Fig 2: Immunohistochemistry identifies the macrophage transformation, cell proliferation, and neovascularization. a Immunohistochemical staining with anti-CD86, CD163, Ki-67, and CD31 antibodies on diabetic wound in each group was performed and analyzed at 14 days post-transplantation. Scar bar: 50 μm. b, c The quantitative data of the total number of CD86-positive M1 macrophages (b) and CD163-positive M2 macrophages (c) per field were analyzed. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, ***p < 0.001. d The quantitative data of the percentage of Ki-67-positive proliferating cells per field were analyzed. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, ***p < 0.001. e The quantitative data of the total number of CD31-positive newly formed blood vessels were analyzed. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 3: Classification of mixed macrophages according to the intensity of expressed markers. (A) Watershed cell segmentation to identify the boundaries of single macrophages. Cochlea were obtained from 4-weeks old mice, and immunostained using the mIHC. The red line denotes the boundaries of each macrophage with F4/80 staining. (B) Quantification of the population in the resting state to identify the percentage of each category in the modiolus including auditory nerve and spiral ganglia. Mid-modiolar sections were analyzed, and macrophages population of each category, M0, M1-like, M2-like and M1/M2, were quantified (n = 5). The M0 non-activated macrophages represented the major percentage among all macrophages, with a significant difference compared to other categories. The mixed M1/M2 macrophages were presented minimally, while the M1-like and M2-like macrophages expressing single marker represented the least percentages. *P < 0.05, ****P < 0.0001, ns: not significant, by one-way ANOVA with Tukey's multiple comparisons test. The error bars represent standard deviation from the mean. (C) Example of mixed macrophages in which M1 markers (CD68, CD86) expression was similar to that of the M2 markers (CD206, CD163). (D) A representative macrophage of a group of mixed macrophages in which the CD86 (M1 marker) showed the highest expression compared to the other markers. (E) Illustration of another group of mixed macrophages in which CD163 (M2 marker) had the highest expression among all markers. (F) Single-cell-based marker intensity for each mixed macrophage in the modiolus including auditory nerve and spiral ganglia. Each dot represents a single mixed macrophage (n = 74 from 7 cochleae); the mean intensity of M1 and M2 markers in each macrophage are plotted against each other. The cut-off value was set at 1, below which most of the macrophages were included, and the different types of macrophages were identified. The red box marks the area of the dominant cell population in which most of the macrophages represent M1 and M2 markers equally within the cut-off value. The numbers indicate the percentages of the cell population of each quadrant; in the lower right quadrant, M1-polarized cells expressing higher M1 marker intensities (>1) than M2 cells, 8.44, 3.90, 9.49, and 5.23%: the upper left quadrant, M2-polarized cells expressing higher M2 marker intensities (>1) than M1 cells, 6.56, 3.90, 10.83, and 9.78%: the upper right quadrant, cells strongly expressing both M1 and M2 markers, 2.56, 5.23, 1.51, and 3.90%.

Fig 4: miR-21 partially mediated the regulation of macrophage polarization by naringin/PPARγ(A) The level of miR-21 in treated BMDMs was detected by quantitative real-time PCR. (B and C) M1 macrophages (F4/80+CD86+) and M2 macrophages (F4/80+CD206+) in BMDMs were determined by flow cytometry. (D and E) The mRNA levels of M1 marker iNOS (D) and M2 marker Arg-1 (E) in treated BMDMs were detected by quantitative real-time PCR. (F) The protein levels of indicated proteins in treated BMDMs were detected by western blotting. Data represent the mean ± SD of three experiments performed in triplicate independently. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 5: EZH2 inhibitors EPZ6438 and GSK126 induced M0 macrophages to differentiate into the M1 phenotype. (A) Effects of GSK126 and EPZ6438 on the proliferation in RAW264.7 cells every 4 h until the end of 48 h. (B) Cellular viability of RAW264.7 cells was detected after EPZ6438 and GSK126 treatment. (C) Schematic illustration of EZH2 inhibitor treatment schedule. (D, E) RAW264.7 cells were treated with EPZ-6438 (10, 20 μM) (D) and GSK126 (5, 10 μM) (E) for 48 h. Control cells were maintained in a medium supplemented with DMSO throughout the entire experimental period. An additional group of cells was treated with LPS (100 ng/ml) for the last 12 h. M1-type macrophage genes (CD86, TNFα, and iNOS) were analyzed by RT-PCR. (F) Schematic illustration of EZH2 inhibitor treatment schedule. EZH2 inhibitor and LPS (100 ng/ml) or IL-4 (20 ng/ml) were added into the culture medium of RAW264.7 cells at the same time. Effects of EPZ6438 and GSK126 on macrophage polarization were detected at time points of 0.5 h, 1 h, and 2 h, respectively. (G, H) M1-type macrophage genes (CD86 and iNOS) (G) and M2-type macrophage genes (CD206 and Arg1) (H) were analyzed by RT-PCR. Data are shown as mean ± SEM and three independent experiments were involved. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance.