Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma

Ligia Lins de Castro, Debora Gonçalves Xisto, Jamil Zola Kitoko, Fernanda Ferreira Cruz, Priscilla Christina Olsen, Patricia Albuquerque Garcia Redondo, Tatiana Paula Teixeira Ferreira, Daniel Jay Weiss, Marco Aurélio Martins, Marcelo Marcos Morales, Patricia Rieken Macedo Rocco, Ligia Lins de Castro, Debora Gonçalves Xisto, Jamil Zola Kitoko, Fernanda Ferreira Cruz, Priscilla Christina Olsen, Patricia Albuquerque Garcia Redondo, Tatiana Paula Teixeira Ferreira, Daniel Jay Weiss, Marco Aurélio Martins, Marcelo Marcos Morales, Patricia Rieken Macedo Rocco

Abstract

Background: Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma.

Methods: C57BL/6 female mice were sensitized and challenged with ovalbumin (OVA). Control (CTRL) animals received saline solution using the same protocol. One day after the last challenge, each group received saline, 105 human AD-MSCs, or EVs (released by 105 AD-MSCs). Seven days after treatment, animals were anesthetized for lung function assessment and subsequently euthanized. Bronchoalveolar lavage fluid (BALF), lungs, thymus, and mediastinal lymph nodes were harvested for analysis of inflammation. Collagen fiber content of airways and lung parenchyma were also evaluated.

Results: In OVA animals, AD-MSCs and EVs acted differently on static lung elastance and on BALF regulatory T cells, CD3+CD4+ T cells, and pro-inflammatory mediators (interleukin [IL]-4, IL-5, IL-13, and eotaxin), but similarly reduced eosinophils in lung tissue, collagen fiber content in airways and lung parenchyma, levels of transforming growth factor-β in lung tissue, and CD3+CD4+ T cell counts in the thymus. No significant changes were observed in total cell count or percentage of CD3+CD4+ T cells in the mediastinal lymph nodes.

Conclusions: In this immunocompetent mouse model of allergic asthma, human AD-MSCs and EVs effectively reduced eosinophil counts in lung tissue and BALF and modulated airway remodeling, but their effects on T cells differed in lung and thymus. EVs may hold promise for asthma; however, further studies are required to elucidate the different mechanisms of action of AD-MSCs versus their EVs.

Keywords: Asthma; Extracellular vesicles; Inflammation; Mesenchymal stromal cells; Remodeling.

