Systemic Administration of Human Bone Marrow-Derived Mesenchymal Stromal Cell Extracellular Vesicles Ameliorates Aspergillus Hyphal Extract-Induced Allergic Airway Inflammation in Immunocompetent Mice

Abstract

An increasing number of studies demonstrate that administration of either conditioned media (CM) or extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) derived from bone marrow and other sources are as effective as the MSCs themselves in mitigating inflammation and injury. The goal of the current study was to determine whether xenogeneic administration of CM or EVs from human bone marrow-derived MSCs would be effective in a model of mixed Th2/Th17, neutrophilic-mediated allergic airway inflammation, reflective of severe refractory asthma, induced by repeated mucosal exposure to Aspergillus hyphal extract (AHE) in immunocompetent C57Bl/6 mice. Systemic administration of both CM and EVs isolated from human and murine MSCs, but not human lung fibroblasts, at the onset of antigen challenge in previously sensitized mice significantly ameliorated the AHE-provoked increases in airway hyperreactivity (AHR), lung inflammation, and the antigen-specific CD4 T-cell Th2 and Th17 phenotype. Notably, both CM and EVs from human MSCs (hMSCs) were generally more potent than those from mouse MSCs (mMSCs) in most of the outcome measures. The weak cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride was found to inhibit release of both soluble mediators and EVs, fully negating effects of systemically administered hMSCs but only partly inhibited the ameliorating effects of mMSCs. These results demonstrate potent xenogeneic effects of CM and EVs from hMSCs in an immunocompetent mouse model of allergic airway inflammation and they also show differences in mechanisms of action of hMSCs versus mMSCs to mitigate AHR and lung inflammation in this model.

Significance: There is a growing experience demonstrating benefit of mesenchymal stromal cell (MSC)-based cell therapies in preclinical models of asthma. In the current study, conditioned media (CM) and, in particular, the extracellular vesicle fraction obtained from the CM were as potent as the MSCs themselves in mitigating Th2/Th17-mediated allergic airway inflammation in a mouse model of severe refractory clinical asthma. Moreover, human MSC CM and extracellular vesicles were effective in this immunocompetent mouse model. These data add to a growing scientific basis for initiating clinical trials of MSCs or extracellular vesicles derived from MSCs in severe refractory asthma and provide further insight into the mechanisms by which the MSCs may ameliorate the asthma.

Keywords: Asthma; Conditioned media; EDCI; Extracellular vesicles; Mesenchymal stromal cells; Mouse.

©AlphaMed Press.

Figures

Figure 1.

EDCI inhibits release of soluble proteins and of extracellular vesicles from cultured hMSCs, mMSCs, and HLFs. (A): Particle size analyses. EVs in the ultracentrifugation pellet collected after 48 hours of incubation from control cells and EDCI-treated HLFs, hMSCs, and mMSCs were analyzed using a NS300 machine and Nanosight NTA 3.0 software using light scatter mode. n = 3-5 for each group. Samples were diluted as needed in PBS to achieve an approximate concentration of 107 to 109 particles per milliliter. Results are presented as mean ± SD of triplicate measurements for each sample. (B): Representative transmission electron micrographs of EVs collected from ultracentrifuged pellets of conditioned media collected from control cells and EDCI-treated cells. Scale bars = 500 nm. (C): Extracellular vesicle concentration quantified on the ultracentrifugation pellet, collected after 48-hour incubation, from control cells and EDCI-treated cells, using the NS300 machine and Nanosight NTA 3.0 software using light scatter mode (n = 3–5 for each group). Data are presented as mean ± SD of triplicate measurements for each sample. Significance compared with control cell is indicated by τ. (D): Total protein content of raw conditioned media and of the ultracentrifuge pellets obtained from control cells and EDCI-treated cells after 48-hour incubation (n = 2 for each group). Data are presented as mean ± SD of triplicate measurements for each sample. Significance compared with control cells is indicated by τ. (E): Representative photomicrographs (contrast phase) of control and EDCI-treated cells after 48-hour incubation. Original magnification ×20. Scale bars = 100 μm. Abbreviations: EDCI, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; EV, extracellular vesicle; HLF, human lung fibroblast; hMSC, human mesenchymal stromal cell; mMSC, mouse mesenchymal stromal cell.

Figure 1.

