Figure 1: Socs1^−/−Rag2^−/− mice develop spontaneous colitis.

(a) The survival curves of Socs1^−/− (black, n=5), Socs1^−/−Tcra^−/− (red, n=14), Socs1^−/−Rag2^−/− (blue, n=12), Socs1-KOTg (green, n=6) and Ifng^−/−Socs1^−/− (green, dotted, n=10) mice. (b) Rag2^−/− and Socs1^−/−Rag2^−/− mice (8-week-old; upper panel). Representative macroscopic observations of the colons of 9-week-old Rag2^−/− and Socs1^−/−Rag2^−/− mice (lower panel). (c) Representative histopathology of the colons of Rag2^−/− (upper left panel) and Socs1^−/−Rag2^−/− mice (other five panels) at 9 weeks of age stained with haematoxylin and eosin. Scale bar, 100 μm. (d) The histological scores of colitis for Rag2^−/− (n=9) and Socs1^−/−Rag2^−/− (n=8) mice at 9 weeks of age. The horizontal lines indicate the mean values. (e) Flow cytometric analyses showing the proportions of CD11b^high and natural killer (NK) (DX5⁺) cells among CD45⁺TER119⁻ cells (upper), Gr-1⁺ and F4/80⁺ cells among CD11b⁺ cells (middle) and CD11c⁺ and F4/80⁺ cells among CD11b⁺ cells (lower) in the spleens of Rag2^−/− and Socs1^−/−Rag2^−/− mice. Data are representative of three independent experiments. (f) Fluorescence immunohistochemistry showing the infiltration of CD45⁺ cells (red) into the colonic lamina propria of Rag2^−/− (upper left panel) and Socs1^−/−Rag2^−/− mice (upper right panel). The infiltrating CD45⁺ cells were predominantly CD11b⁺ cells (green), although NK1.1⁺ cells (red) were also significantly increased (lower panels). Nuclei of cells are stained with Hoechst 33342 dye (blue). Data are representative of three independent experiments using samples from different mice. Scale bar, 100 μm. (g) RNAs were extracted from the colons of 9-week-old Rag2^−/− and Socs1^−/−Rag2^−/− mice, and the indicated gene expression was analysed by RT-PCR. Three mice per group are shown. Data are representative of three independent experiments using different samples from one to three mice per group. (h) CD11b^highNK1.1⁻ and NK1.1⁺ cells were sorted by FACSAria from the spleens of Rag2^−/− and Socs1^−/−Rag2^−/− mice, and RNA was purified from the cells. The relative expression levels of the indicated genes were normalized against Hprt1 using Ct values determined by quantitative real-time RT-PCR. Error bars represent +s.d. of triplicate measurements for each sample. *P<0.05 and **P<0.01 compared with Rag2^−/− cells. Data are representative of two independent experiments. (i) Proteins were extracted from the colons of 9-week-old Rag2^−/− and Socs1^−/−Rag2^−/− mice, and western blot analyses were performed using indicated Abs. Three mice per group are shown. PY, tyrosine phosphorylated; PS, serine phosphorylated. Data are representative of two independent experiments using different samples from two to three mice per group. ND, not determined.