Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jul 13;12(1):397.
doi: 10.1186/s13287-021-02469-5.

Human umbilical cord mesenchymal stromal cells attenuate pulmonary fibrosis via regulatory T cell through interaction with macrophage

Zan Tang #  1   2 , Junxiao Gao #  1 , Jie Wu  2 , Guifang Zeng  2 , Yan Liao  2 , Zhenkun Song  2 , Xiao Liang  2 , Junyuan Hu  2 , Yong Hu  3 , Muyun Liu  4 , Nan Li  5
Affiliations

Human umbilical cord mesenchymal stromal cells attenuate pulmonary fibrosis via regulatory T cell through interaction with macrophage

Zan Tang et al. Stem Cell Res Ther. .

Abstract

Background: Pulmonary fibrosis (PF) is a growing clinical problem with limited therapeutic options. Human umbilical cord mesenchymal stromal cell (hucMSC) therapy is being investigated in clinical trials for the treatment of PF patients. However, little is known about the underlying molecular and cellular mechanisms of hucMSC therapy on PF. In this study, the molecular and cellular behavior of hucMSC was investigated in a bleomycin-induced mouse PF model.

Methods: The effect of hucMSCs on mouse lung regeneration was determined by detecting Ki67 expression and EdU incorporation in alveolar type 2 (AT2) and lung fibroblast cells. hucMSCs were transfected to express the membrane localized GFP before transplant into the mouse lung. The cellular behavior of hucMSCs in mouse lung was tracked by GFP staining. Single cell RNA sequencing was performed to investigate the effects of hucMSCs on gene expression profiles of macrophages after bleomycin treatment.

Results: hucMSCs could alleviate collagen accumulation in lung and decrease the mortality of mouse induced by bleomycin. hucMSC transplantation promoted AT2 cell proliferation and inhibited lung fibroblast cell proliferation. By using single cell RNA sequencing, a subcluster of interferon-sensitive macrophages (IFNSMs) were identified after hucMSC infusion. These IFNSMs elevate the secretion of CXCL9 and CXCL10 following hucMSC infusion and recruit more Treg cells to the injured lung.

Conclusions: Our study establishes a link between hucMSCs, macrophage, Treg, and PF. It provides new insights into how hucMSCs interact with macrophage during the repair process of bleomycin-induced PF and play its immunoregulation function.

