon receptor deficient mice

ABSTRACT: To assess the role of the aryl hydrocarbon receptor (AHR) receptor in dendritic epidermal T cells (DETC), we sorted DETC from 2 weeks old mice homozygous and heterozygous for AHR-knockout. While DETC are not maintained in the epidermis of mice with a homozygous AHR-knockout, those in heterozygous mice devellop normally. The age at 2 weeks is critical for the DETC establishment and the peak time of the so-called proliferation burst of DETC in wildtype mice. DETC were identified in epidermal cell suspension by expression of the gamma-delta T cell receptor. The DETC proportion of live epidermal cells was between 10-15 % in Ahr-het and 2-4 % in Ahr-ko mice. After FACS-sorting to a purity of 90-98 %, DETC were lysed and their RNA was extracted. Three RNA samples for each genotype were generated, by pooling the RNA of 2-3 mice for each sample. RNA was processed and hybridized to Applied BiosystemsTM ClariomTM S Mouse Gene Expression Microarrays. Using the Software package R the data were normalized using the Robust Multichip Average algorithm (RMA) and significance of differentially regulated genes was assessed by the False Discovery Rate (FDR) using the Benjamini and Hochberg’s method.

ORGANISM(S): Mus musculus

PROVIDER: GSE142437 | GEO | 2020/04/10

REPOSITORIES: GEO

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Project description:Dendritic epidermal T cells (DETC) reside in murine skin and participate in homeostasis and wound repair. Upon wounding, DETC become activated through the recognition of an unidentified ligand expressed by keratinocytes proximal to sites of injury. Such DETC activation is mediated through a monoclonal T cell receptor (TCR). Using a soluble form of this monoclonal TCR, we have shown that keratinocytes upregulate DETC TCR ligands in wounded tissue within 2 hours following wounding. Down-modulation of the ligand is seen 3 hours following wounding, and no expression is evident in non-wounded skin. In vitro studies on cell lines which express this unknown ligand indicate that antigen recognition by the DETC TCR is dependent upon N-linked glycosylation of the ligand. Given the glycosylation sensitivity of the ligand and the restricted expression following wounding, we are interested in pursuing microarray analysis to identify genes that are modulated in keratinocytes in response to wounding. Keratinocytes represent 90% of the cells in the epidermis (DETC and Langerhan’s cells make up the remaining 10%). As such, we propose to isolate RNA from whole epidermis under either wounded or resting conditions. In addition to comparing RNA from wounded and non-wounded epidermis, we would like to compare RNA from tissue that has been wounded for different times. Initially, we would like to analyze 4 sampes (non-wounded epidermis, and epidermal cells isolated 30 minutes, 2 hours, and 4 hours following wounding). These time points would correlate to a period prior to cell surface expression of ligand (30 minutes), during cell surface expression (2 hours), and following down regulation of cell surface expression (4 hours). In addition to providing possible identification of the unknown DETC TCR ligand, such analysis would provide novel information about early responses by keratinocytes in response to physical wounding in vivo. We propose to isolate RNA from whole epidermis under either wounded or resting conditions. In addition to comparing RNA from wounded and non-wounded epidermis, we would like to compare RNA from tissues that has been wounded for different times.

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RNA microarray of sorted dendritic epidermal T cells of aryl hydrocarbon receptor deficient mice

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