Mitochondrial Transfer via Tunneling Nanotubes is an Important Mechanism by Which Mesenchymal Stem Cells Enhance Macrophage Phagocytosis in the In Vitro and In Vivo Models of ARDS

Abstract

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in preclinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the antimicrobial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC antimicrobial effect in the in vivo model of Escherichia coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct coculture of MSC with monocyte-derived macrophages enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through tunneling nanotubes (TNT)-like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the antimicrobial effect of MSC in vivo. Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the antimicrobial effect of MSC in ARDS. Stem Cells 2016;34:2210-2223.

Keywords: ARDS; Macrophages; Mesenchymal stem cells; Mitochondrial transfer; Phagocytosis.

Figure 1

Effect of alveolar macrophage (AM) depletion on MSC antimicrobial and anti‐inflammatory properties in mouse E. coli pneumonia. (A) AM‐depleted mice had significantly higher E. coli CFU counts in BALF compared to nondepleted mice treated with PBS (*, p < .05 vs. control PBS, 2‐way ANOVA (Bonferroni)). MSC administration had no effect on bacterial clearance in the AM‐depleted group although significantly reducing E. coli CFU in control mice compared to PBS controls (*, p = .02, Student's t‐test). (B) Cytokine profile of BALF samples from normal mice. (C) Cytokine profile of BALF samples from AM‐depleted mice. (D‐F) AM‐depleted mice had significantly reduced levels of BALF TNF‐α, IL‐10, and IL‐6 compared to nondepleted animals treated with PBS. MSC administration had no effect on cytokine levels in AM‐depleted animals (*, p < .05 vs. control PBS, 2‐way ANOVA (Bonferroni)). MSC treatment significantly decreased TNF‐α and IL‐10 levels in nondepleted animals (*, p = .02 and **, p = .007 respectively vs. PBS treated group, Student's t‐test). (G, H) BALF total WBC counts and absolute neutrophil counts were significantly abrogated in the AM‐depleted group. MSC administration had no effect in AM‐depleted animals, while although reducing absolute neutrophil counts in nondepleted mice (***, p < .001, *, p < .05 vs. control PBS, 2‐way ANOVA (Bonferroni)). (I) BALF protein influx was significantly decreased in the AM‐depleted group versus nondepleted mice treated with PBS. MSC treatment significantly reduced BALF protein concentration in nondepleted mice and had no effect in AM‐depleted animals (*, p = .03, Student's t‐test). All data expressed as mean ± SD for each condition (at least n = 4 mice/condition). Abbreviations: BALF, broncho‐alveolar lavage fluid; MSC, mesenchymal stem cells; PBS, phosphate buffered saline; WBC, white blood cells.

Figure 2

MSC enhance macrophage phagocytosis. (A) In the in vivo E. coli pneumonia model, MSC treatment significantly increased the percentage of alveolar macrophage positive for pHrodo E. coli bioparticles compared to PBS treated mice ((n = 7 mice/condition), *, p = .01, Student's t‐test). (B, C) In vitro human MDM were infected with E. coli (MOI 10) with or without direct coculture with MSC (1/20 ratio). (B) MSC coculture significantly reduced extracellular E. coli CFU counts coupled with (C) significantly elevated levels of intracellular CFU (n = 3 in triplicate, *, p < .05, **, p< .01, Student's t‐test). Data are shown as mean $\pm$ SD for each condition. Abbreviations: MDM, monocyte‐derived macrophage; MSC, mesenchymal stem cells; PBS, phosphate buffered saline.

Figure 3

Mitochondrial transfer from MSC to macrophages. (A) Transfer of mitochondria from MSC to primary human macrophages through TNT‐like structures (Ai) human MDM uniformly express CD45 (blue) (Aii) MSC mitochondria are labeled with MitoRed (red) (Aiii) In coculture with MSC for 24 hours, colocalization of blue and red, indicates robust transfer of mitochondria from MSC to MDM. Network of mitochondria‐positive TNT emerging from the MSC and connecting to several distant macrophages (up to 200 µm) is also observed (arrows) (images were taken at a magnification of 10 × 63; scale bar = 50 µm). (B) Population of MDM cultured alone, stained with CD45‐PE but negative for MitoRed‐APC. (C) Population of MSC cultured alone, stained with MitoRed‐APC but negative for CD45‐PE. (D) After 4 hours in coculture, more than 90% of CD45⁺MDM demonstrate acquisition of MitoRed fluorescence (APC⁺), indicating extensive mitochondrial transfer from MSC. (E) Intensity of MitoRed fluorescence of MSC population decreased after coculture with MDM (blue histogram). Data representative of at least three independent experiments. (F) E. coli‐infected mice were treated with MitoRed‐labeled MSC IN, AM were gated as Gr‐1⁻F4/80⁺CD11c^hiCD11b^low and analyzed for their expression of MitoRed fluorescence at 24 and 48 hours after treatment. Ninety‐three percent and sixty‐five percent of AM were positive for MitoRed at 24 and 48 hours, respectively. Plot representative of 3 mice/condition. Abbreviations: MDM, monocyte‐derived macrophage; MSC, mesenchymal stem cells.

