Fig. 5
GRIM19 inhibits IL-17 by downregulating STAT3-NFATc1. a Recipients (B/c mice) were intravenously injected with 5 × 106 donor (WT or GRIM19 Tg) bone marrow cells and 1 × 107 WT or GRIM19 Tg splenocytes after lethal irradiation. The expression levels of intracellular signaling molecules p-NFATc1 and RCNA3 in the spleen were determined by confocal microscopy on day 14 after allogeneic BMT. All confocal microscopy images were obtained for each mouse (n = 6 per group). Representative images are shown. b Immunohistochemical staining for RCAN3 in spleen tissue from GVHD mice. Positive immunoreactivity appears as a brown color. It is counterstained with blue or green. Original magnification, ×400. c Fourteen days after BMT, Rcan3 mRNA levels in splenocytes were determined by real-time PCR. d Splenic CD4+ T cells from B6 mice were transfected with mock or GRIM19 expression vector. Mock or GRIM19-transfected CD4+ T cells were activated by stimulation with anti-CD3/CD28 (Th0 condition) for 3 days. GRIM9, Il-17, and Rcan3 mRNA levels were determined by real-time PCR. e Splenic CD4+ T cells from B6 mice were transfected with mock or Grim19 siRNA under Th0 conditions. Il-17 and Rcan3 mRNA levels were determined by real-time PCR. f Splenic CD4+ T cells from B6 mice were transfected with GRIM19-overexpression vector, Grim19 siRNA, or STAT5 inhibitor. IL-17 levels in supernatants were measured by ELISA. Data are expressed as mean ± SD of three independent experiments