Fig. 6 |. TNF and IL-17A induce IL-33 in adipose stromal cells.

a, IL-33 protein from SVF eWAT lysates of WT male mice 5–8, 15–16, and 20+ weeks of age, determined by enzyme-linked immunosorbent assay (ELISA; n = 4 mice per time point) and normalized to total SVF protein. b, IL-33 (left) and IL-2 (right) protein from SVF eWAT and splenic lysates of 14-week-old WT and Tcrd^−/− male mice (n ≥ 3, pooled). c, IL-33 protein from SVF eWAT lysates of 16-week-old WT, Vg4/6^−/−, and Il17a^−/− male mice (n ≥ 4), normalized to total SVF protein. d, Representative flow cytometry plots for stromal cell subsets in the eWAT SVF in male WT mice. e, Cell-surface IL-17R, CD26, CD9, and Cdh11 MFI of CD45⁻Pdpn^hiPDGFRα⁻ and CD45⁻Pdpn^loPDGFRα⁺ stromal cells. f, Quantitative real-time PCR for Il33 mRNA normalized to Tbp mRNA from sorted Pdpn^hi, PDGFRα⁺, CD31⁺, and CD45⁺ cells from WT mice (n = 6). g, 3T3L1 or primary adipose stromal cells derived from eWAT SVF were unstimulated (unstim) or stimulated with TNF^lo (0.1 ng/mL), TNF (1 ng/mL), IL-17A^lo (0.1 ng/mL), IL-17A (1 ng/mL), or TNF (1 ng/mL) + IL-17A (1 ng/mL) for 18 h. Il33 transcript levels were measured with quantitative real-time PCR (left), and protein levels were measured with ELISA (right). h, Primary human stromal cells derived from visceral (Lonza) and subcutaneous (ATCC) adipose tissues were unstimulated (unstim) or stimulated with TNF^lo (0.1 ng/mL), TNF (1 ng/mL), IL-17A^lo (0.1 ng/mL), IL-17A (1 ng/mL), or TNF (1 ng/mL) + IL-17A (1 ng/mL) for 18 h. Cell lysates were collected, and IL-33 protein was measured with ELISA. In plots, each symbol represents an individual mouse; small horizontal lines indicate the mean. NS, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way ANOVA in a–c,f–h). Data are pooled across three experiments (a–c; mean ± s.e.m.) or representative of two experiments (d–h; mean ± s.e.m. in f–h).