Mesenchymal stem cells exert their anti-asthmatic effects through macrophage modulation in a murine chronic asthma model

Abstract

Despite numerous previous studies, the full action mechanism of the pathogenesis of asthma remains undiscovered, and the need for further investigation is increasing in order to identify more effective target molecules. Recent attempts to develop more efficacious treatments for asthma have incorporated mesenchymal stem cell (MSC)-based cell therapies. This study aimed to evaluate the anti-asthmatic effects of MSCs primed with Liproxstatin-1, a potent ferroptosis inhibitor. In addition, we sought to examine the changes within macrophage populations and their characteristics in asthmatic conditions. Seven-week-old transgenic mice, constitutively overexpressing lung-specific interleukin (IL)-13, were used to simulate chronic asthma. Human umbilical cord-derived MSCs (hUC-MSCs) primed with Liproxstatin-1 were intratracheally administered four days prior to sampling. IL-13 transgenic mice demonstrated phenotypes of chronic asthma, including severe inflammation, goblet cell hyperplasia, and subepithelial fibrosis. Ly6C⁺M2 macrophages, found within the pro-inflammatory CD11c⁺CD11b⁺ macrophages, were upregulated and showed a strong correlation with lung eosinophil counts. Liproxstatin-1-primed hUC-MSCs showed enhanced ability to downregulate the activation of T helper type 2 cells compared to naïve MSCs in vitro and reduced airway inflammation, particularly Ly6C⁺M2 macrophages population, and fibrosis in vivo. In conclusion, intratracheal administration is an effective method of MSC delivery, and macrophages hold great potential as an additional therapeutic target for asthma.

Figure 1

Liproxstatin-1-primed hUC-MSCs alleviated airway inflammation mitigated pulmonary fibrosis in vivo. (a) Shows the result of total cell counts and (b) shows differential counts of macrophages, neutrophils, eosinophils, and lymphocytes in BAL fluid. (c) Shows microscopic images of Diff-Quick-stained slides of cytocentrifuged BAL fluid (× 40). Samples from IL-13 TG groups were diluted with doubled amount of PBS compared with the samples from the wild type groups. (d–e) shows histological comparison between the groups at a power of 20. H&E (d) and PAS (e) stains were used. (f) Shows Masson’s Trichrome-stained slides of each group at low power (× 20). (g–h) shows semi-quantitative grading results of H&E staining (g) and PAS staining (h). Sircol collagen assay was performed to quantify the soluble collagen contents in the homogenates of the lungs (i). (j–n) represents relative gene expressions of Muc5ac (j), Fgf1 (k), Fn1 (l), Mmp-9 (m), and Mmp-12 (n) compared to the housekeeping, Gapdh. Each value in these panels is from a different individual and the mean ± SEM are illustrated. All results are representative of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. (by Kruskal–Wallis test using GraphPad Prism 7, https://www.graphpad.com).

Figure 2

Liproxstatin-1-primed hUC-MSCs altered macrophage populations in the lung. (a) Flow cytometry data showing two distinct macrophage populations that differ by levels of CD11b and F4/80 expression. Numbers represent percentages among total CD45⁺ leukocytes, excluding SiglecF⁺CD11c⁻ eosinophils. Based on the flow cytometry representations shown in (a), percentages of CD11b^highF4/80^int macrophages (b) and CD11b^intF4/80^high macrophages (c) are each portrayed in graphs. (d) Represents a histogram of Ly6C and CX₃CR1 expression levels of CD11b^intF4/80^high macrophages and CD11b^highF4/80^int macrophages. (e) Represents the MFI of Ly6C of CD11b^highF4/80^int macrophages and (f) shows the MFI of CX₃CR1 of CD11b^intF4/80^high macrophages. (g) Shows the percentages of Ly6⁺ macrophages among CD11b^highF4/80^int macrophages. Flow cytometry data were analyzed by the FlowJo Software v10.6, https://www.flowjo.com. Each value in these panels is from a different individual and the mean ± SEM are illustrated. All results are representative of at least three independent experiments. *p < 0.05, ns not significant. (by Kruskal–Wallis test using GraphPad Prism 7, https://www.graphpad.com).

Figure 3

Liproxstatin-1-primed hUC-MSCs caused a reduction in the number of CD11c⁺CD11b⁺ pro-inflammatory macrophages. (a) shows a flow cytometric representation of macrophage populations divided into four groups by their expression of CD11b and CD11c. (b, c) show percentages of CD11c⁺CD11b⁻ (b) and CD11c⁺CD11b⁺ macrophages (c) among total macrophages. (d) Percentage of lung eosinophils out of total CD45⁺ cells are represented in (d). (e) shows the absolute counts of CD11c⁺CD11b⁺SiglecF⁺ macrophages and (f) represents a histogram of SiglecF expressions of CD11c⁺CD11b⁺, CD11c⁻CD11b⁺, CD11c⁺CD11b⁻, and CD11c⁻CD11b⁻ macrophages. Flow cytometry data were analyzed by the FlowJo Software v10.6, https://www.flowjo.com. Each value in these panels is from a different individual and the mean ± SEM are illustrated. All results are representative of at least three independent experiments. *p < 0.05, **p < 0.01, ns, not significant. (by Kruskal–Wallis test using GraphPad Prism 7, https://www.graphpad.com).

Figure 4

Ly6C⁺M2 macrophages are key mediators of asthmatic phenotypes. (a) Flow cytometric data on M1 and M2 macrophages of CD11c⁺CD11b⁺ macrophages. The percentages of CD86⁺CD206⁻ M1 (b), CD86⁻CD206⁺ M2 macrophages (c), and CD86⁺CD206⁺ macrophages (d) are represented in scatter plots. (e) the MFI of IL-5 in CD86⁺CD206⁻ M1 and CD86⁻CD206⁺ M2 macrophages in IL-13 TG mice. (f) Shows Ly6C expression levels in M1 and M2 macrophages of WT and IL-13 TG mice. (g, h) shows the ratio of Ly6C⁺M2 and LY6C⁻M2 macrophage populations among the CD11c⁺CD11b⁺ macrophages. Flow cytometry data were analyzed by the FlowJo Software v10.6, https://www.flowjo.com. Each value in these panels is from a different individual and the mean ± SEM are illustrated. All results are representative of at least three independent experiments. *p < 0.05, **p < 0.01, ns not significant. (by Kruskal–Wallis test using GraphPad Prism 7, https://www.graphpad.com).

Figure 5

Effects of Liproxstatin-1-primed hUC-MSCs on murine macrophages. (a–c) Murine macrophages were co-cultured with hUC-MSCs in a trans-well system and ex vivo alveolar macrophages (a) and bone marrow-derived macrophages (b, c) were subjected to RT-qPCR analysis for mRNA evaluation of markers of polarized macrophages. Gapdh and Hprt1 were used as internal controls. Values in all the panels are mean ± SEM. All results are representative of at least three independent experiments. *p < 0.05, **p < 0.01, ns stands for not significantly different (by Kruskal Wallis test using GraphPad Prism 7, https://www.graphpad.com). Il-13 interleukin 13, Mmp-12 matrix metallopeptidase 12, Tgf-β transforming growth factor beta.