Figures

Fig. 1
Fig. 1
Characterization of EVs. a Representative graph of the intensity and hydrodynamic diameter of extracellular vesicle samples analyzed using the dynamic light scattering technique. Graph showing two populations of extracellular vesicles obtained from human adipose tissue-derived mesenchymal stromal cells, one of lower intensity and medium size, characteristic of exosomes, and another with greater intensity and average size, characteristic of microvesicles. b, c, and d Scanning electron microscopy of mesenchymal stromal cells. b Mesenchymal stromal cells before induction of cellular stress (fetal bovine serum deprivation), showing the presence of few extracellular vesicles. c Image obtained 12 hours after cellular stress induction, showing increase in the number of vesicles in the cell surface. d Higher magnification of image C, showing extracellular vesicles with some indicative sizes
Fig. 2
Fig. 2
Experimental design. C57BL/6 female mice were divided into two groups: CTRL (sensitized and challenged with saline) and OVA (sensitized and challenged with ovalbumin). Seven intraperitoneal (i.p.) sensitizations were performed. On days 40, 43, and 46 after first sensitization, an intratracheal challenge (i.t.) was performed. Treatment was administered intravenously (i.v.); 1 day after the last challenge and 7 days after this treatment, the animals were euthanized for data acquisition. The treatment consisted of saline (SAL), human adipose-derived mesenchymal stromal cells (AD-MSC), or extracellular vesicles (EV) at a dose equivalent to 105 AD-MSC (37 μg of total protein)
Fig. 3
Fig. 3
Lung mechanics. a Static lung elastance (Est,L). b Lung resistive (ΔP1, white bar) and viscoelastic (ΔP2, gray bar) pressures. Data are presented as means ± SD of seven animals/group. CTRL mice sensitized and challenged with saline, OVA mice sensitized and challenged with ovalbumin, OVA-SAL OVA mice treated with saline, OVA-AD-MSC OVA mice treated with mesenchymal stromal cells derived from adipose tissue (AD-MSCs), OVA-EV OVA mice treated with extracellular vesicles derived from AD-MSCs. CTRL versus OVA-SAL (Est,L p = 0.04, ΔP1, p = 0.003, ΔP2, p = 0.005)
Fig. 4
Fig. 4
Representative photomicrographs of lung parenchyma (upper panels) and airway (lower panels) stained with Sirius Red under polarization. Percentage of collagen fiber content in lung parenchyma and airway. Levels of transforming growth factor beta (TGF-β) in lung tissue. Data are presented as means + SD of seven animals/group. CTRL mice sensitized and challenged with saline, OVA mice sensitized and challenged with ovalbumin, OVA-SAL OVA mice treated with saline, OVA-AD-MSC OVA mice treated with mesenchymal stromal cells derived from adipose tissue (AD-MSCs), OVA-EV OVA mice treated with extracellular vesicles derived from AD-MSCs. CTRL versus OVA-SAL (parenchyma p < 0.0001, airway p = 0.0002, TGF-β p = 0.0007)
Fig. 5
Fig. 5
Representative photomicrographs of lung parenchyma stained with Sirius Red. Squares indicate peribronchial area. Lower panel: number of eosinophils in the peribronchial area. Data are presented as means + SD of seven animals/group. aCTRL mice sensitized and challenged with saline, bOVA-SALOVA mice sensitized and challenged with ovalbumin and then treated with saline, cOVA-AD-MSC OVA mice treated with mesenchymal stromal cells derived from adipose tissue (AD-MSCs), dOVA-EV OVA mice treated with extracellular vesicles derived from AD-MSCs, e Number of eosinophils in lung parenchyma. CTRL versus OVA-SAL (p < 0.0001)
Fig. 6
Fig. 6
Total cell count and percentage of T cells (CD3+CD4+), Treg cells (CD4+CD25+Foxp3+), and eosinophils (Siglec-F+) in bronchoalveolar lavage fluid (BALF). Data are presented as means + SD of ten animals/group. CTRL mice sensitized and challenged with saline, OVA mice sensitized and challenged with ovalbumin, OVA-SAL OVA mice treated with saline, OVA-AD-MSC OVA mice treated with mesenchymal stromal cells derived from adipose tissue (AD-MSCs), OVA-EV OVA mice treated with extracellular vesicles derived from AD-MSCs. CTRL versus OVA-SAL (total cell count p < 0.0001, eosinophils p = 0.0006, Treg cells p < 0.0001, T cells p = 0.034)
Fig. 7
Fig. 7
Levels of interleukin (IL)-4, IL-5, IL-13, eotaxin, IL-10, and interferon (IFN)-γ. Data are presented as median and interquartile range of seven animals/group. CTRL mice sensitized and challenged with saline, OVA mice sensitized and challenged with ovalbumin, OVA-SAL OVA mice treated with saline, OVA-AD-MSC OVA mice treated with mesenchymal stromal cells derived from adipose tissue (AD-MSCs), OVA-EV OVA mice treated with extracellular vesicles derived from AD-MSCs. CTRL versus OVA-SAL (IL-13 p = 0.004, eotaxin p = 0.0011)
Fig. 8
Fig. 8
Total cell count and percentage of T cells (CD3+CD4+) counts in thymus and mediastinal lymph nodes. Data are presented as means + SD of ten animals/group. CTRL mice sensitized and challenged with saline, OVA mice sensitized and challenged with ovalbumin, OVA-SAL OVA mice treated with saline, OVA-AD-MSC OVA mice treated with mesenchymal stromal cells derived from adipose tissue (AD-MSCs), OVA-EV OVA mice treated with extracellular vesicles derived from AD-MSCs. CTRL versus OVA-SAL (thymus p = 0.0002)

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