Fig 1: PD-L1 knockout promoted pro-inflammatory polarization of macrophages/microglia after SCI. (A–F) IF staining of the CD11b (green), and M1-like biomarker, iNOS (red) and M2-like biomarker, Arg1 (red), in damage area on day 14 post-SCI. Nuclei were counterstained with DAPI (blue). Bar = 100 µm. (A, D) The number of CD11b+ macrophages/microglia (B, E) and the number of iNOS+ CD11b+ and Arg1+ CD11b+ macrophages/microglia (C, F) were determined (n = 6 mice per group, values are the mean ± SD, ns indicates no significance, *p < 0.05, **p < 0.01, two-tailed Student’s t tests). (G) mRNA expression levels of marker genes (M1-like: iNOS, TNF-a and IL-1ß, M2-like: CD206, YM1/2 and Arg1) in the damage area from PD-L1 WT and KO mice on day 14 post-SCI (n = 3 each group, *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t tests). (H, I) The protein expression level of iNOS and Arg2 detected by western blot in PD-L1 WT and KO mice at day-14 post SCI. (n = 6 mice per group, values are the mean ± SD, **p < 0.01, ***p < 0.001, one-way ANOVA). (J–M) Flow cytometry analysis of isolated CD11b+ cells in PD-L1 WT and KO mice at day 14 post SCI. The percentages of CD11b+ iNOS+ and CD11b+ CD206+ cells were calculated (n = 6 per group, values are the mean ± SD, **p < 0.01, ***p < 0.001, two-tailed Student’s t tests).
Fig 2: Intrathecal injection of PD-L1 promotes recovery and attenuate inflammation response. (A–C) Statistical analysis of the Louisville Swim Scale and BMS score of mice in different groups after SCI (n = 5 mice per group at each time point, values are the mean ± SD, *p < 0.05, **p < 0.01 vs. PD-L1 Protein, #p < 0.05, ##p < 0.01 vs. Anti-PD-L1 mAb, repeated measurement analysis of variance). (D, E) Statistical analysis of the mechanical and thermal threshold of mice in different groups after SCI. (n = 5 mice per group at each time point, values are the mean ± SD, *p < 0.05, **p < 0.01 vs. PD-L1 Protein, #p < 0.05, ##p < 0.01 vs. Anti-PD-L1 mAb, repeated measurement analysis of variance). (F–I) Flow cytometry analysis of CD11b+ iNOS+ and CD11b+ CD206+ cells in different groups at day 14 post-SCI (n = 6 per group, values are the mean ± SD, *p < 0.05, **p < 0.01, one-way ANOVA).
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