Fig 2: Accumulated ASCs decreased M1 macrophages while increasing M2 macrophages and Tregs in the spleen.a Experimental scheme for analyzing the distribution of administered ASCs in the body. DiD-dye labeled ASCs were administered to nephritis-induced WKY/NCrj rats on day 2. Tissues were harvested on day 3. b The number of DiD-positive cells with large DiD-dye particles and small DiD-dye particles in the liver, lung, spleen, and kidney is shown (n = 3 per group). Scale bars, 100 μm. c, d T-distributed stochastic neighbor embedding (tSNE) implementation for flow cytometry data visualization of leukocyte and T-cell subsets. The data are presented as color-coded leukocyte population overlays (left) or cell number density color-coded (right) in the tSNE space. e Frequency of splenic leukocyte subsets in CD45+ cells analyzed by flow cytometry on days 3 and 7 (day 3, n = 5 per group; day 7, n = 8 per group). f The population of Ki67-positive cells in splenic Treg subset on day 3 (n = 3 per group). All data are shown as means ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 as determined by ANOVA.

Fig 3: EVs from administered ASCs were predominantly transferred to M2 macrophages.a Experimental scheme for analyzing the organ and cellular distribution of MSC membrane components by flow cytometry. The spleen and kidney were analyzed one day after the DiD-dye labeled ASC administration. The left graph shows the percentage of CD45-positive or negative subsets in DiD+ cells. The right graph shows the number of DiD+ cells in the spleen and kidney (n = 7 per group). b The confocal micrograph of CD11b/c+DiD+ cells sorted by flow cytometry. DiD particles were localized on the leukocyte membrane. c Representative images of ASC, Apoptotic ASC, and EV. Scale bars, 10 μm. The right graph shows the frequency of three DiD+ populations in the spleen and kidney (n = 3 per group). d tSNE plot from the flow cytometry analysis shows DiD+ leukocyte subsets. e Pie charts show the proportion of each subset in DiD+ leukocytes in the spleen and kidney (n = 7 per group). f Representative images and results of flow cytometry analysis show DiD+ cell frequency in renal M2 macrophages after administration of DiD-dye labeled ASCs or BMMSCs into nephritis rats (n = 7). All data are shown as means ± SD. *p ≤ 0.05 as determined by Welch’s t test.

Fig 4: Splenectomy abolished the therapeutic potential of ASCs.a Experimental scheme for analyzing the therapeutic potential of ASCs in rats treated with splenectomy. The rats underwent splenectomy 1 week before the nephrotoxic antibody injection. ASCs were administered on days 0, 2, and 4. b, c Serum creatinine level and histological crescent score on days 3 and 7 with and without splenectomy in each MSC treatment. d The tSNE plot shows the renal T-cell subset on day 3. e Frequency of renal leukocyte subsets in CD45+ cells analyzed by flow cytometry on days 3 and 7 (day 3, n = 4 per group; day 7, n = 7–8 per group). All data are shown as means ± SD. *p ≤ 0.05, ***p ≤ 0.001 as determined by Welch’s t test.

Fig 5: Effects of LXA4 treatment on inflammatory cell infiltration in alkali-burned corneas. (A,B) show that treatment for 14 days with LXA4 significantly reduced the number of CD45+ cells, especially neutrophils (CD11b+HIS-48+), in the alkali-burned corneas (Mean ± SEM, * p < 0.05). There were no significant differences in the number of macrophages (CD68+/CD11b+) in corneas treated with LXA4 (n = 8 corneas per group). (C) Immunofluorescence showed fewer inflammatory cells in LXA4-treated corneas than in vehicle-treated corneas. (D) Effects of LXA4 treatment on the phenotype of macrophages from the blood of alkali-burned corneas. Isolated monocytes from the blood of rat corneas treated with LXA4 or vehicle were stained with CD68, CD86, and CD163 and analyzed by flow cytometry. There was a higher percentage of macrophages compared to vehicle and non-injured (control) rats. Percentage of M2 cells (CD68+/CD163+) was higher than M1 (CD68+/CD86+) in LXA4-treated corneas. Data were expressed as Mean ± SEM; * p < 0.05; *** p < 0.001, ANOVA with Tukey’s multiple comparison.