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. 2018 Jan 29;9(1):17.
doi: 10.1186/s13287-018-0774-8.

Mesenchymal stem cell-derived extracellular vesicles attenuate influenza virus-induced acute lung injury in a pig model

Mahesh Khatri  1 , Levi Arthur Richardson  2 , Tea Meulia  3
Affiliations

Mesenchymal stem cell-derived extracellular vesicles attenuate influenza virus-induced acute lung injury in a pig model

Mahesh Khatri et al. Stem Cell Res Ther. .

Abstract

Background: Mesenchymal stem (stromal) cells (MSCs) mediate their immunoregulatory and tissue repair functions by secreting paracrine factors, including extracellular vesicles (EVs). In several animal models of human diseases, MSC-EVs mimic the beneficial effects of MSCs. Influenza viruses cause annual outbreaks of acute respiratory illness resulting in significant mortality and morbidity. Influenza viruses constantly evolve, thus generating drug-resistant strains and rendering current vaccines less effective against the newly generated strains. Therefore, new therapies that can control virus replication and the inflammatory response of the host are needed. The objective of this study was to examine if MSC-EV treatment can attenuate influenza virus-induced acute lung injury in a preclinical model.

Methods: We isolated EVs from swine bone marrow-derived MSCs. Morphology of MSC-EVs was determined by electron microscopy and expression of mesenchymal markers was examined by flow cytometry. Next, we examined the anti-influenza activity of MSC-EVs in vitro in lung epithelial cells and anti-viral and immunomodulatory properties in vivo in a pig model of influenza virus.

Results: MSC-EVs were isolated from MSC-conditioned medium by ultracentrifugation. MSC-EVs were round-shaped and, similarly to MSCs, expressed mesenchymal markers and lacked the expression of swine leukocyte antigens I and II. Incubation of PKH-26-labeled EVs with lung epithelial cells revealed that MSC-EVs incorporated into the epithelial cells. Next, we examined the anti-influenza and anti-inflammatory properties of MSC-EVs. MSC-EVs inhibited the hemagglutination activity of avian, swine, and human influenza viruses at concentrations of 1.25-5 μg/ml. MSC-EVs inhibited influenza virus replication and virus-induced apoptosis in lung epithelial cells. The anti-influenza activity of MSC-EVs was due to transfer of RNAs from EVs to epithelial cells since pre-incubation of MSC-EVs with RNase enzyme abrogated the anti-influenza activity of MSC-EVs. In a pig model of influenza virus, intratracheal administration of MSC-EVs 12 h after influenza virus infection significantly reduced virus shedding in the nasal swabs, influenza virus replication in the lungs, and virus-induced production of proinflammatory cytokines in the lungs of influenza-infected pigs. The histopathological findings revealed that MSC-EVs alleviated influenza virus-induced lung lesions in pigs.

Conclusions: Our data demonstrated in a relevant preclinical large animal model of influenza virus that MSC-EVs possessed anti-influenza and anti-inflammatory properties and that EVs may be used as cell-free therapy for influenza in humans.

Keywords: Acute lung injury; Extracellular vesicles; Influenza; Large animal model; Mesenchymal stem cells; Stem cell therapy.

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Conflict of interest statement

