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. 2022 Dec 16:13:945234.
doi: 10.3389/fimmu.2022.945234. eCollection 2022.

Tumor necrosis factor-α-primed mesenchymal stem cell-derived exosomes promote M2 macrophage polarization via Galectin-1 and modify intrauterine adhesion on a novel murine model

Jingman Li  1 , Yuchen Pan  1   2 , Jingjing Yang  1 , Jiali Wang  1 , Qi Jiang  1 , Huan Dou  1   3 , Yayi Hou  1   3
Affiliations

Tumor necrosis factor-α-primed mesenchymal stem cell-derived exosomes promote M2 macrophage polarization via Galectin-1 and modify intrauterine adhesion on a novel murine model

Jingman Li et al. Front Immunol. .

Abstract

Background: Intrauterine adhesion (IUA) is a condition caused due to damage or infection of the endometrium. It is characterized by continuous inflammation and following fibrosis and dysfunction. However, the current animal IUA models have several disadvantages, including complex operation, high mortality, and many extra distractions owing to opening of the abdominal cavity to expose the uterus. Mesenchymal stem cells (MSCs), which have been used in treatment of IUA, are heterogeneous and immunosuppressive. However, their therapeutic effect is not as good as expected.

Methods: Here, we successfully built a new murine IUA model, called electric tool-scratching IUA model, and applied it in our experiments to investigate the efficacy of tumor necrosis factor-α (TNF-α) primed MSCs (T-MSCs). In the new model, we used a self-made electric tool that can cause mechanical damage to the endometrium without opening the abdominal cavity. ELISA and histological staining analysis were performed to evaluate pathological features of IUA. qRT-PCR, flow cytometry and immunofluoresence staining were performed to detect the phenotypes of macrophages. TMT proteomics quantification and western blotting assay were performed to analyze the differentially expressed proteins of MSC exosomes.

Results: Based on the new IUA model, we found TNF-α pretreatment could enhance the ability of MSCs to relieve inflammation and reduce endometrium fibrosis. Mechanistically, T-MSC promoted macrophage polarization to M2 phenotype through exosomes. Subsequently, we found the expression of Galectin-1 was increased in T-MSC exosomes. Finally, we analyzed the gene expression pattern of Galectin-1 treated macrophages and found Galectin-1 promoted macrophage polarization to M2 phenotype mainly through the Jak-STAT signaling pathway.

Conclusions: Our studies proposed an innovative mouse model and a better MSC treatment strategy for IUA.

Keywords: Galectin-1; exosomes; intrauterine adhesions; macrophages; mesenchymal stem cells.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The details of electric tool-scratching IUA model. (A) The photos of our electric tool. (B) The schematic diagram of modeling process, meaning the needle probe inserted into the uterus of mice. (C) The photograph of modeling process. Female Balb/c mice that were 8–10 weeks old were used. IUA, intrauterine adhesion.
Figure 2
Figure 2
Pathological features of electric tool-scratching IUA mice model. (A) The schematic diagram of modeling process. IUA mice model was established by electric tool-scratching way mentioned before. The early inflammatory phase and late fibrosis period respectively are 0.5 and 7 days after modeling. (B) H&E staining of murine uterine tissues. (C) The statistical figure of the endometrial thickness. (D) The statistical figure of gland numbers. (E) Relative expression of mRNAs of IL-1β, IL-6, and TNF-α in murine uterine tissues detected by qRT-PCR assay. (F) The level of IL-1β, IL-6, and TNF-α in serum detected by ELISA. (G) Masson’s trichrome staining of murine uterine tissues and statistical figure of collagen volume fraction. (H) The α-SMA protein expression detected by immunohistochemistry and statistical figure of numbers of α-SMA+ cells. (I) The PCNA protein expression detected by immunohistochemistry. (J) The CK19 protein expression detected by immunohistochemistry. Bar = 0.1 mm. The measurement data are presented as the means ± SEM, n = 6; *p < 0.05, **p < 0.01, ***p < 0.001. IUA, intrauterine adhesion; α-SMA, α-smooth muscle actin. ns, no significance.
Figure 3
Figure 3
TNF-α pretreatment enhances the therapeutic efficacy of MSCs in IUA mice. (A) The schematic diagram of modeling process. MSCs were stimulated by TNF-α (10 ng/ml) for 24 h to prepare T-MSCs. Both N-MSCs and T-MSCs (1 × 106 cells in 200 μl of PBS) were injected through the tail vein 2 h after the operation. (B) Relative expression of mRNAs of IL-1β, IL-6, and TNF-α in murine uterine tissues. (C) The expression levels of IL-1β, IL-6, and TNF-α in serum detected by ELISA. (D) (i) The top row: H&E staining of murine uterine tissues. (ii) The middle row: Masson’s trichrome staining of murine uterine tissues. (iii) The bottom row: α-SMA protein expression detected by immunohistochemistry. (E) The statistical figure of the endometrial thickness. (F) The statistical figure of gland numbers. (G) The statistical figure of collagen volume fraction. (H) The statistical figure of numbers of α-SMA+ cells. Bar = 0.1 mm. The measurement data are presented as the means ± SEM, n = 6; *p < 0.05, **p < 0.01, ***p < 0.001. TNF-α, tumor necrosis factor-α; MSCs, mesenchymal stem cells; IUA, intrauterine adhesion; T-MSCs, tumor necrosis factor-α-primed MSCs; N-MSCs, naïve MSCs; PBS, phosphate-buffered saline. ns, no significance.
Figure 4
Figure 4
T-MSCs promote macrophage polarization to M2 phenotype through exosomes. (A) Immunofluorescence staining of F4/80 and CD86 proteins in murine uterine tissues after IUA modeling for 0.5 days. On the right is the statistical figure of numbers of F4/80+ CD86+ cells. (B) Immunofluorescence staining of F4/80 and CD206 proteins in murine uterine tissues after IUA modeling for 0.5 days. On the right is the statistical figure of numbers of F4/80+ CD206+ cells. (C) The schematic diagram of four ways for MSCs to play the immunosuppressive roles on macrophages including direct contact, efferocytosis, paracrine, and exosomes. (D–G) The CD206 expression level on F4/80+ macrophages was detected by flow cytometry assay. (D) MSCs were added into mouse peritoneal macrophage culture environment (1:10) directly for 24 h. (E) CFSE-marked MSCs were added into mouse peritoneal macrophage culture environment (1:10) directly for 24 h. The CFSE+ macrophages were considered macrophages that have engulfed MSCs. (F) MSCs were added into macrophage culture environment (1:10) with transwell for 24 h. (G) The MSC-derived exosomes (25 μg/ml) were added into mouse peritoneal macrophage culture environment for 24 h. (H) Relative expression of mRNAs of CD206, IL-10, Arg-1, CD163, and HO-1 in exosome-treated mouse peritoneal macrophages. Bar = 0.1 mm. The measurement data are presented as the means ± SEM, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001. MSCs, mesenchymal stem cells; T-MSCs, tumor necrosis factor-α-primed MSCs; IUA, intrauterine adhesion; CFSE, carboxyfluorescein succinimidyl ester. ns, no significance.
Figure 5
Figure 5
The upregulated Galectin-1 in exosomes participates in immunosuppressive function of T-MSCs. (A, B) Proteomic analysis of exosomes from T-MSC group and N-MSC group. (A) Volcano plots for the distribution of differentially expressed proteins. The blue part represents downregulation of expression, the red part represents upregulation, and the gray part represents no significance. n = 3. (B) Heatmap of 37 differentially expressed proteins between T-MSCs and N-MSCs. The blue part represents downregulation of expression. The red part represents upregulation. (C) The protein expression of CD63, Galectin-1 (Gal-1), and GAPDH were detected by Western blotting assay. (D) Relative expression of mRNAs of CD206, IL-10, Arg-1, CD163, and HO-1 in exosome-treated macrophages for 24 h. The ON-MSCs and OT-MSCs mean the N- or T-MSCs with OTX008 (60 μM) pretreatment for 1 h, followed by stimulation with or without TNF-α (10 ng/ml) for 24 h. (H) Relative expression of mRNAs of CD206, IL-10, Arg-1, CD163, and HO-1 in rhGalectin-1 protein (1 μg/ml)-treated mouse peritoneal macrophages for 24 h. The measurement data are presented as the means ± SEM, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001. MSCs, mesenchymal stem cells; T-MSCs, tumor necrosis factor-α-primed MSCs; N-MSCs, naïve MSCs. ns, no significance.
Figure 6
Figure 6
Galectin-1 promoted macrophages to M2 mainly through Jak-STAT signaling pathway. (A, B) RNA-seq analysis of mouse peritoneal macrophages treated with or without rhGalectin-1 protein (1 μg/ml) for 24 h. (A) Volcano plots for the distribution of differentially expressed genes (Galectin-1 group/control group). The blue part represents downregulation of expression, the red part represents upregulation, and the gray part represents no significance. n = 3. (B) The KEGG pathways analysis of differentially expressed genes between Galectin-1 group and control group. The red part indicates the differential gene is extremely significantly enriched KEGG classification (p < 0.01); the blue part indicates the differential gene is significantly enriched KEGG classification (p < 0.05). The size of the circle represents the number of genes. (C) Mouse peritoneal macrophages were pretreated with or without ruxolitinib (1 μmol/L), BAY 11-7082 (1 μmol/L), VX-11e (100 nmol/L), and rapamycin (10 μmol/L) for 1 h, followed by stimulation with rhGalectin-1 protein (1 μg/ml) for 24 h. The mRNA expression levels of CD206, IL-10, Arg-1, CD163, and HO-1 in mouse peritoneal macrophages were determined by qRT-PCR. (D) Full-text summary diagram. The measurement data are presented as the means ± SEM, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001. KEGG, Kyoto Encyclopedia of Genes and Genomes. ns, no significance.
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