Keywords: Human umbilical cord mesenchymal stromal cell; Macrophage; Regulatory T cell; Single cell RNA sequencing.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
hucMSCs alleviated bleomycin-induced pulmonary fibrosis. A Scheme of the experimental timeline for the bleomycin-induced pulmonary fibrosis model and hucMSC infusion. B H&E-stained lung sections from saline-treated mice, bleomycin-treated mice, and hucMSC-treated mice at various time points. C Hydroxyproline contents of saline-treated mice lungs, bleomycin-treated mice lungs, and hucMSC-treated mice lungs at various time points (mean ± SD, n = 3 mice per group). D Survival curves of saline-treated mice, bleomycin-treated mice, and hucMSC-treated mice (n = 12 mice per group). *p < 0.05, **p < 0.01, n.s. no significant difference, Student’s t-test
Fig. 2
Fig. 2
hucMSC infusion promoted lung regeneration. A Day 7 after bleomycin treatment, lung sections were stained with antibodies against proSPC and Ki67. White arrowheads indicate proliferating alveolar type 2 cells. B The percentage of Ki67-positive alveolar type 2 cells (mean ± SD, n = 3 mice per group). C Flow cytometry analysis of EdU incorporation in alveolar type 2 cells in saline-treated mice lungs, bleomycin-treated mice lungs, and hucMSC-treated mice lungs on day 7 and day 14. D The percentage of EdU-positive alveolar type 2 cells (mean ± SD, n = 3 mice per group). E Flow cytometry analysis of EdU incorporation in lung fibroblast cells in saline-treated mice lungs, bleomycin-treated mice lungs, and hucMSC-treated mice lungs on day 7 and day 14. F The percentage of EdU-positive lung fibroblast cells (mean ± SD, n = 3 mice per group). *p < 0.05, **p < 0.01, n.s. no significant difference, Student’s t-test
Fig. 3
Fig. 3
hucMSCs interacted with macrophages after infusion. A Immunofluorescent labeling of lung sections after GFP-labeled hucMSC infusion in bleomycin-treated lungs at various time points. B Lung sections were stained with antibodies against GFP and F4/80. C–I Nos2, Cxcl9, Cxcl10, Mrc1, Arg1, Chil3, and Tgm2 expression levels in bleomycin-treated lungs and hucMSC-treated lungs on day 7 and day 14 by whole lung qRT-PCR. Nos2, Cxcl9, and Cxcl10 expression levels (mean ± SD, n = 3 mice per group) were significantly increased in hucMSC-treated lungs compared to bleomycin-treated lungs at day 7 (C–E). Mrc1, Arg1, Chil3, and Tgm2 expression levels (mean ± SD, n = 3 mice per group) were significantly decreased in hucMSC-treated lungs compared to bleomycin-treated lungs at day 14 (F-I). The expression level of markers and cytokines in bleomycin-treated lungs and hucMSC-treated lungs was compared to saline-treated mice lungs. *p < 0.05, **p < 0.01, n.s. no significant difference, Student’s t-test
Fig. 4
Fig. 4
Single cell RNA sequencing revealed an interferon-sensitive macrophage population in hucMSCs infused lungs. A UMAP plot of macrophages from bleomycin- and hucMSC-treated mouse lungs. B, C The proportions of different macrophage clusters. D Heatmap of some differentially expressed genes between each macrophage cluster. E t-SNE visualization overlaid with the expression of Spp1, H2-Eb1, Rsad2, F13a1, Car4, and Nr4a1. F Violin plots of Rsad2, Isg15, Ifit1, Ifit2, Ifit3, Ifi204, Ifi205, and Irf7 expression in each macrophage cluster
Fig. 5
Fig. 5
Macrophage and hucMSCs interaction increased interferon-responsive gene expression in macrophages. A Diagram of macrophage and hucMSCs co-culture. B Rsad2, Isg15, Ifit1, Ifit2, Ifit3, Ifi204, Ifi205, and Irf7 expression levels in macrophage co-cultured hucMSCs (mean ± SD, n = 3 wells per group). C Violin plots of Cxcl9 and Cxcl10 in each macrophage cluster. D Cxcl9 and Cxcl10 expression levels in macrophages co-cultured with hucMSCs (mean ± SD, n = 3 wells per group). E Flow cytometry analysis of Foxp3 regulatory T cells in saline-treated mice lungs, bleomycin-treated mice lungs, and hucMSC-treated mice lung. F Quantification of Foxp3-positive Tregs in saline-treated mice lungs, bleomycin-treated mice lungs, and hucMSC-treated mice lungs at day 7 (mean ± SD, n = 3 mice per group). G Working model showing that hucMSCs interact with macrophage to recruit Tregs into lung. *p < 0.05, ***p < 0.001, n.s. no significant difference, Student’s t-test
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aran D, Looney AP, Liu L, Wu E, Fong V, Hsu A, Chak S, Naikawadi RP, Wolters PJ, Abate AR, Butte AJ, Bhattacharya M. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat Immunol. 2019;20(2):163–172. doi: 10.1038/s41590-018-0276-y. - DOI - PMC - PubMed
    1. Atabai K, Jame S, Azhar N, Kuo A, Lam M, McKleroy W, Dehart G, Rahman S, Xia DD, Melton AC, et al. Mfge8 diminishes the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages. J Clin Invest. 2009;119(12):3713–3722. doi: 10.1172/JCI40053. - DOI - PMC - PubMed
    1. Barkauskas CE, Cronce MJ, Rackley CR, Bowie EJ, Keene DR, Stripp BR, Randell SH, Noble PW, Hogan BL. Type 2 alveolar cells are stem cells in adult lung. J Clin Invest. 2013;123(7):3025–3036. doi: 10.1172/JCI68782. - DOI - PMC - PubMed
    1. Baumgartner KB, Samet JM, Stidley CA, Colby TV, Waldron JA. Cigarette smoking: a risk factor for idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 1997;155(1):242–248. doi: 10.1164/ajrccm.155.1.9001319. - DOI - PubMed
    1. Byrne AJ, Maher TM, Lloyd CM. Pulmonary macrophages: a new therapeutic pathway in fibrosing lung disease? Trends Mol Med. 2016;22(4):303–316. doi: 10.1016/j.molmed.2016.02.004. - DOI - PubMed

Publication types