Background ARDS remains a major cause of respiratory failure in critically ill patients with no specific therapy. MSC based cell therapy is a promising candidate and is being used in clinical trials for ARDS. However the mechanisms of MSC effect in lung injury are not very well understood. Islam et al., 2012 showed mitochondrial transfer from MSC to alveolar epithelial cells was protective in the mouse model of LPS induced pneumonia. Pathophysiology of ARDS is underpinned by dysregulated inflammation and pulmonary macrophages are key cellular mediators of the lung immune response. This study was undertaken to test if MSC could transfer their mitochondria to macrophages and to investigate the effects of MSC mitochondria transfer on macrophage function in the in vivo and in vitro models of ARDS.

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Abstract T3 Figure 1

Mitochondrial transfer from MSC to macrophages can enhance macrophage phagocytic activity in vivo. (A) MSC use tunnelling nano tubules (TNT) structures to transfer mitochondria (arrows). MSC were pre-stained with MitoTracker Red before co-culture with macrophages, 6 hr later sides were fixed and stained for(blue) to visualise macrophages. Almost allpositive cells demonstrate acquisition of red mitochondria from MSC. (B) In the in vivo model, MSC (MitoTracker)-treated mice BALF was taken and alveolar macrophages assessed for phagocytic activity using fluorescent E.coli bioparticles by flow cytometry. Macrophages that had acquired MSC mitochondria showed a higher phagocytic index in comparison to those without. This was assessed by an increase in Mean fluorescence Intensity (MFI). Some of the materials employed in this work were provided by the Texaz A&M Health Science Centre College of Medicine Institute for Regenerative Medicine at Scott and White through a grant from NCRR of the NIH, Grant #P40RR017447

Methods In vivo studies were performed using a mouse model of E.coli pneumonia induced ARDS. C56BL/6 mice were infected with E.coli, human bone marrow-derived MSC or PBS instilled intra-nasally 4 h after infection. For in vitro studies primary human monocyte-derived macrophages (MDM) were infected with E.coli and co-cultured with MSC in contact. MSC mitochondria were pre-stained with MitoTracker Red and MDM stained for CD45 expression. Double positive cells were visualised with confocal microscopy and quantified using flow cytometry. Phagocytosis was assessed using fluorescent E.coli bioparticles by flow cytometry.

Results When co-cultured with MSC >90% of MDMs acquired MitoRed fluorescence, indicating mitochondrial transfer from BM-MSC. Confocal imaging revealed presence of Mito-Red positive tunnelling nanotubules (TNTs) formed by MSC. In vivo >78% of CD11c^hi/F4–80+ alveolar macrophages retained MSC mitochondria at 24 hr post infection. Alveolar macrophages that had acquired MSC mitochondria had a significantly higher phagocytic index compared to those without suggesting enhancement of phagocytic capacity. Inhibition of TNT formation in MSC resulted in decreased transfer to macrophages by 60%, coupled with significant abrogation of MSC effect on macrophage phagocytosis in vitro and anti-microbial effect seen with MSC in vivo.

Conclusions Our findings suggest that anti-microbial activity of macrophages is enhanced at least partially by transfer of BM-MSC mitochondria through TNTs, representing an important mechanism of MSC effect in ARDS.

Supported by: MRC MR/L017229/1.

Reference 1 Islam MN, Das SR, Emin MT, et al. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med. 2012;18:759–65