Adenovirus-IL-10 relieves chronic rejection after mouse heart transplantation by inhibiting miR-155 and activating SOCS5

Abstract

Objective: Chronic rejection remains the main factor that influence long-term survival of patients after heart transplantation. Interleukin-10 (IL-10) play critical role in macrophages-mediated transplant immune responses. We investigated the mechanism of IL-10 in macrophage related chronic rejection after mouse heart transplantation. Methods: Mouse heart transplant chronic rejection model was established to evaluate pathological changes in the allograft. Myocardial interstitial fibrosis, apoptosis, and inflammatory factor levels were detected in ad-IL-10-treated mice. The positive iNOS⁺ and Arg-1⁺ expressions, macrophage subset changes, and the proportion of regulatory T-cells (Tregs) and TIGIT⁺ Tregs were quantified by flow. In in vitro experiments, ad-IL-10 was transfected into macrophages followed by detection of apoptosis, phagocytosis, and CD163, CD16/32, and CD206 expression. The expression and relationships between IL-10, miR-155, and SOCS5 were also detected and verified. A rescue experiment was performed to evaluate macrophage function through the combined treatment of ad-IL-10 and overexpression of miR-155. Results: Significantly decreased IL-10 expression in chronic rejection during mouse heart transplantation was observed. Ad-IL-10-treated mice showed decreased pathological injury, perivascular fibrosis, apoptosis, inflammation, and iNOS⁺ and CD16/32⁺ expression, and increased Treg/TIGIT⁺ Treg cell, Arg-1⁺ and CD206⁺ cell proportion. Ad-IL-10-treated macrophages in vitro showed reduced apoptosis, improved phagocytosis, and M2 polarization. Mechanically, IL-10 negatively regulated miR-155 to activate SOCS5. Overexpression of miR-155 reversed IL-10 mediated-positive regulation of macrophage function. Conclusion: IL-10 downregulated miR-155 and activated SOCS5, thereby promoting macrophage M2 polarization to relieve chronic rejection after heart transplantation.

Keywords: Chronic rejection after heart transplantation; IL-10; Macrophage; Macrophage function; SOCS5; microRNA-155.

Figure 1

IL-10 expression is downregulated in heart tissues and peritoneal macrophages of mice in chronic heart transplantation rejection model. The chronic rejection of heart transplantation model was established with bm12 mice as donors and B6 mice as recipients. HE (A) and EVG staining (B) were used to observe the pathological changes of the hearts of the model mice in the 2^nd, 4^th, and 8^th week after transplantation, and the pathological injury was quantitatively analyzed. Vascular intimal hyperplasia was marked by arrows. Mice in the sham-operated group were used as controls. Expression of IL-10 in mouse heart tissues and peritoneal macrophages in the 2^nd, 4^th, and 8^th week after transplantation was evaluated using qRT-PCR (C). IL-10 positive expression in mouse heart tissue was evaluated in the 8^th week after transplantation using immunohistochemical staining (D). A/B/D (n = 6), C (n = 3). The data were expressed as mean ± standard deviation and were representative of three independent experiments. Data in panels A/B/D were analyzed using the t-test, and data in panel C was analyzed using two-way ANOVA, followed by Tukey's multiple comparison test. ** p < 0.01.

Figure 2

ad-IL-10 relieves pathological injury of heart transplantation chronic rejection in mice and regulates the balance of pro-inflammatory and anti-inflammatory factors. ad-IL-10 was injected into the model mice, and expression of IL-10 mRNA was detected using qRT-PCR (A). Texture and hardness of the transplanted heart tissue were observed (B). Fibrosis of the heart tissue was evaluated using Masson staining and the bar graph of heart width messured by vernier caliper (C). Pathological conditions of transplanted heart vessels in each group were analyzed using EVG staining. Vascular intimal hyperplasia was marked by arrows. (D). Positive expression of TUNEL (E) was analyzed quantitatively. Expressions of IFN-γ, TNF-α, IL-1β, IL-17A, IL-4, and TGF-β1 in serum of mice were detected using ELISA (F). A/B/C/D/E (n = 6); F (n = 15). Data were representative of three independent experiments. Data in panels A/D/E were analyzed using one-way ANOVA and data in panel F was analyzed using two-way ANOVA, followed by Tukey's multiple comparison test. ** p < 0.01.

Figure 3

ad-IL-10 promotes macrophage M2 polarization in chronic rejection after mouse heart transplantation. ad-IL-10 was injected into the model mice, and the changes of macrophage subsets in the mice spleen were detected using flow cytometry (A). Positive expressions of iNOS and Arg-1 in the mice heart tissue were evaluated using immunohistochemistry (B) and analyzed quantitatively. Treg and TIGIT+ Treg proportions were detected using flow cytometry (C-D). A/B/C/D (n = 6). Data were representative of three independent experiments. Data in panel D was analyzed using one-way ANOVA and data in panels A/B/C were analyzed using two-way ANOVA, followed by Tukey's multiple comparison test. ** p < 0.01.

Figure 4

ad-IL-10 enhances macrophage phagocytosis and promotes macrophage differentiation to M2 type. Cell morphology was observed by a microscope (A), and flow cytometry (B) was used to detect the purity of isolated macrophages. ad-IL-10 was transfected into macrophages, and the transfection efficiency was detected using qRT-PCR (C). Macrophages treated with ad-IL-10 were transfected with miR-155 mimic, and then flow cytometry was used to detect apoptosis (D), phagocytosis (E), CD163⁺ expression (F), CD16/32 (M1) expression (G), and CD206 (M2) expression (H). Data were presented as means ± SD (n = 6) and were representative of three independent experiments. Data in panel C was analyzed using the t-test, and data in the rest panels were analyzed using two-way ANOVA, followed by Tukey's multiple comparison test. ** p < 0.01.

Figure 5

IL-10 activates SOCS5 expression by downregulating miR-155. Expression of miR-155 in mouse heart tissue and macrophages were detected using qRT-PCR (A). It was predicted that there was a targeted binding relationship between miR-155 and SOCS5 through the biological website, which was verified using dual-luciferase reporter gene assay (B) and co-immunoprecipitation assay (C). Expressions of SOCS5 mRNA and protein were detected using qRT-PCR and western blot analysis (D-E). Data were presented as means ± SD (n = 6) and were representative of three independent experiments. Data in panels A/D/E were analyzed using one-way ANOVA, and data in panels B/C were analyzed using two-way ANOVA, followed by Tukey's multiple comparison test. ** p < 0.01.

Figure 6

Overexpression of miR-155 reverses the positive regulation of IL-10 on macrophage function. Overexpressed miR-155 was transfected into the macrophages treated with ad-IL-10. Flow cytometry was used to detect apoptosis, phagocytosis, CD163⁺ expression, CD16/32 (M1) expression, and CD206 (M2) expression (A-E). Three independent repeated tests were conducted. The data were expressed as mean ± standard deviation and were representative of three independent experiments. The data were analyzed using two-way ANOVA, followed by Tukey's multiple comparison test. ** p < 0.01.