Fig 2: Immune complexes induce M2 polarization in DKO MØ. Peritoneal macrophages were not treated (NT) or stimulated with immune complexes (IC) or LPS+IC (6 hours). RNA was isolated and quantified by qRT-PCR. B, Protein levels of IL-10 and IL-12 from the media of samples in (A). Apoe-/- white bars, DKO black bars (n=3). Significance was determined by independent sample t test. n.d., not detected. *P<0.05; ***P<0.001. Apo indicates apolipoprotein; Arg-1, arginase 1; DKO, double knockout; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; qRT-PCR, quantitative real-time polymerase chain reaction; TNF-a, tumor necrosis factor alpha.

Fig 3: (A) Representative Western blots showing the protein expression of IL-6 and Arg1 in BV2 cells cultured or not in the presence of AZE (1000 µM) for 6 or 24 h. (B,C) Violin plots of bands densities demonstrating the effects of AZE on the expression of IL-6 and Arg1 at the indicated times. Data reported is the mean ± SEM, from two independent experiments using separate batches of cells (n = 6). GAPDH was used as loading control. Ns = not significant. *** p < 0.001 or **** p < 0.0001 vs. Ctrl at the indicated time, as determined by ANOVA followed by Sidak’s post hoc test.

Fig 4: Depletion of anti-inflammatory microglia inhibits reduction of lesion area. (A-C) Representative images showing GFAP (red) and MR (green) staining in spinal cord sagittal sections at 4 weeks post-transplantation. A1-C1 are higher magnification images of boxed areas in A-C. Scale bars: 200 µm (A-C), 20 µm (A1-C1). (D) Quantification of MR+ cells at 4 weeks post-transplantation (n = 5 per group). Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. Data are mean ± SD; ***p < 0.001. (E) Schematic of depletion of anti-inflammatory microglia using mannosylated clodronate liposomes. (F) Experimental design: evaluation of lesion area at 4 weeks and axon regeneration at 8 weeks after MCL injection. (G) Representative images showing Arg1(red) staining in sagittal sections of spinal cord lesion area at 4 weeks post-transplantation. Scale bar: 20 µm. (H) Quantification of efficiency of anti-inflammatory microglia depletion (n = 5 per group). Unpaired t test with Welch's correction. Data are mean ± SD; ***p < 0.001. (I, K) Representative images showing five consecutive parasagittal sections centered at the spinal cord midline at 4 weeks post-transplantation. Sections are stained for GFAP (red) to demarcate glial scar borders and lesion areas plus either PDGFR-ß to label pericyte (I, green) or fibronectin to label fibroblast (K, green). Scale bars: 200 µm. (J, L) Quantification of PDGFR-ß+ (J) and fibronectin+ (L) area (n = 5 per group). Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. Data are mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Validation Experiments: immunoprecipitation of PP2A (Panel A and B) and ARG-1. HNE modifications levels detected by immunoprecipitation of PP2A and ARG-1 (n = 6/group) in Ts65Dn Veh vs Eu Veh and Ts65Dn InRapa vs Ts65Dn Veh. Panels A and B show the Immunoprecipitation blots and graph relative bar graph of HNE-modified PP2A between Ts65Dn Veh vs Eu Veh comparison group (A) and Ts65Dn InRapa vs Ts65Dn Veh comparison group (B). Panels C and D show the Immunoprecipitation blots and graph relative bar graph of HNE-modified ARG-1 between Ts65Dn Veh vs Eu Veh comparison group (C) and Ts65Dn InRapa vs Ts65Dn Veh comparison group (D). Densitometry values shown in the bar graph are the mean of 6 samples per each group, normalized on expression, and are given as percentage of Eu Veh, set as 100%. Statistical significance was determined using one–way ANOVA and Student t-test analysis (*p < 0.05, **p < 0.01, ***p < 0.001).