Fig 1.
Prophylactic treatment with imatinib reduces MSU crystal-induced inflammation in the air pouch model.
(A) Experimental outline. Mice were anesthetized, shaved on the back, and injected with 3 ml of sterile air subcutaneously (s.c.) into the back to form an air pouch. Three days after the first injection, an additional 3 ml of sterile air were injected into the pouch. Three days later, we injected 3 mg of MSU crystals in 1 ml PBS, or PBS alone, into the air pouches. Mice received intra-peritoneal (i.p.) injections of imatinib (100 mg/kg i.p. in 200 μl PBS) or vehicle (PBS) twice a day starting 24 h before the injection of MSU crystals. (B) Number of total cells, neutrophils (Ly6G+; CD11b+), eosinophils (SSChigh; Siglec-F+) and monocytes (Ly6G-; CD11b+) in air pouch exudates collected 6 h after injection of MSU crystals or PBS. (C) Levels of IL-1β in air pouch exudates collected 6 h after injection of MSU crystals or PBS. Data in B and C are depicted as values from individual mice, with histograms indicating medians + quartiles of data pooled from the three independent experiments performed with a total of n = 8 (‘PBS’ group) or n = 9 (‘MSU’ and ‘MSU + Imatinib’ groups) mice. *, ** or *** = P < 0.05, 0.01 or 0.001 vs. indicated group by Mann-Whitney U test.
Fig 2.
Prophylactic treatment with imatinib reduces MSU crystal-induced ankle inflammation.
(A) C57BL/6J mice received intra-peritoneal (i.p.) injections of imatinib (30 or 100 mg/kg i.p. in 200 μl PBS) or vehicle (PBS) twice a day starting 24 h before intra-articular (i.a.) injection of MSU crystals (0.5 mg in 10 μL) in one ankle and vehicle (10 μl PBS) in the contra-lateral ankle. (B and C) Time course (B) and representative photographs (C) (at 24 h) of MSU crystal-induced ankle swelling in mice pre-treated with imatinib or PBS (n = 12-15/group). Data in B are shown as means ± SEM of data pooled from the three independent experiments performed. *** = P < 0.001 vs. indicated group by repeated measures two-way ANOVA. (D and E) Image of photon emission, pseudocolor, superimposed over grayscale reference image (D) and quantification (E) of myeloperoxidase (MPO)-induced bioluminescence in the ankle joint 24 h after i.a. injections of MSU crystals or PBS in mice pre-treated with imatinib or PBS (n = 10/group). Bioluminescence intensity was calculated in defined regions of interest (ROI) around the ankle joints (red circles in D). Data are shown as means ± SEM of data pooled from the four independent experiments performed. * = P < 0.05 vs. indicated group; N.S.: not significant (P > 0.05) by unpaired Student t test. (F) H&E-stained sections of ankles 24 h after injection of 0.5 mg MSU crystals in mice pre-treated with imatinib or PBS. Lower panels in F are enlargement of the areas marked (*) in the upper panels, with solid arrowheads depicting a few of the many neutrophils present at sites of MSU crystal injection, especially in the section from the mouse not treated with imatinib. Original magnification: x1 for upper panels, x20 for lower panels.
Fig 3.
Intraperitoneal treatment with imatinib suppresses IL-1-independent MSU crystal-induced acute arthritis.
(A) Changes in ankle thickness after i.a injection of 2.0 mg MSU or PBS in IL-1R1-/- mice (n = 8-9/group) pre-treated with imatinib (100 mg/kg) or PBS twice a day starting 24 h before the injection of MSU crystals. Data are shown as means ± SEM of data pooled from the two independent experiments performed. *** = P < 0.001 vs. indicated group by repeated measures two-way ANOVA. (B) H&E-stained sections of ankle showing MSU injection sites 24 h after injection of 2 mg MSU crystals in IL-1R1-/- pre-treated with imatinib (100 mg/kg) or PBS. Original magnification: x20.
Fig 4.
Intraperitoneal treatment with imatinib suppresses mast cell-independent MSU crystal-induced acute arthritis.
(A) Changes in ankle thickness after i.a injection of 0.5 mg MSU in mast cell-deficient KitW-sh/W-sh mice (n = 6/group) pre-treated with imatinib (100 mg/kg) or PBS twice a day starting 24 h before the injection of MSU crystals. Data are shown as means ± SEM of data pooled from the two independent experiments performed. *** = P < 0.001 vs. indicated group by repeated measures two-way ANOVA. (B) H&E-stained sections of ankle showing MSU injection sites 24 h after injection of 0.5 mg MSU crystals in KitW-sh/W-sh mice pre-treated with imatinib (100 mg/kg) or PBS. Original magnification: x20.
Fig 5.
Intraperitoneal treatment with imatinib reduces MSU crystal-induced ankle inflammation in a therapeutic protocol.
(A) C57BL/6J mice received imatinib (100 mg/kg, i.p.) or vehicle (PBS) 1 and 6 h after i.a. injection of MSU crystals (0.5 mg in 10 μl) in one ankle and vehicle (10 μl PBS) in the contra-lateral ankle. (B) Time course of MSU crystal-induced ankle swelling in this therapeutic protocol (n = 8-9/group). Data are shown as means ± SEM of data pooled from the three independent experiments performed. *** = P < 0.001 vs. indicated group by repeated measures two-way ANOVA. (C) H&E-stained sections of ankle showing MSU injection sites 24 h after injection of 0.5 mg MSU crystals in the therapeutic protocol. Lower panels in C are enlargement of the areas marked (*) in the upper panels. Original magnification: x20.
Fig 6.
Oral treatment with imatinib reduces MSU crystal-induced acute arthritis.
(A) C57BL/6J mice received imatinib (100 mg/kg in 100 μl) or vehicle (100 μl PBS) by gavage twice a day starting 24 h before the i.a. injection of MSU crystals (0.5 mg in 10 μl) in one ankle and vehicle (10 μl PBS) in the contra-lateral ankle. (B) Ankle swelling 5 h and 24 h after i.a. injection of MSU crystals or vehicle (PBS). Data are depicted as values from individual mice, with histograms indicating medians + quartiles pooled of data pooled from the two independent experiments performed with a total of n = 8 mice/group. ** or *** = P < 0.01 or 0.001 vs. corresponding PBS-treated group, and ## = P < 0.01 vs. indicated group by Mann-Whitney U test.
Fig 7.
Intra-articular treatment with imatinib-loaded PLGA nanoparticles reduces MSU crystal-induced acute arthritis in a therapeutic protocol.
(A) C57BL/6J mice were injected i.a. with MSU crystals (0.5 mg in 10 μl) in both ankles at time 0. One hour and 6 hours after MSU crystal injection, mice were injected i.a. with imatinib-loaded PLGA nanoparticles (PLGA-imatinib; 20 μl at 0.5 mg/ml) in one ankle and control nanoparticles (PLGA-vehicle; 20 μl at 0.5 mg/ml) in the contra-lateral ankle. (B) Ankle swelling 5 h and 24 h after i.a. injection of MSU crystals (n = 7/group). Data are depicted as values from individual mice pooled from the two independent experiments performed. * or ** = P < 0.05 or 0.01 vs. indicated group using a paired Student’s t test.