Journal: Science immunology

Article Title: Aire drives steroid hormone biosynthesis by medullary thymic epithelial cells

doi: 10.1126/sciimmunol.abo7975

Figure Lengend Snippet: (A) Gene expression of glucocorticoid biosynthetic enzyme genes in mTEC cells of WT and Aire−/− mice. Bulk RNA-seq data from Aire−/− mTEC are shown for each gene relative to WT expression of the same gene. (B) Gene expression of glucocorticoid biosynthetic enzyme genes in mTEC cells of WT and Fezf2−/− mice. Bulk RNA-seq data from Fezf2−/− mTEC are shown for each gene relative to WT expression of the same gene. (C) Expression of glucocorticoid biosynthetic enzyme genes in bulk mTEC subset samples of WT and Aire−/− mice. The expression level of each gene is presented as a proportion of its maximal expression (1.0) across analyzed cell types as indicated by the color scale. (D) Single-cell transcriptomes obtained from deep sequencing of 174 mTECs were analyzed for the coincident expression of glucocorticoid synthesis genes (Star, Cyp11a1, Hsd3b6, Cyp21a1, and Cyp11b1) with each other or with Aire- and Fezf2-dependent TRA genes (Ins1, Spt1, Mup4, S100a8, Resp18, Rabp9, Krt10, and Maoa) in the same cell (see Materials and Methods for calculation details). Data are presented as means ± SEM. (E) Thymi from 2-week-old WT, KI, and KI Aire−/− mice were digested and cells analyzed by flow cytometry. Representative contour plots show each subset, and numbers indicate the percentage of cells within the Cyp11b1mScarlet+ gate. (F) Data collected and analyzed as in (E), pooled from two experiments with 2- and 3-week-old mice, presented as means ± SEM. Data in (E) and (F) are representative or pooled from two independent experiments with N = 7 WT, 9 KI, and 7 KI Aire−/− mice. (G) WT, KI, and KI Aire−/− thymi from 1-week-old mice were fixed, sectioned, and stained for UEA-1 (mTEC, FITC) and Cyp11b1mScarlet (Atto 565). Confocal images were taken at ×60 oil immersion magnification and are representative of two independent experiments. Scale bars, 100 μm. (H) Total RNAwas extracted from whole thymus lobes of WT and Aire−/− mice, and RT-qPCR was used to quantify Cyp11b1 gene expression. Expression was normalized to 18S RNA and is shown relative to WT expression. Data are pooled from four independent experiments, shown as means ± SEM with N = 17 WT and 9 Aire−/− thymi. (I) Thymus lobes were dissected, minced, and cultured for 72 hours in steroid-free medium. Supernatants were collected, and corticosterone was quantified by a commercial ELISA kit. Data are pooled from four independent experiments, with N = 19 WT and 11 Aire−/− thymi. Statistical significance in (D), (H), and (I) was determined by one-tailed t tests and that in (F) was determined by a mixed linear model with post hoc unpaired t tests applying the Holm-Šidák correction for multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant.

Article Snippet: Steroids were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (corticosterone, Arbor Assays, K014; progesterone, Cayman Chemical, 582601; testosterone, Cayman Chemical, 582701; estradiol, Cayman Chemical, 501890) according to the manufacturer’s protocol, with standards diluted in steroid-free culture medium.

Techniques: Gene Expression, RNA Sequencing, Expressing, Sequencing, Flow Cytometry, Staining, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Science immunology

Article Title: Aire drives steroid hormone biosynthesis by medullary thymic epithelial cells

doi: 10.1126/sciimmunol.abo7975

Figure Lengend Snippet: (A) Single-cell RNA-seq analysis of mTEC expression of androgen (Hsd17b3) and estrogen (Cyp19a1) biosynthetic enzymes, with relative expression, as indicated by the color scale, mapped onto a UMAP plot. Numbered populations indicate different cTEC and mTEC cell populations as defined in fig. S4. (B) mTEC specificity and Aire dependence of gene expression in biosynthetic pathways of progestins, corticosteroids (glucocorticoids and mineralocorticoids), androgens, and estrogens. Relative gene expression in bulk Aire+ versus Aire−/− mTEC samples is presented to show Aire-dependent expression, and relative expression in mTECs versus cTECs is presented to show mTEC specificity. Each point represents one gene in the biosynthetic pathway, and the gray box highlights genes with at least twofold higher expression in mTECs versus cTECs and twofold higher expression in Aire+ vs Aire−/− mTEC. A large pink point at the top right represents two or more genes. (C) Total RNAwas extracted from whole thymus lobes of WT and Aire−/− mice, and RT-qPCR was used to quantify gene expression. Ins2 and Spt1 are Aire-dependent TRA genes, and others encode steroidogenic enzymes. Expression was normalized to 18S RNA and is shown relative to WT expression. Data are pooled from four independent experiments, shown as means ± SEM with N = 17 WT and 9 Aire−/− thymi. (D) Thymus lobes were dissected, minced, and cultured for 72 hours in steroid-free medium. Supernatants were collected, and steroids were quantified by commercial ELISA kits. Data were pooled from four independent experiments, with N = 19 WT and 11 Aire−/− thymi. (E) mTEC specificity and Aire dependence of enzyme gene expression in biosynthetic pathways of lipid signaling molecules (prostaglandins, retinoic acid, and thyroid hormone) and amino acid–derived molecules (catecholamines). Relative expression in bulk Aire+ versus Aire−/− mTEC samples is presented to show Aire-dependent expression, and relative expression in mTECs versus cTECs is presented to show mTEC specificity. The gray box highlights genes with at least a twofold expression difference on both axes. Statistical significance in (C) was determined using unpaired t tests with the family-wise error rate set to 0.05 and determined using the correction of Benjamini, Krieger, and Yekutieli and in (D) using one-tailed t tests. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant.

Article Snippet: Steroids were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (corticosterone, Arbor Assays, K014; progesterone, Cayman Chemical, 582601; testosterone, Cayman Chemical, 582701; estradiol, Cayman Chemical, 501890) according to the manufacturer’s protocol, with standards diluted in steroid-free culture medium.

Techniques: RNA Sequencing, Expressing, Gene Expression, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Derivative Assay, One-tailed Test