Fig 2: Anti-inflammatory effects of IFN-γ-treated MSCs in IRI rats. Infiltration of T cells and macrophages into injured kidney of MSC-injected IRI rats was assessed at 7 days after IRI by immunostaining. (a) Representative immunohistochemical staining of CD3, CD68, CD163 and CD206 in rat kidney sections (scale bar, 100 µm). (b) Quantification of CD3-, CD68-, CD163- and CD206-positive cells (n = 5 in each group). Data are presented as the mean ± SD. **p < 0.01. Abbreviations are as in Fig. 2.

Fig 3: miR-106b-5p mediated M2 polarization of TAMs. (A) miR-106b-5p expression was significantly up-regulated in THP-1 cells treated with conditioned medium from human U251 glioma cells, and human astrocytes HA cells served as a control (**P<0.01 vs control). (B) Expressions of M2 macrophage markers (Arg1, IL-10, CD163 and CD206) were significantly up-regulated in THP-1 cells treated with conditioned medium from human U251 glioma cells (*P<0.05 vs control). (C) miR-106b-5p expression was downregulated in M1 subset, but upregulated in M2 subset (*P<0.05, ** P<0.01 vs M0). (D) The CD163 protein expression increased in M2 macrophages as compared to M0 and M1 macrophages (**P<0.01 vs M0). (E) CD206 protein expression increased in M2 macrophages as compared to M0 and M1 macrophages (*P<0.05 vs M0). (F) Expressions of M2 macrophage markers (Arg1, IL-10, Fizz1, CD163, CD206) were upregulated (qRT-PCR; *P<0.05, ** P<0.01 vs control). (G) Expressions of M2 macrophage markers (IL-10, CD163 and Arg1) were upregulated after transfection with miR-106b-5p mimics (*P<0.05 vs control). (H) mRNA expressions of M2 macrophage markers (IL-10, CD163 and Arg1) were down-regulated after transfection with miR-106b-5p inhibitor (*P<0.05 vs control). (I) The expression level of miR-106b-5p was decreased when PMs were stimulated with LPS in M1, and the level was markedly increased stimulated with IL-4 in M2 (*P<0.05 vs M0).

Fig 4: MiR-375 decreased MΦ LDHB in mouse and human breast carcinoma. (A - D) Female NMRI-Foxn1nu mice were pre-treated with 17β-estradiol pellets followed by subcutaneous injection of 1 × 107 MCF-7 control or decoy cells in the right and left flank. Tumors were harvested after 35 days or after a maximum tumor volume of 1.5 cm3 has been reached. (A) Experimental layout. (B) Infiltrating murine MΦ were FACS-sorted out of tumors and analyzed for Ldhb and Ldha mRNA expression. (C, D) Immunohistochemical staining of tumor sections. (C) LDHB protein expression in whole tumor tissue and in infiltrating MΦ. (D) Representative pictures of MCF-7 control and decoy tumor sections with arrowheads in magnification showing colocalization of CD163 and LDHB. (E - H) Human invasive mammary carcinoma TMA sections were analyzed for miR-375 abundance by in situ hybridization, followed by staining of CD163 and LDHB. Bright-field signal of miR-375 was converted to fluorescence image using InForm2.0. (E) Mean LDHB intensity in human invasive breast cancer sections compared with normal breast (n = 156 breast tumors; n = 49 normal breasts). (F) Mean LDHB intensity in human ductal carcinoma in situ (DCIS) sections compared with normal breast (n = 16 DCIS; n = 49 normal breasts). (G) Correlation between miR-375 mean intensity and LDHB expressing cells in invasive breast tumor sections (n = 155). (H) Representative pictures of invasive breast cancer section with arrowheads in magnification showing miR-375 colocalization with CD163 and LDHB. Data are represented as mean ± SEM of n ≥ 3 and p-values were calculated using Wilcoxon rank-sum test (B, C) and two-tailed Student's t-test (E, F). *, p < 0.05, ***, p < 0.001.

Fig 5: Pn with exon 21 reduces M1 macrophage markers but enhances M2 macrophage markers. U937 M0 macrophages were treated with recombinant full-length Pn protein, Pn-1. (A,B) RT-qPCR analyses showing upregulation of M2 TAM markers induced by recombinant Pn-1 in THP-1 (A) or U937 (B) macrophages. PMA-treated macrophages were stimulated with recombinant Pn (500 ng/mL) in RPMI media supplemented with 0.1% BSA for 24 h. RT-qPCR analysis indicated downregulation of M1 markers (iNOS, IL-1β and IL-12a) and up-regulation of M2 markers (CD206, CD163 and Fizz1). Data are shown in mean ± SD, n = 4 per group, * p < 0.05 vs. BSA. Student’s t-test. (C,D) Immunohistochemical staining of CD31 (C) or vWF (D), an endothelial cell marker. CD31- or vWF-positive area in tumors was quantitated in at least 4 view fields per tumor by using Image J program. (n = 6–7). *, ** p < 0.05 vs. CTRL, IgG, respectively. Box plot showing mean % of capillary area in tumors. Scale bars represent 100μm. p values were calculated by using the one-way ANOVA test and Tukey–Kramer’s post hoc test.