Inflammatory cytokine-primed MSC-derived extracellular vesicles ameliorate acute lung injury via enhanced immunomodulation and alveolar repair

Abstract

Background: Acute lung injury (ALI) is characterized by excessive inflammation and alveolar damage, arising from pathogens or systemic insults such as sepsis, and can progress to severe acute respiratory distress syndrome (ARDS). Despite its severity, effective pharmacological treatments remain unavailable, and current clinical interventions are limited to supportive care such as mechanical ventilation. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as promising candidates for lung repair, but insufficient immunosuppressive capacity often limits their efficacy.

Methods: Human adipose-derived mesenchymal stem cells (hADMSCs) were primed with IFN-γ and TNF-α to enhance the immunomodulatory properties of their secreted EVs. We characterized unprimed control MSC-EVs (C-MEVs) and primed MSC-EVs (P-MEVs) by transmission electron microscopy, nanoparticle tracking analysis, and western blotting for EV markers. Functional assays in THP-1 and A549 cells examined anti-inflammatory potency and barrier regeneration against lipopolysaccharide (LPS)-induced damage. A preclinical mouse model of LPS-induced ALI was used to evaluate inflammatory cytokine expression, immune cell infiltration, pulmonary edema, and vascular leakage. Finally, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Vero E6 cells were tested whether P-MEVs could mitigate the inflammatory damage characteristic of virus-triggered acute lung injury.

Results: Primed hADMSCs exhibited elevated expression of immunosuppressive molecules (e.g., COX-2, IDO, TSG-6), without changing EV morphology or yield. P-MEVs mitigated LPS-induced inflammation more effectively than C-MEVs in THP-1 and A549 cells. In vivo, P-MEVs more robustly attenuated inflammatory cytokines, immune cell recruitment, and lung injury markers in mice challenged with LPS. In SARS-CoV-2-infected Vero E6 cells, P-MEVs suppressed cytopathic effects and inflammatory responses more potently than C-MEVs. Mechanistic analyses revealed that these enhancements were associated with elevated miRNA levels, including miR-221-3p, involved in inhibiting inflammatory pathways.

Conclusion: Inflammatory cytokine priming substantially augments the immunomodulatory and tissue-regenerative efficacy of hADMSC-derived EVs, offering superior therapeutic effects in ALI models and promising activity against SARS-CoV-2-induced lung damage. These findings underscore the therapeutic potential of P-MEVs as an innovative, cell-free platform for treating severe pulmonary disorders, including ARDS.

Keywords: Acute lung injury (ALI); Extracellular vesicles (EVs); Mesenchymal stem cells (MSCs); Priming.

Fig. 1

Characterization of hADMSC-derived EVs (a) Schematic diagram showing the steps involved in the production of EVs isolated from control (C-MEVs) or primed hADMSCs (P-MEVs). Created using BioRender.com. (b) Representative TEM image of EVs derived from control and primed hADMSCs. Black scale bars represent 200 nm (left) and 100 nm (right). (c) The average diameter of EVs measured from the images in (b). n = 150. (d) NTA depicting the size distribution and representative video images of C-MEVs and P-MEVs. The observed size range of EVs was 50–200 nm. (e) Immunoblotting for representative cell and EV markers in cell lysate and EVs isolated from the indicated samples (Full-length blots are presented in Supplementary Figure S9a). (f) Time-course analysis of DiR-labeled EV uptake in A549 and THP-1 cells. Cells were incubated with 1000 EVs/cell, and intracellular fluorescence was quantified by flow cytometry at 0, 8, 16, and 24 h. n = 4. Data are presented as the mean ± standard error of the mean (SEM), analyzed by unpaired two-tailed Student’s t-test for (c), and by two-way ANOVA followed by the Holm–Sidak multiple comparison test for (f). Statistical differences in post hoc tests are indicated as ns = not significant, and ****p < 0.0001

Fig. 2

Evaluation of the anti-inflammatory effects of P-MEVs against LPS-induced inflammation in macrophages and lung epithelial cells Macrophages and lung epithelial cells were pretreated with MSC-EVs (C-MEVs or P-MEVs) or PBS for 2 h, followed by exposure to the indicated concentration of LPS for the indicated periods. (a) qRT-PCR analysis of relative mRNA expression levels of TNF-α, IL-1β, and IL-6 in THP-1 cells 6 h post LPS exposure (5 µg/mL). n = 3. (b) qRT-PCR analysis of relative mRNA expression levels of TNF-α, IL-1β, and IL-6 in A549 cells 6 h post LPS exposure (50 µg/mL). n = 3. (c) ELISA analysis of TNF-α, IL-1β, and IL-6 levels in THP-1 cells 24 h after LPS exposure (5 µg/mL). n = 3. (d) ELISA analysis of TNF-α, IL-1β, and IL-6 levels in A549 cells 24 h after LPS exposure (50 µg/mL). n = 3. C, control; P, primed. Data are presented as the mean ± SEM, analyzed by one-way ANOVA, followed by the Holm–Sidak multiple comparison test. Statistical differences in post hoc tests are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 3

Evaluation of the tissue regenerative effects of P-MEVs against LPS-induced damage in lung epithelial cells Lung epithelial cells were pretreated with MSC-EVs (C-MEVs or P-MEVs) or PBS for 2 h, followed by exposure to LPS (500 µg/mL) for 24 h. (a, b) Assessment of cell viability in A549 cells by (a) MTS assay (n = 5) and (b) in vitro bioluminescence assay (n = 6). (c, d) Assessment of epithelial permeability in A549 cells by measuring the fluorescence intensity of FITC–dextran. (c) Experimental scheme (created using BioRender.com) and (d) relative permeability (n = 4). C, control; P, primed; C-MEV, control MSC-EV; P-MEV, primed MSC-EV. Data are presented as the mean ± SEM, analyzed by one-way ANOVA, followed by the Holm–Sidak multiple comparison test. Statistical differences in post hoc tests are indicated as ***p < 0.001 and ****p < 0.0001

