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. 2022 Sep 27;22(5):e40.
doi: 10.4110/in.2022.22.e40. eCollection 2022 Oct.

Mesenchymal Stem Cells Attenuate Asthmatic Inflammation and Airway Remodeling by Modulating Macrophages/Monocytes in the IL-13-Overexpressing Mouse Model

Yosep Mo  1   2 , Yujin Kim  1   2 , Ji-Young Bang  1   2 , Jiung Jung  3 , Chun-Geun Lee  4 , Jack A Elias  4 , Hye-Ryun Kang  1   2   3   5
Affiliations

Mesenchymal Stem Cells Attenuate Asthmatic Inflammation and Airway Remodeling by Modulating Macrophages/Monocytes in the IL-13-Overexpressing Mouse Model

Yosep Mo et al. Immune Netw. .

Abstract

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs, focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations, gene expression, and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma, including severe type 2 inflammation, airway fibrosis, and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF-CD11c-CD11b+ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages, especially M2a and M2c. Furthermore, MSCs downregulated the excessive accumulation of Ly6c- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c- monocytes promoted the infiltration of MoM and Th2 inflammation, that of MSC-exposed Ly6c- monocytes did not. Ex vivo Ly6c- MoMs upregulated M2-related genes, which were reduced by MSC treatment. Molecules secreted by Ly6c- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts, which were also suppressed by MSC treatment. In conclusion, intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c- monocytes could be a therapeutic target for asthma management.

Keywords: Asthma; Interleukin-13; Macrophages; Mesenchymal stem cells; Monocytes.

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Conflict of interest statement

