Panel A shows a schematic diagram illustrating the generation of Ifnlr1 f/f; Col1a2-Cre mice through tamoxifen-induced fibroblast-specific deletion of Ifnlr1. Panel B shows flow cytometry plots and bar graphs with the y-axis representing Vimentin positive cells (percent) or Ly6G positive CD11b positive cells (percent) and the x-axis representing Ifnlr1 f/f and Ifnlr1 f/f; Col1a2-Cre groups before and after purification. Panel C shows immunoblot images displaying phosphorylated STAT1, STAT1, and GAPDH protein levels in renal fibroblasts, renal TECs, and neutrophils treated with phosphate-buffered saline or IFN-lambda2. Panel D shows bar graphs with the y-axis representing Relative mRNA levels (fold change) and the x-axis representing Ifnlr1 f/f plus phosphate-buffered saline, Ifnlr1 f/f plus IFN-lambda2, Ifnlr1 f/f; Col1a2-Cre plus phosphate-buffered saline, and Ifnlr1 f/f; Col1a2-Cre plus IFN-lambda2 groups for renal fibroblasts, renal TECs, and neutrophils.
Generation and validation of mice with conditional Ifnlr1 deletion in fibroblasts. (A) Strategy for generating mice with fibroblast-specific Ifnlr1 deletion (designated Ifnlr1f/f; Col1a2-Cre). (B) Representative flow cytometry plots demonstrating the successful enrichment of renal vimentin+ fibroblasts and splenic Ly6G+ CD11b+ neutrophils from Ifnlr1f/f mice and Ifnlr1f/f; Col1a2-Cre mice. Cells were gated on forward and side scatters, then single cell, live cells, and finally on vimentin+ fibroblasts or Ly6G+ CD11b+ neutrophils were gated (n = 3). (C) Primary renal fibroblasts, renal TECs, and splenic neutrophils isolated from Ifnlr1f/f mice and Ifnlr1f/f; Col1a2-Cre mice were treated with 100 ng/ml of IFN-λ2 or PBS for 1 h. Western blot was used to assess STAT1 expression and phosphorylation. GAPDH was used as a loading control. (D) After 24 h of IFN-λ2 or PBS exposure, RT-qPCR was used to determine the levels of Isg15 and Mx1 mRNA in primary renal fibroblasts, renal TECs, and neutrophils from Ifnlr1f/f and Ifnlr1f/f; Col1a2-Cre mice. n = 4 biologically independent samples per group. Data are representative of two independent experiments. Data are shown as mean ± SEM. ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test. ns, no significant difference. Source data are available for this figure: SourceData FS3.