Mesenchymal Stromal Cells Modulate Macrophages in Clinically Relevant Lung Injury Models by Extracellular Vesicle Mitochondrial Transfer

Abstract

Rationale: Acute respiratory distress syndrome (ARDS) remains a major cause of respiratory failure in critically ill patients. Mesenchymal stromal cells (MSCs) are a promising candidate for a cell-based therapy. However, the mechanisms of MSCs' effects in ARDS are not well understood. In this study, we focused on the paracrine effect of MSCs on macrophage polarization and the role of extracellular vesicle (EV)-mediated mitochondrial transfer.

Objectives: To determine the effects of human MSCs on macrophage function in the ARDS environment and to elucidate the mechanisms of these effects.

Methods: Human monocyte-derived macrophages (MDMs) were studied in noncontact coculture with human MSCs when stimulated with LPS or bronchoalveolar lavage fluid (BALF) from patients with ARDS. Murine alveolar macrophages (AMs) were cultured ex vivo with/without human MSC-derived EVs before adoptive transfer to LPS-injured mice.

Measurements and main results: MSCs suppressed cytokine production, increased M2 macrophage marker expression, and augmented phagocytic capacity of human MDMs stimulated with LPS or ARDS BALF. These effects were partially mediated by CD44-expressing EVs. Adoptive transfer of AMs pretreated with MSC-derived EVs reduced inflammation and lung injury in LPS-injured mice. Inhibition of oxidative phosphorylation in MDMs prevented the modulatory effects of MSCs. Generating dysfunctional mitochondria in MSCs using rhodamine 6G pretreatment also abrogated these effects.

Conclusions: In the ARDS environment, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype through EV-mediated mitochondrial transfer. MSC-induced changes in macrophage phenotype critically depend on enhancement of macrophage oxidative phosphorylation. AMs treated with MSC-derived EVs ameliorate lung injury in vivo.

Keywords: acute respiratory distress syndrome; extracellular vesicles; macrophages; mesenchymal stromal cells; mitochondria.

Figure 1.

Human mesenchymal stromal cell (MSC) modulation of human monocyte–derived macrophage (MDM) phenotype and function. (A) MSCs in noncontact coculture reduced the production of tumor necrosis factor (TNF)-α by MDMs after 24 hours of LPS treatment. One-way analysis of variance (ANOVA) with Bonferroni’s post hoc test was used (n = 5 per group). (B) IL-8 production by MDMs was reduced by MSC coculture after LPS treatment. One-way ANOVA with Bonferroni’s post hoc test was used (n = 4 per group). (C) MSC coculture increased the percentage of MDMs expressing CD206 on their surface in the (Ci) absence or (Cii) presence of LPS. Unpaired t test (n = 3 per group) and one-way ANOVA with Bonferroni’s post hoc test (n = 4 per group), respectively, were used. (D) MSCs increased the proportion of MDMs that had phagocytosed Escherichia coli pHrodo (Thermo Fisher Scientific, Carlsbad, CA) dye-stained fluorescent bioparticles after LPS. One-way ANOVA with Bonferroni’s post hoc test was used (n = 5 per group). Data are presented as mean ± SD. *P < 0.05; **P < 0.01.

Figure 2.

Human mesenchymal stromal cells (MSCs) modulate human monocyte–derived macrophages (MDMs) in the presence of bronchoalveolar lavage fluid (BALF) from patients with acute respiratory distress syndrome (ARDS). (A) MSCs reduced the production of tumor necrosis factor (TNF)-α by MDMs treated with 30% ARDS BALF for 24 hours. Kruskal-Wallis test with Dunn’s post hoc test was used (healthy volunteer [HV] BALF, n = 3; other groups, n = 5). (B) MSCs increased the proportion of MDMs expressing the M2 macrophage surface marker CD206 after 72 hours. One-way analysis of variance with Bonferroni’s post hoc test was used (n = 3 for all groups). (C) MSCs increased the proportion of phagocytic MDMs in the presence of ARDS BALF. One-way analysis of variance with Bonferroni’s post hoc test was used (n = 4 for all groups). Data are presented as mean ± SD. *P < 0.05; **P < 0.01.

Figure 3.