Ethics approval and consent to participate

All experimental procedures involving pigs were conducted in accordance with the guidelines of the Institutional Laboratory Animal Care and Use Committee, The Ohio State University (protocol #2014A00000040).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Characteristics of MSC-derived EVs. Extracellular vesicles (EVs) were isolated from the conditioned medium of porcine bone marrow-derived mesenchymal stem cells (BM-MSCs) by ultracentrifugation. Morphology and size of MSC-EVs was examined by TEM. EVs were round-shaped and approximately 100 nm in size (×25 K). Expression of mesenchymal markers on MSCs and MSC-EVs was examined by flow cytometry. MSCs expressed the mesenchymal markers CD29, CD44, and CD90, and swine leukocyte antigen (SLA)-I, but SLA-II was not expressed. Similarly to MSCs, EVs expressed the mesenchymal markers but lacked the expression of SLA-I and SLA-II (black line: isotype staining; red line: specific staining)
Fig. 2
Fig. 2
Mesenchymal stem cell extracellular vesicles (MSC-EVs) express EV markers. EV-coated latex beads were examined for the expression of EV markers by flow cytometry. EVs expressed the specific EV markers CD9, CD63, and CD81 (broken line: isotype staining; solid line: specific staining)
Fig. 3
Fig. 3
MSC-EVs incorporate into lung epithelial cells. PKH-26 labeled extracellular vesicles (EVs) were incubated with LECs for 24 h. Incorporation of EVs in LECs was examined by fluorescent microscope and flow cytometry. The inset in the middle panel shows the internalization of EVs in the cytoplasm of LECs. Cellular cytoplasm was stained using β-tubulin antibody (green); EVs (red) were found to be localized inside the LECs (×200)
Fig. 4
Fig. 4
MSC-EVs inhibit influenza virus replication in lung epithelial cells. Swine/MN/08; H1N1 (SwIV; MOI = 1) was incubated with Dulbecco’s modified Eagle’s medium (DMEM) or 10 μg/ml MSC extracellular vesicles (EVs) for 20 min at room temperature. After the incubation, LECs were inoculated with virus-EV mixture or virus alone and incubated for 1 h at 37 °C. Influenza virus NP was detected at 8 h after infection (a) and SwIV-induced cytopathology was observed at 48 h after infection (b). SwIV-infected cells expressing NP were counted at 8 h after infection. Each bar represents mean ± SD of virus-infected cells in five microscopic fields (20×) (c). Virus titers in supernatants of SwIV-infected and MSC-EV-treated cells at 48 h after infection were determined by titration in MDCK cells. Data are expressed as mean ± SD from three independent experiments using EVs derived from BM-MSCs from three different pigs (d). *P < 0.05
Fig. 5
Fig. 5
MSC-EVs inhibit influenza virus-induced apoptosis. a Swine/MN/08; H1N1 (SwIV; MOI = 1) was incubated with Dulbecco’s modified Eagle’s medium (DMEM) or 10 μg/ml MSC extracellular vesicles (EVs) for 20 min at room temperature. After the incubation, pig lung epithelial cells were incubated for 1 h with SwIV or SwIV pre-incubated with MSC-EVs. At 24 h after infection, apoptotic cells were detected by TUNEL assay using the ApopTag Fluorescein Apoptosis Detection Kit (EMD Millipore). b TUNEL-positive cells in SwIV-infected and MSC-EV-treated LECs were counted at 24 h after infection. Values are expressed as mean ± SD of apoptotic cells in five microscopic fields (20×). *P < 0.05
Fig. 6
Fig. 6
MSC-EVs inhibit influenza virus replication after virus entry in lung epithelial cells. a Pig lung epithelial cells were infected with swine/MN/08; H1N1 (SwIV; MOI = 1) for 1 h; after the adsorption, cells were washed and cultured in media only or media containing 10 μg/ml MSC extracellular vesicles (EVs) or RNase-treated MSC-EVs. Influenza virus NP was detected 8 h after infection. b Each bar represents mean ± SD of virus-infected cells in five microscopic fields (20×). Experiments were repeated three times using EVs derived from BM-MSCs from three different pigs. *P < 0.05. DMEM Dulbecco’s modified Eagle’s medium
Fig. 7
Fig. 7
Effect of MSC-EV administration on microscopic lung lesions in pigs infected with SwIV. Eight-week-old pigs were mock infected or infected with swine/MN/08; H1N1 (SwIV). After 12 h, pigs were administered with Dulbecco’s modified Eagle’s medium (DMEM) or MSC extracellular vesicles (EVs). Three days after EV administration, pigs were euthanized and microscopic lung lesions and levels of total protein in bronchoalveolar lavage (BAL) were examined. a Control uninfected lung, showing normal alveolar walls, clear air space, and absence of exudation into the alveolar space. b SwIV induced exudative interstitial pneumonia characterized by thickened alveolar walls, collapsed alveolar spaces, and infiltration of inflammatory cells, whereas c lungs of pigs inoculated with MSC-EVs 12 h after SwIV infection show mild infiltration of inflammatory cells. Hematoxylin and eosin stain. Magnification × 200. d,e Histopathological scores. Lung tissue slides were examined for bronchiolar epithelial changes, peribronchiolar inflammation, and interstitial pneumonia, and lesions were scored from 0–3 (d). Values in each bar indicate mean microscopic lung lesions of three pigs ± SD. e MSC-EV administration decreased the levels of total protein in the BAL of SwIV + EV administered pigs as compared with SwIV + DMEM inoculated pigs. Data are expressed as mean levels of total protein in BAL of three pigs ± SD
Fig. 8
Fig. 8
Effect of MSC-EV administration on virus titers in nasal swabs and lungs of pigs infected with SwIV. Eight-week-old pigs were mock infected or infected with swine/MN/08; H1N1 (SwIV). After 12 h, pigs were administered intratracheally with Dulbecco’s modified Eagle’s medium (DMEM) or MSC extracellular vesicles (EVs). Nasal swabs were collected from infected pigs at 1 and 3 days after EV administration (1DPEV and 3DPEV). At 3 days after EV administration, pigs were euthanized and lungs were harvested. Lung tissues were homogenized to prepare 10% lung lysate. Influenza virus shedding in nasal swabs and virus titers in lungs were determined by titration in MDCK cells. Values in each bar indicate mean virus titers of three pigs ± SD
Fig. 9
Fig. 9
Effect of MSC-EV administration on cytokine production in lungs of SwIV-infected pigs. Eight-week-old pigs were mock infected or infected with swine/MN/08; H1N1 (SwIV). After 12 h, pigs were administered intratracheally with Dulbecco’s modified Eagle’s medium (DMEM) or MSC extracellular vesicles (EVs). Three days after EV administration, pigs were euthanized and cytokine production in lung lysate was analyzed by ELISA. Each bar represents mean concentrations of cytokines ± SD from three pigs. IL interleukin, TNF tumor necrosis factor
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