EDCI inhibits release of soluble proteins and of extracellular vesicles from cultured hMSCs, mMSCs, and HLFs. (A): Particle size analyses. EVs in the ultracentrifugation pellet collected after 48 hours of incubation from control cells and EDCI-treated HLFs, hMSCs, and mMSCs were analyzed using a NS300 machine and Nanosight NTA 3.0 software using light scatter mode. n = 3-5 for each group. Samples were diluted as needed in PBS to achieve an approximate concentration of 107 to 109 particles per milliliter. Results are presented as mean ± SD of triplicate measurements for each sample. (B): Representative transmission electron micrographs of EVs collected from ultracentrifuged pellets of conditioned media collected from control cells and EDCI-treated cells. Scale bars = 500 nm. (C): Extracellular vesicle concentration quantified on the ultracentrifugation pellet, collected after 48-hour incubation, from control cells and EDCI-treated cells, using the NS300 machine and Nanosight NTA 3.0 software using light scatter mode (n = 3–5 for each group). Data are presented as mean ± SD of triplicate measurements for each sample. Significance compared with control cell is indicated by τ. (D): Total protein content of raw conditioned media and of the ultracentrifuge pellets obtained from control cells and EDCI-treated cells after 48-hour incubation (n = 2 for each group). Data are presented as mean ± SD of triplicate measurements for each sample. Significance compared with control cells is indicated by τ. (E): Representative photomicrographs (contrast phase) of control and EDCI-treated cells after 48-hour incubation. Original magnification ×20. Scale bars = 100 μm. Abbreviations: EDCI, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; EV, extracellular vesicle; HLF, human lung fibroblast; hMSC, human mesenchymal stromal cell; mMSC, mouse mesenchymal stromal cell.

Figure 2.

Systemic administration of human or mouse MSCs or their respective conditioned media or extracellular vesicles significantly ameliorates the airway hyperresponsiveness induced by Aspergillus hyphal extract. (A): Analysis of Rn, G, and H according to methacholine dose of N and A treated with P, human lung fibroblast (C), E, CM, or EV (n = 6 for all combinations except the following: 17 N and 15 A-P). (B): Analysis of Rn, G, and H of N and A treated with the vehicle P, hMSCs (C), E, CM, or EV (n = 6 for all combinations except the following: 17 N, 15 A-P, and 10 A-hMSC-C). (C): Analysis of Rn, G, and H of naive and AHE-exposed mice treated with the vehicle P, mMSCs (C), E, CM, or EV (n = 6 for all treatment combinations except the following: 17 N and 15 A-P). Data are presented as peak response normalized to the baseline, and then expressed as percent increase over the baseline ± SD. Statistical significance set at p ≤ .05. ∗, significantly different from N; #, significantly different from A-P; τ, significantly different from each of the three cell types. Abbreviations: A, Aspergillus hyphal extract-exposed mice; C, cells; CM, conditioned media; E, EDCI-treated cells; EV, extracellular vesicle; G, overall tissue resistance; H, lung elasticity; HLF, human lung fibroblast; N, naïve mice; P, phosphate-buffered saline; Rn, airway resistance.

Figure 3.

Systemic administration of human or mouse MSCs or their respective conditioned media or extracellular vesicles significantly reduces histologic lung inflammation provoked by Aspergillus hyphal-extract sensitization and challenge. (A): Representative photomicrographs of H&E-stained lung section. (B): Inflammation score (range: 0–3 ± SEM) of airways in N and A mice treated with HLF, hMSCs, and mMSCs (C), CM, or EVs (n = 6 for all treatment combinations except the following: 17 N, 15 A-P, and 10 A-hMSC-C). Data are presented as mean ± SD. Statistical significance set at p ≤ .05. ∗, significantly different from N; #, significantly different from A-P; τ, significantly different from each of the three cell types. Original magnification ×10; scale bars = 100 μm. Abbreviations: A, Aspergillus hyphal extract-exposed mice; C, cells; CM, conditioned media; E, EDCI-treated cells; EV, extracellular vesicle; HLF, human lung fibroblast; hMSC, human mesenchymal stromal cell; mMSC, mouse mesenchymal stromal cell; N, naïve mice; P, phosphate-buffered saline.

Figure 4.