Fig. 4

P-MEVs alleviate lung inflammation in a mouse model of LPS-induced ARDS C57BL/6 mice were challenged with LPS (5 mg/kg, intravenously) and then received daily injections of PBS or MSC-EVs (6 × 10⁹ particles, intravenously). After 72 h, the mice were sacrificed and subjected to functional analysis. (a) Experimental scheme for establishing an LPS-induced ALI mouse model and administration of MSC-EVs. Created using BioRender.com. (b) Daily monitoring of mouse body weight changes. Values are calculated as a percentage of body weight from day 0. n = 4–5. (c) qRT-PCR analysis of relative mRNA expression levels of TNF-α, IL-1β, and IL-6 in the lung tissues. n = 5. (d) ELISA analysis of TNF-α, IL-1β, and IL-6 concentrations in the lung tissues. n = 5. (e) Western blot analysis of lung homogenate (left, Full-length blots are presented in Supplementary Figure S10). Phosphorylated bands were normalized to the respective total bands and are shown as fold changes relative to the control. n = 5. (f) Flow cytometric analysis of neutrophils (CD11b + Ly6G+), monocyte-derived macrophages (Siglec F- CD11b + on CD11c + F4/80+), and alveolar macrophages (Siglec F + CD11b- on CD11c + F4/80+) accumulation in lung tissue. n = 5. Con, control mice with PBS injections; LPS, mice with LPS injection only; LPS + C-MEVs, mice with LPS and control MSC-EVs injections; LPS + P-MEVs, mice with LPS and primed MSC-EVs injections. Data are presented as the mean ± SEM, analyzed by two-way ANOVA for (b) and analyzed with a one-way ANOVA for (c), (d), (e), and (f), followed by the Holm–Sidak multiple comparison test. Statistical differences in post hoc tests are indicated as ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 5

P-MEVs attenuate LPS-induced lung injury, pulmonary edema, and vascular leakage C57BL/6 mice were administered LPS (5 mg/kg, intravenously) and received daily injections of PBS or MSC-EVs (6 × 10⁹ particles, intravenously). After 72 h, the mice were sacrificed and subjected to functional analysis. (a) Representative H&E staining of lung tissue. Black scale bars represent 250 μm at 40x and 50 μm at 200x. (b) Evaluated lung index (%; lung weight (g)/body weight (g) x 100). n = 4–5. (c) Lung W/D ratio for pulmonary edema evaluations. n = 4–5. (d) Evans blue index (µg/g) for assessing lung permeability. Mice received a tail vein injection of 1% Evans blue 2 h before euthanasia. n = 4–5. One mouse in the LPS group died before data collection could be performed and was excluded from the study. Con, control mice with PBS injections; LPS, mice with LPS injection only; LPS + C-MEVs, mice with LPS and control MSC-EVs injections; LPS + P-MEVs, mice with LPS and primed MSC-EVs injections. Data are presented as the mean ± SEM, analyzed by one-way ANOVA, followed by the Holm–Sidak multiple comparison test. Statistical differences in post hoc tests are indicated as ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 6

P-MEVs ameliorate the SARS-CoV-2 infection-induced damage in vitro Vero E6 cells were infected with SARS-CoV-2 (MOI = 0.003) for 1 h, followed by exposure to C-MEVs or P-MEVs for 48 h. (a) Representative images of CPE were observed under a microscope. Black scale bars represent 25 μm. (b) qRT-PCR analysis of relative mRNA expression levels of TNF-α, IL-1β, IL-6, CCL2, and CXCL10. n = 3. C-MEVs, control MSC-EVs; P-MEVs, primed MSC-EVs; C, control; P, primed. Data are presented as the mean ± SEM, analyzed by one-way ANOVA, followed by the Holm–Sidak multiple comparison test. Statistical differences in post hoc tests are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7

P-MEV-enriched miRNAs suppress LPS-induced inflammation in macrophages (a) Scatter plot of differential expression of miRNAs between C-MEVs (𝑥-axis) and P-MEVs (𝑦-axis). The red and green lines represent the threshold for a 2-fold increase and decrease in miRNA levels in P-MEVs compared to C-MEVs, respectively. (b) The normalized data of C-MEVs (blue circle) and P-MEVs (red circle) are shown on the left 𝑦-axis with their corresponding fold change (black triangle on the right 𝑦-axis) of 23 miRNAs in negative regulation of inflammation. (c–e) Protein expressions of TNF-α (c), IL-1β (d), and IL-6 (e) in THP-1 cells at 24 h after transfection with each of the seven miRNA candidates (100 nM), followed by treatment with 5 µg/mL LPS using ELISA. n = 3. (f-h) qRT-PCR analysis of relative mRNA expression levels of TNF-α (c), IL-1β (d), and IL-6 (e) in THP-1 cells 6 h post LPS exposure (5 µg/mL). Cells were transfected with a miR-221-3p mimic or miR-221-3p inhibitor or Control (100 nM) for 24 h prior to LPS and EV treatment. n = 3. C-MEVs, control MSC-EVs; P-MEVs, primed MSC-EVs. Data are presented as the mean ± SEM, analyzed by one-way ANOVA, followed by the Holm–Sidak multiple comparison test. Statistical differences in post hoc tests are indicated as *p < 0.05 and ****p < 0.0001