Figure 4.

Systemic IFN signaling in Regnase-3^−/− mice. (A) CD19⁺ B cells were isolated from enlarged lymph nodes of Regnase-3^−/− mice and their Regnase-3^+/+ littermates (n = 3/3), and RNA was isolated and subjected to RNA sequencing. Heatmap of RNA sequencing data for all significantly up-regulated (≥2 log2 fold) genes in Regnase-3^−/− B cells is shown. GO term association to “response to IFNβ” and “response to IFNγ” is indicated for each gene. (B) Serum cytokines in Regnase-3^−/− mice and Regnase-3^+/+ littermates at 6 mo of age (n = 8/8), assessed by multiplex assay. (C) Stat1 mRNA expression in tissues from Regnase-3^+/+ and Regnase-3^−/− mice at 8 mo of age, assessed by quantitative RT-PCR, normalized to Hprt relative (rel.) to their expression in Regnase-3^+/+ mice (n = 5/5). (D) Left: Immunohistochemical analysis of MHC-II in liver sections of Regnase-3^−/− mice with lymphadenopathy and Regnase-3^+/+ controls (representative images). Magnification of images is indicated in brackets. Bars, 250 µm. Right: Frequency of strong positive pixels in MHC-II immunohistochemical sections of lung, kidney, and liver were determined by Definiens software (n = 6 Regnase-3^−/− mice and 6 Regnase-3^+/+ littermate controls). (E) Frequency of MHC-II–positive macrophages (CD68⁺) of all CD68⁺ macrophages in the lung of Regnase-3^−/− mice with lymphadenopathy and their Regnase-3^+/+ littermate controls, assessed in immunohistochemical, consecutive sections (n = 5/6). (F) Left: Immunohistochemical analysis of pSTAT1 in skin-draining lymph nodes from Regnase-3^−/− mice with lymphadenopathy and Regnase-3^+/+ littermate controls (representative images). Right: Frequency of strong positive pixels in pSTAT1 immunohistochemical sections of lymph nodes, determined by Definiens software (n = 6 Regnase-3^−/− mice and 6 Regnase-3^+/+ littermate controls). Bars, 500 µm. Data are represented as mean ± SEM and were compared by Mann–Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant).