Figure 4. Irg1 expression and itaconate influence new bone formation in arthritis. (A) Area of osteoproliferative lesions (in mm2) in hind paws of mice injected with K/BxN serum at indicated time points after injection (n=7–26). (B) Area of osteoproliferative lesions (in mm2) in hind paws from Irg1+/+ or Irg1−/− mice injected with K/BxN serum (n=10–12). (C) Representative images of TRAP-stained histological slides from hind paws of mice shown in (B). Black arrows indicate the boundaries of the osteoproliferative lesions. Scale bars represent 2 mm and 500 µm. (D, E) Relative mRNA expression of Irg1 (D) and itaconate levels (E) in paws isolated from mice injected with serum from K/BxN mice harvested at indicated time points (n=3–4). (F) Representative images of histological slides of an inflamed paw (K/BxN serum transfer arthritis model) stained for TRAP (purple staining indicates TRAP+ cells) or Irg1 expression as detected with RNA ISH (brown staining indicates Irg1 expressing cells). Scale bars represent 500 µm, 200 µm and 100 µm. (G) Itaconate levels in paws from bone marrow transplanted mice injected with serum from K/BxN mice on day nine post injection. Lethally irradiated Irg1+/+ and Irg1−/− mice were transplanted with Irg1+/+ and Irg1−/− bone marrow (Irg1+/+ bone marrow to Irg1+/+ mice (Irg1+/+>Irg1+/+), Irg1−/− bone marrow to Irg1+/+ mice (Irg1−/−>Irg1+/+), Irg1+/+ bone marrow to Irg1−/− mice (Irg1+/+>Irg1−/−) (n=4–7). (H) Area of osteoproliferative lesions (in mm2) of hind paws from wildtype or CD11c-DTR mice injected with serum from K/BxN mice and treated with diphtheria toxin (n=8). The data are represented as means±SDs. One-way ANOVA with Šidàk’s correction (A, D, E, G) and unpaired t-test (B, H). ANOVA, analysis of variance; Irg1, immunoresponsive gene 1; ISH, in situ hybridisation.
