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. (29th April 2016)

• Record Type: Journal Article
• Authors: Jackson, Megan V.; Morrison, Thomas J.; Doherty, Declan F.; McAuley, Daniel F.; Matthay, Michael A.; Kissenpfennig, Adrien; O'Kane, Cecilia M.; Krasnodembskaya, Anna D.
• Is Part Of: Stem cells. Volume 34:Number 8(2016:Aug.)
• Page Start: 2210
• Page End: 2223
• Publication Date: 2016-04-29
• Subjects: Mesenchymal stem cells -- Macrophages -- Mitochondrial transfer -- ARDS -- Phagocytosis; Cloning -- Periodicals; Clone cells -- Periodicals; Stem cells -- Periodicals; Cell Differentiation -- Periodicals; Cell Division -- Periodicals; Clone Cells -- Periodicals; Hematopoietic Stem Cells -- Periodicals; Stem Cells -- Periodicals; 571.84
• DOI: 10.1002/stem.2372
• Languages: English
• ISSNs: 1066-5099

Abstract

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. Abstract : Antimicrobial effect of mesenchymal stromal cells (MSC) in acute respiratory distress syndrome (ARDS) is mediated in part by enhanced alveolar macrophage phagocytosis through TNT-dependent mitochondrial transfer leading to improved macrophage bioenergetics. (A) In mouse E. coli pneumonia MSC treatment improved bacterial clearance in the lung ( p = .02), alveolar macrophage(AM) depletion by liposomal clodronate significantly increased bacterial burden ( p < .05) and abrogated MSC antimicrobial effect. (B) In E. coli pneumonia MSC treatment significantly increased the percentage of phagocytic AM compared to PBS suggesting improved AM phagocytosis ( p = .01). (C) (i-iii) transfer of mitochondria from MSC (MitoRed+) to primary human macrophages (MDM) (CD45+) through TNT-like structures (arrows) (scale bar = 50 μm) (iv) Flow cytometry showing more than 90% of CD45+ MDM acquired MSC specific MitoRed fluorescence (APC+), indicating extensive mitochondrial transfer from MSC (4 hours of coculture) (v) In vivo 93% and 65% of AM were positive for MitoRed at 24 and 48 hours after MSC administration, respectively. (D) AM that had internalized MSC mitochondria (Mito+) showed a significantly higher phagocytic index in comparison to those without ( p = .003). (E) Pretreatment of MSC with Cytochalasin B (500 nM) inhibited TNT formation and partially blocked mitochondrial transfer (scale bar = 50 μm). By flow cytometry pretreatment of MSC with Cytochalasin B resulted in approximately 50% abrogation in the MitoRed MFI of macrophages ( p < .05). (F) Coculture with untreated but not Cytochalasin B pretreated MSC significantly enhanced MDM levels of mitochondrial ATP turnover ( p < .05). (G) Pretreatment of MSC with Cytochalasin B abrogated MSC antibacterial effect in the in vivo E. coli pneumonia model ( p < .05).