Fig 2: IL-34 promoted macrophage M2 polarization but not the differentiation of regulatory T cells (Tregs) in vitro. (A) Representative plot of CD4+CD62+ naïve T cells sorted from spleen with CD4 microbeads. (B) Representative plots of CD25+Foxp3+ Tregs gated from CD4+ population. (C) Graphic presentation showing the percentages of CD25+Foxp3+ Tregs. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showing the mRNA abundance of Foxp3 in T cells. (E) Representative plot of F4/80+ macrophages analyzed by flow cytometry. (F, G) Western blot assay (F) and quantitative analysis (G) showing the protein abundance of CD206 in BMDMs treated with or without IL-34 (50 ng/ml). (H) qRT-PCR analysis of CD206 mRNA expression in BMDMs treated with or without IL-34 (50 ng/ml). (I) qRT-PCR analysis of CD80, iNOS and CD86 mRNA expression in BMDMs treated with or without IL-34 (50 ng/ml). *P < 0.05, **P < 0.01. Data are presented as means ± SD.

Fig 3: IL-34 overexpression ameliorated the progress of TCMR. (A) Representative micrographs for hematoxylin and eosin (HE), periodic acid–Schiff (PAS), CD4 and CD8 staining in grafts (scale bar = 20 μm). (B–D) Graphic presentation showing the i (B), t (C) and v (D) scores in grafts among groups as indicated. (E, F) Graphic presentation showing the CD4 positive area (E) and CD8 positive area (F) in grafts among groups as indicated. *P < 0.05, **P < 0.01. Data are presented as means ± SD.

Fig 4: IL-34 overexpression protected grafts by upregulating regulatory T cells (Tregs) and M2 macrophages and inhibiting pro-inflammatory cytokines. (A) Representative immunohistochemistry (IHC) staining for Foxp3 and CD206 in grafts (scale bar = 20 μm). (B–C) Foxp3 positive cells (B) and CD206 positive cells (C) in grafts among groups as indicated. (D, E) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showing the mRNA abundance of Foxp3 (D) and CD206 (E) in grafts. (F–H) Graphic presentation showing the concentrations of serum interferon-gamma (IFN-γ), IL-17 and tumor necrosis factor-alpha (TNF-α) among groups. *P < 0.05, **P < 0.01. Data are presented as means ± SD.

Fig 5: IL-34 expression was decreased in TCMR allografts. (A) Representative immunohistochemistry (IHC) staining images of interleukin-34 (IL-34) in grafts (magnification: 100×, scale bar = 100 μm; magnification: 400×, scale bar = 20 μm) (B) Graphic presentation showing the quantification of IL-34 positive area in grafts. (C, D) Western blot (C) and quantitative analysis (D) showing the protein abundance of IL-34 in grafts. (E) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showing the mRNA of IL-34 in grafts. *P < 0.05, **P < 0.01. Data are presented as means ± SD.