Conflict of Interest: The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. Intravenous MSCs administration attenuated the chronic IL-13 overexpression-associated changes, including Th2 inflammation. (A) MSCs (1×105 cells) were intravenously administered to 7–8 weeks old littermate WT and IL-13 Tg mice four days before sacrifice. (B) The number of inflammatory cells including macrophages, neutrophils, eosinophils, and lymphocytes in BAL fluid. (C) The number of eosinophils and neutrophils in the lungs by flow cytometry analysis. (D) Lung histology and semi-quantitative grading of lung inflammation score (H&E staining, 400× magnification). (E) Lung histology and semi-quantitative grading of mucus score (PAS staining, 200× magnification). (F) Semi-quantitative grading of fibrosis score (MT staining, 100× magnification). (G) The relative gene expression of Muc5ac in the lung. (H) The quantification of soluble collagen in the lung tissue by Sircol collagen assay.
PAS, periodic acid schiff; MT, masson’s trichrome; Muc5ac, mucin 5ac. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 2
Figure 2. Intravenous MSCs administration alleviated airway Th2 inflammation and promoted Tregs. (A) The percentages of IL-5+ CD4+ T cells, IL-13+ CD4+ T cells, IFN-γ+ CD4+ T cells and IL-17+ CD4+ T cells in the lung. (B) The percentages of Foxp3+CD25+ Tregs among CD4+ T cells and the number of IL-10+ Tregs in the lung. (C) Isolated Tregs from the spleen were co-cultured with MSCs (1×104 cells) or PBS directly/indirectly for two days and the number of Foxp3+CD25+ Tregs were analyzed by flow cytometry.
*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 3
Figure 3. Intravenous MSCs administration decreased the ILC2 population. (A) The percentages of IL-5+ ILCs, IL-13+ ILCs, IFN-γ+ ILCs and IL-17+ ILCs in the lung. (B) Isolated ILCs from the lung were co-cultured with MSCs (1×104 cells) or PBS directly/indirectly for one day and the percentages of the ILC2 population expressed with each of IL-5+, IL-13+, ST2+, and GATA3+ were analyzed by flow cytometry.
*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 4
Figure 4. Intravenous MSCs administration altered lung macrophage populations. (A) The FACS gating strategy of measuring macrophages subpopulation within total macrophages. (B) The number of total macrophages in the lung. (C) The percentages of SiglecF+CD11c+CD11b- AM, SiglecF+CD11C+CD11b+ transient AM, and SiglecF+CD11C-CD11b+ macrophages among total macrophages. The number of SiglecF-CD11c-CD11b+ MoMs among total macrophages. (D) The number of CD206+ M2 and CD86+ M1 among MoMs and in the lung. The number of CD206+CD86-MHCII+ M2a in the M2 subpopulation. The percentages of CD206+CD86-MHCII+ M2a, CD206+CD86-MHCII- M2c, and CD206-CD86+MHCII+ M2b among SiglecF-CD11c-CD11b+ MoMs in the lung. (E) Isolated CD11b- AMs and CD11b+ MoMs from the lung were co-cultured with MSCs (1×104 cells) or PBS directly/indirectly for one day and the gene expression of M2 phenotype-related Cd163, Tgfb1, and Ym1 were analyzed by qRT-PCR analysis.
*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 5
Figure 5. Effects of intravenous MSCs administration on non-classical Ly6c- monocytes in the lung. (A) The total number of total monocytes, Ly6c+ monocytes, and Ly6c- monocytes in the lung. (B) The Ly6c+/Ly6c- monocytes ratio in the lung. (C) Isolated Ly6c+ and Ly6c- monocytes from the bone marrow were co-cultured with MSCs (1×104 cells) or PBS on M-CSF stimulation for five days and harvested one day after IL-13 stimulation. The gene expression of Itgam and M2 phenotype-related Tgfb1, Mrc1, Ym1, and H2-Ab were measured using qRT-PCR. (D) Lung fibroblast cell lines were cultured on TGF-β stimulation for three days in the culture medium of Ly6c- monocytes, which were co-cultured with/without MSCs and were stimulated by the M-CSF. The gene expression of fibrosis-related Fgf1 and Fn1 was analyzed using qRT-PCR.
*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 6
Figure 6. MSC-exposed Ly6c- monocytes suppress the infiltration of monocyte into the lung and Th2 inflammation. (A) MSC-exposed Ly6c- monocytes were adoptively transferred into HDM-stimulated mice via the tail vein. (B) The number of inflammatory cells including macrophages, neutrophils, eosinophils, and lymphocytes in BAL fluid. (C) Lung histology and semi-quantitative grading of lung inflammation score (H&E staining, 400× magnification); Lung histology and semi-quantitative grading of mucus score (PAS staining, 200× magnification); Semi-quantitative grading of fibrosis score (MT staining, 200× magnification). (D) The percentages of IL-5+ CD4+ T cells and IL-13+ CD4+ T cells in the lung and their dot plot. The percentages of Foxp3+CD25+ Tregs among CD4+ T cells and IL-10+ Tregs in the lung and their dot plot. (E) The percentages of SiglecF-CD11c-CD11b+ MoMs and SiglecF+CD11c+CD11b- AMs among total macrophages. The percentages of CD86+ M1 and CD206+ M2 among MoMs in the lung. The percentages of CD206-CD86+MHCII+ M2b MoMs, CD206+CD86-MHCII+ M2a MoMs, and CD206+CD86-MHCII- M2c MoMs among total macrophages in the lung. The percentages of Ly6c- non-classical macrophage and Ly6c+ classical macrophage among total macrophages in the lung. PAS, periodic acid schiff; MT, masson’s trichrome.
*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 7
Figure 7. Co-culture with human MSC suppressed the differentiation of M2 macrophages and Th2 cells of hPBMCs. (A) Isolated hPBMCs from allergic asthma patients cultured with hM-CSF for five days and stimulated with IL-13 for one day to induce M2 macrophages. Then, hMSCs (1×104 cells) were treated for one day and harvested for analysis. (B) The M2 phenotype-related MRC1, HLA-DR, and TGFB1 were measured using qRT-PCR. (C) A cell plate was coated with 1 μg/ml CD3 Monoclonal Antibodies for one day. After coating, 1 x 105 hPBMCs were seeded with CD28 Monoclonal Antibodies and stimulated with recombinant IL-2, IL-4, and IL-13 for 3 days. Finally, following one day of MSCs (1×104 cells) exposure, cells were harvested for analysis. (D) Th2 cytokines, including IL4, IL5, and IL13 were analyzed using qRT-PCR.
*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
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