Systemic administration of human or mouse MSCs or their respective conditioned media or extracellular vesicles significantly reduces increases in BALF inflammatory cells provoked by Aspergillus hyphal-extract sensitization and challenge. (A): Total cell number within the BALF in N and A mice treated with HLF, hMSCs, and mMSCs (C), CM, or EVs. (B): Differential cell population within the BALF normalized to total cell numbers of neutrophils, eosinophils, macrophages and lymphocytes (n = 6 for all treatment combinations except the following: 17 N, 15 A-P, and 0 A-hMSC-C). Data are presented as mean ± SD. Statistical significance set at p ≤ .05. ∗, significantly different from N; #, significantly different from A-P; τ, significantly different from each of the three cell types. Abbreviations: A, Aspergillus hyphal extract-exposed mice; BALF, bronchoalveolar lavage fluid; C, cells; CM, conditioned media; E, EDCI-treated cells; EV, extracellular vesicle; HLF, human lung fibroblast; hMSC, human mesenchymal stromal cell; mMSC, mouse mesenchymal stromal cell; N, naïve mice; P, phosphate-buffered saline.

Figure 5.

Systemic administration of human or mouse mesenchymal stromal cells or their respective conditioned media or extracellular vesicles significantly reduces the increased BALF content of proinflammatory soluble cytokines and chemokines provoked by Aspergillus hyphal-extract sensitization and challenge. (A): Soluble BALF cytokines associated with Th2 (IL-4, IL-5, IL-13), Th17 (IL-6, IL-17a), and Th1 inflammation (IFN-γ). (B): Soluble BALF further Th17 inflammation-associated cytokines (IL-12, KC), alternate inflammatory cytokines (IL-3, RANTES), and cytokines previously identified as secreted by MSCs in immunomodulation (IL-1A, IL-10) (n = 6 for all treatment combinations except the following: 17 N, 15 A-P, 10 A-hMSC-C). Data are presented as mean ± SD. Statistical significance set at p ≤ .05. ∗, significantly different from N; #, significantly different from A-P; τ significantly different from each of the three cell types. Abbreviations: A, Aspergillus hyphal extract-exposed mice; BALF, bronchoalveolar lavage fluid; CM, conditioned media; E, EDCI-treated cells; EV, extracellular vesicle; HLF, human lung fibroblast; hMSC, human mesenchymal stromal cell; IL, interleukin; INF, interferon; KC, keratinocyte chemoattractant; mMSC, mouse mesenchymal stromal cell; N, naïve mice; P, phosphate-buffered saline.

Figure 5.

Systemic administration of human or mouse mesenchymal stromal cells or their respective conditioned media or extracellular vesicles significantly reduces the increased BALF content of proinflammatory soluble cytokines and chemokines provoked by Aspergillus hyphal-extract sensitization and challenge. (A): Soluble BALF cytokines associated with Th2 (IL-4, IL-5, IL-13), Th17 (IL-6, IL-17a), and Th1 inflammation (IFN-γ). (B): Soluble BALF further Th17 inflammation-associated cytokines (IL-12, KC), alternate inflammatory cytokines (IL-3, RANTES), and cytokines previously identified as secreted by MSCs in immunomodulation (IL-1A, IL-10) (n = 6 for all treatment combinations except the following: 17 N, 15 A-P, 10 A-hMSC-C). Data are presented as mean ± SD. Statistical significance set at p ≤ .05. ∗, significantly different from N; #, significantly different from A-P; τ significantly different from each of the three cell types. Abbreviations: A, Aspergillus hyphal extract-exposed mice; BALF, bronchoalveolar lavage fluid; CM, conditioned media; E, EDCI-treated cells; EV, extracellular vesicle; HLF, human lung fibroblast; hMSC, human mesenchymal stromal cell; IL, interleukin; INF, interferon; KC, keratinocyte chemoattractant; mMSC, mouse mesenchymal stromal cell; N, naïve mice; P, phosphate-buffered saline.

Figure 6.

Systemic administration of human or mouse MSCs or their respective conditioned media or extracellular vesicles significantly alters IL-4, IL-5, IL-17, and INF-γ production in ex vivo restimulation of mediastinal lymphocytes. Shown is the assessment of IL-4, IL-5, IL-17, and INF-γ levels in supernatants from pooled mixed mediastinal lymph node cell populations restimulated ex vivo for 48 hours with Aspergillus hyphal-extract antigen (n = 6 for all treatment combinations except the following: 17 N, 15 A-P, 10 A-hMSC-C). Data are presented as mean ± SD. p ≤ .05. Abbreviations: A, Aspergillus hyphal extract-exposed mice; CM, conditioned media; E, EDCI-treated cells; EV, extracellular vesicle; HLF, human lung fibroblast; hMSC, human mesenchymal stromal cell; IL, interleukin; INF, interferon; mMSC, mouse mesenchymal stromal cell; N, naïve mice; P, phosphate-buffered saline.