CD44-expressing extracellular vesicles (EVs) from human mesenchymal stromal cells (MSCs) are partially responsible for their modulatory effects. (A) Flow cytometry demonstrates that MSC-derived EVs are (Ai) smaller in diameter than latex beads of 4-μm diameter and (Aii) positive for CD44 expression on their surface. (B) Preincubation of MSC conditioned medium (CM) with anti-CD44 neutralizing antibody partially reversed MSC suppression of tumor necrosis factor (TNF)-α secretion by human monocyte–derived macrophages (MDMs; n = 4 for all groups) and completely prevented MSC enhancement of (Ci) the proportion of phagocytic MDMs as well as (Cii) their phagocytic index (Phago; n = 5 for all groups). One-way analysis of variance with Bonferroni’s post hoc test was used. Data are presented as mean ± SD. *P < 0.05; **P < 0.01. APC = allophycocyanin; Cy = cyanine; FITC = fluorescein isothiocyanate; FSC = forward scatter; MFI = median fluorescence intensity; PE = phycoerythrin; PerCP = peridinin-chlorophyll-protein complex; SSC = side scatter.

Figure 4.

Human mesenchymal stromal cell extracellular vesicle (EV)-treated alveolar macrophages (AMs) reduce LPS-induced lung injury. (A) Treatment of LPS-injured mice with EV-treated AMs reduced total cell counts and neutrophilic cell counts as well as the amount of tumor necrosis factor (TNF)-α and protein in the bronchoalveolar lavage fluid (BALF). One-way analysis of variance with Bonferroni’s post hoc test was used for total cell and neutrophil counts and BALF protein (n = 4 phosphate-buffered saline [PBS]; n = 5 LPS; n = 3 LPS + EV-treated AMs). Student’s unpaired t test was used for TNF-α (n = 3 LPS; n = 3 LPS + EV-treated AMs). (B) Untreated (unt) AMs had no effect on total or neutrophil cell counts and had no effect on BALF TNF-α or protein levels. One-way analysis of variance with Bonferroni’s post hoc test was used for total cell and neutrophil counts and BALF protein (n = 4 PBS; n = 4 LPS; n = 5 LPS + untreated AMs). Student’s unpaired t test was used for TNF-α (n = 5 LPS; n = 5 LPS + untreated AMs). (C) Cytospins of BALF preparations demonstrated inflammatory cell recruitment to the airspaces after LPS injury and reduced cell numbers after treatment with EV-treated AMs but not after treatment with untreated AMs (original magnification, ×20). Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5.

Human mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) transfer functional mitochondria to human monocyte-derived macrophages (MDMs), which enhances macrophage oxidative phosphorylation. (A) Flow cytometry of culture medium taken from MitoTracker Red–prestained MSCs showed that 25% of EVs were positive for mitochondria. (B) EVs contained in MSC culture medium (CM) transfer mitochondria (red) to MDMs (MitoTracker Green) (white arrows). MSC mitochondria are integrated into the MDM mitochondrial network (yellow, yellow arrows) (original magnification, ×20). (C) Treatment of MDMs with MSC CM resulted in increases in (Ci) basal mitochondrial respiration and (Ciii) mitochondrial ATP turnover, as determined by using the Seahorse metabolic analyzer. (Cii) The increase in maximal mitochondrial respiration did not reach statistical significance. Kruskal-Wallis test with Dunn’s post hoc test was used (n = 4 for all groups). Data are presented as mean ± SD. *P < 0.05. APC = allophycyanin; FSC = forward scatter; OCR = oxygen consumption rate; SSC = side scatter.

Figure 6.

Mitochondrial transfer via human mesenchymal stromal cell (MSC)-derived extracellular vesicles modulates human monocyte-derived macrophage (MDM) function through enhancement of macrophage oxidative phosphorylation. (A) Addition of oligomycin (olig; reversible ATP synthase inhibitor) prevented the suppression of tumor necrosis factor (TNF)-α by MSC culture medium (CM) (n = 5 for all groups but dimethyl sulfoxide [DMSO; n = 4]). (B) Oligomycin also prevented MSC CM enhancement of the proportion of (Bi) phagocytic MDMs and (Bii) phagocytic index (Phago; n = 7 for all groups). Pretreatment of MSCs with rhodamine 6G (an irreversible ATP synthase inhibitor) similarly reversed MSC CM capacity to (C) suppress TNF-α production (n = 4 all groups), (D) enhance the phagocytic capacity of MDMs (n = 4 all groups), and (E) promote M2 marker CD206 expression (n = 5 for all groups). All analyses were done by one-way analysis of variance with Bonferroni’s post hoc test. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. FITC = fluorescein isothiocyanate; MFI = median fluorescence intensity.