Fig. 1

PLX3397 treatment during cGVHD progression attenuates clinical and histopathological outcomes. A Experimental timeline for irradiation, cell transplantation and subsequent PLX3397 treatment. Lethally irradiated B6D2F1 mice were transplanted with 5 × 10⁶ bone-marrow cells and 0.5 × 10⁶ mature splenic T cells from either B6D2F1 (syngeneic controls) or C57Bl/6 (allogeneic) donor mice to induce cGVHD. From day 30 post-transplant, mice either continued to receive standard control chow, or were switched to ‘treatment chow’ that incorporated 150 ppm or 300 ppm PLX3397. All mice were scored for pathology weekly until the end of experiment (n = 6–12 mice/group). B Temporal overview of clinical scores for all experimental mice until sacrifice. cGVHD mice scored higher than syngeneic controls throughout the course of the experiment. PLX3397 treatment at 150 ppm or 300 ppm reduced clinical scores in the cGVHD group. Green asterisks are cGVHD ‘control chow’ versus cGVHD PLX3397 300 ppm treatment on days 56, 63 and 70 post-transplant. Magenta asterisks are cGVHD ‘control chow’ versus cGVHD PLX3397 150 ppm and 300 ppm, and versus ‘syngeneic control chow’ treatment on day 70 post-transplant, respectively. C Clinical scores in syngeneic and cGVHD (allotransplant) mice on day 28 post-transplant, prior to the commencement of PLX3397 treatment. Asterisks indicate post-hoc results. D Clinical scores in syngeneic mice and cGVHD mice on day 70 post-transplant, after 40 days of control or PLX3397 treatment at 150 ppm or 300 ppm. Note that PLX3397 treatment at either concentration significantly reduced clinical pathology scores in the cGVHD group. Asterisks indicate post-hoc results. E Quantification of IBA1^pos immunoreactivity (proportional area) in the skin of syngeneic and cGVHD mice, with and without PLX3397 treatment. Asterisks indicate post-hoc results. F Quantification of the mean fluorescence intensity (MFI) of IBA1^pos cells in the skin of syngeneic and cGVHD mice, with and without PLX3397 treatment. Values shown are in arbitrary units (A.U.). Asterisks indicate post-hoc results. G, H Quantification of CD68^pos immunoreactive area (G) and CD68^pos MFI (I) in skin tissue from syngeneic and cGVHD mice, with and without PLX3397 treatment. Asterisks indicate post-hoc results. I Representative confocal images of IBA1^pos (green) and CD68^pos (red) macrophages in the skin of syngeneic and cGVHD mice, with and without PLX3397 treatment. DAPI (blue) was used to label cell nuclei. J Masson’s trichrome stained skin samples of syngeneic and cGVHD treated with control or PLX3397 chow. K, L Fibrosis score (K) and dermal thickness (L) of skin tissue stained with Masson’s trichrome staining from syngeneic and cGVHD mice with and without PLX3397. M Quantification of CD3^pos T-cell numbers in the epidermal layers of the skin from syngeneic and cGVHD mice, with and without PLX3397 treatment. Note that cGVHD mice had an increased density of CD3^pos T cells compared to syngeneic controls, an effect that was ameliorated by PLX3397 treatment. Asterisks indicate post-hoc results. N Representative confocal images of CD3^pos T cells from syngeneic and cGVHD mice, with and without PLX3397 treatment. Statistics: repeated measure two-way ANOVA with Bonferroni’s multiple comparisons (B) one-way ANOVA (C–H, K–M) followed by Bonferroni post hoc comparison. Dots in box plots represent individual mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar = 50 µm (I, N), 100 µm (J)