Figure 4

Internalized by macrophages, mesenchymal stem cells (MSC) mitochondria enhance their phagocytic activity. (A) In E. coli pneumonia MSC (MitoRed)‐treated mouse BALF was harvested and phagocytic activity of alveolar macrophage was assessed using fluorescent E. coli bioparticles by flow cytometry. Macrophages that had internalized MSC mitochondria (Mito+) showed a significantly higher phagocytic index in comparison to those without (Mito‐) (n = 12, **, p = .003, Student's t‐test). This was assessed by an increase in pHRodo median fluorescence intensity (MFI). (B) Isolated mitochondria taken from MitoRed‐treated MSC were added to human MDM and internalization was confirmed after 24 hours by flow cytometry. (C) In vitro addition of isolated MSC mitochondrial fraction to E. coli infected MDM significantly reduced extracellular CFU counts (n = 3 in triplicate, *, p < .05, Student's t‐test) coupled with an increase in intracellular CFU (D). Abbreviations: MDM, monocyte‐derived macrophages; Mito, mitochondria.

Figure 5

Inhibition of MSC tunneling nanotubes (TNT) formation by pretreatment with Cytochalasin B partially blocks mitochondrial transfer differentially affecting MSC modulation of MDM. (A) Confocal microscopy demonstrates normal spindle‐shape morphology of MitoRed MSC (red) in coculture with MDM (CD45+, blue), where TNT are present and mitochondrial transfer is evident (images were taken at a magnification of 10 × 63; scale bar = 50 µm). Cytochalasin B (500 nM) pretreated MSC appear rounded and TNT are no longer visible, however mitochondrial transfer still takes place as shown by colocalization of staining. (B) Coculture of MDM with Cytochalasin B pretreated MSC resulted in approximately 50% abrogation in the MitoRed MFI of macrophages (*, p < .05, Mann‐Whitney U test). (C, D) Mitochondrial respiration of human macrophages and human MSC was measured as oxygen consumption rate (OCR) using the SeaHorse technology. Macrophage mitochondrial function was analyzed during coculture with or without human MSC in the presence or absence of Oligomycin, FCCP, and Rotenone/Antimycin A to differentiate ATP‐linked respiration from proton leak. Coculture with untreated but not Cytochalasin B pretreated MSC significantly enhanced MDM levels of mitochondrial basal respiration and mitochondrial ATP turnover (n = 5–6, *, p < .05, Mann‐Whitney U test). (E) MSC pretreated with Cytochalasin B significantly restored cell viability of MDM post E. coli infection (***, p < .001 vs. MDM, ANOVA (Bonferroni)). (F) Both intact and Cytochalasin B pretreated MSC coculture significantly decreased LPS‐induced TNF‐α levels in culture medium (CM) (*, p < .05, **, p < .01, ***, p < .001 vs. MDM+LPS, **, p < .05 vs. MDM, ANOVA (Bonferroni). (G) Extracellular E. coli CFU were significantly decreased in coculture with untreated but not Cytochalasin B pretreated MSC compared to MDM alone (*, p < .05, **, p < .01 vs. MDM, ANOVA [Bonferroni]). Data shown as mean ± SD, n = 3–4 in triplicate for each condition. Abbreviations: MDM, monocyte‐derived macrophages; MFI, median fluorescence intensity; MSC, mesenchymal stem cells.

Figure 6

Inhibition of tunneling nanotubes formation by MSC abrogates the antimicrobial effect of MSC in E. coli pneumonia. Pretreatment of MSC with Cytochalasin B inhibited the therapeutic effect of MSC in bacterial clearance in the BALF (A) and lung homogenate (B). Administration of the MSC mitochondrial fraction did not significantly affect the bacterial burden in the BALF compared to the PBS group (in BALF *, p < .05 MSC vs. PBS, Kruskal Wallis test, in lung homogenate *, p < .05 MSC vs. CytoB, Kruskal Wallis test, PBS vs. MSC, Student's t‐test, n = 3–5 mice per group). Data shown as mean $\pm$ SD for each condition. Abbreviations: BALF, broncho‐alveolar lavage fluid; MSC, mesenchymal stem cells.

Figure 7

Mitochondrial transfer from MSC to human MDM via noncontact dependent mechanism. MDM were cocultured with MSC‐CM or MSC (pretreated with MitoRed) in a Transwell system without cell contact for 24 hours in the presence of LPS. (A) The extent of mitochondrial transfer to MDM was measured by flow cytometry (n = 3–5/group, *, p < .05, **, p < .01, ***, p < .001, ANOVA [Bonferroni]) (B) The MDM were also given pHRodo particles to quantify phagocytosis (n = 5/group, *, p < .05, ANOVA (Bonferroni)). (C) Phagocytic MDM were divided into two groups, with and without internalization of MSC mitochondria, and their phagocytic indexes were determined by median fluorescence intensity (MFI) (n = 5/group *, p < .05, Student's t‐test). Abbreviations: CM, culture medium; MDM, monocyte‐derived macrophages; MSC, mesenchymal stem cells.