Thomas Morrison

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

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

T3 Mitochondrial transfer is an important mechanism by which Mesenchymal Stromal Cells (MSC) facilitate macrophage phagocytosis in the in vitro and in vivo models of Acute Respiratory Distress Syndrome (ARDS)

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.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 CD11chi/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

S80 Mesenchymal stromal cells (MSC) modulate human macrophages in acute respiratory distress syndrome (ARDS) via secretion of extracellular vesicles (EV) which enhance oxidative phosphorylation and regulate jak/stat signalling

Background ARDS remains a major cause of respiratory failure in critically ill patients with no effective treatment. MSC are a promising candidate for therapy. However the mechanisms of MSC effects in lung injury are not well understood. We have recently shown that alveolar macrophages are critical cellular mediators of the therapeutic effect of MSC in the mouse model of E.coli pneumonia.1 Here we focused on the paracrine effect of MSC on macrophage polarisation and intracellular signalling. Methods Primary human macrophages were co-cultured with human bone marrow derived-MSC, without contact, at a 5:1 ratio, MSC-conditioned medium (CM) or EV with or without LPS or bronchoalveolar lavage fluid (BALF) from ARDS patients. A phospho-kinase array was performed on lysates for analysis of signalling cascades. Levels of pSTAT, Suppressor of Cytokine Signalling (SOCS) 1 and 3 proteins were tested by Western blot. Cell metabolism was investigated using Seahorse technology. Results Cytokine and surface marker expression show that MSC promote an M2-like macrophage phenotype with enhanced phagocytic activity. MSC-CM enhanced mitochondrial respiration in macrophages and oligomycin inhibited the effect of MSC-CM on cytokine secretion and phagocytosis, suggesting that MSC-CM induced a metabolic switch to oxidative phosphorylation, characteristic of M2 macrophages. Consistently with the M2 phenotype, MSC induced a high SOCS1:SOCS3 protein expression ratio, accompanied with activation of STAT6 and inhibition of STAT1 phosphorylation. MSC effects were reversed by anti-CD44 antibody (important for internalisation of MSC-derived EV) suggesting that EV in MSC-CM are mediators of their effect. Importantly, adoptive transfer of EV-treated alveolar macrophages conferred protection in the mild model of murine LPS-induced pneumonia. EV contents responsible for these effects are currently being investigated. Conclusion MSC promote M2-like macrophage polarisation via secretion of EV. This effect is associated with enhanced oxidative phosphorylation and altered JAK/STAT signalling, potentially regulated by differential expression of SOCS1 and 3 proteins. Reference Jackson MV, Morrison TJ, Doherty DF, et al. Mitochondrial Transfer via Tunnelling Nanotubes (TNT) is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ARDS. Stem Cells 2016;34(8):2210–23.

S63 Human Mesenchymal Stromal Cell (hMSC) regulation of human macrophages in in vitro models of the Acute Respiratory Distress Syndrome (ARDS)

Background Currently there is no effective therapy which targets the mechanisms underlying the development of ARDS. MSCs present a promising candidate therapy and are being tested in clinical trials for ARDS however their mechanisms of effect in ARDS are not fully understood. Since the alveolar macrophage is key to orchestrating the alveolar inflammatory response, it was hypothesised that hMSCs induce an anti-inflammatory M2-like phenotype in human macrophages. The aim of this study therefore was to determine the effect of MSCs on macrophage phenotype and function and to elucidate the mechanisms of these effects. Methods Using an in vitro non-contact co-culture system, human MSCs and human monocyte-derived-macrophages (MDMs) were stimulated with E.coli lipopolysaccharide (LPS). Cytokine and marker expression profiles were examined using ELISAs, multiplex and flow cytometry. Phagocytic capacity of MDMs was measured using fluorescent E.coli bioparticles by flow cytometry. For additional clinical relevance, the ARDS microenvironment was mimicked by using bronchoalveolar lavage fluid (BALF) obtained from patients with ARDS to examine the effect of MSCs. Results MSCs suppress the production of both pro-inflammatory and anti-inflammatory cytokines by MDMs stimulated with LPS. MSCs increase expression of M2 markers CD163 and CD206 and have no effect on M1 markers CD80 and ICAM-1. Importantly, in spite of the immunosuppressive effect on macrophages, MSCs increase their phagocytic capacity. MSC effects on cytokine secretion and marker expression were maintained in the presence of BALF from patients with ARDS (Figure 1).Abstract S63 Figure 1 MSCs decrease secretion of pro-inflammatory cytokines TNF-α (A) and IL-8 (B) and increase expression of M2 macrophage marker CD206 (C) by MDMs stimulated with BALF from non-septic (NS) or septic (S) patients of ARDS. (A + B, n = 3–7, Kruskal Wallis *p < 0.05) (C, n = 4, ANOVA *p < 0.05) Conclusions Human bone marrow-derived MSCs induce an M2-like phenotype and suppress cytokine secretion in primary human MDMs stimulated with LPS or ARDS patient BALF. Importantly, these effects are coupled with augmentation of macrophage phagocytosis which may be important in the clearance of bacteria and apoptotic cells. Uncovering the paracrine mechanisms responsible for the MSC effects on human macrophages remain the focus of ongoing work. Supported by MRC MR/L017229/1, Department of Employment and Learning. Some of the materials employed in this work were provided by the Texas 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.

Mesenchymal stromal cells for treatment of the acute respiratory distress syndrome: The beginning of the story

In spite of decades of research, the acute respiratory distress syndrome (ARDS) continues to have an unacceptably high mortality and morbidity. Mesenchymal stromal cells (MSCs) present a promising candidate for the treatment of this condition and have demonstrated benefit in preclinical models. MSCs, which are a topic of growing interest in many inflammatory disorders, have already progressed to early phase clinical trials in ARDS. While a number of their mechanisms of effect have been elucidated, a better understanding of the complex actions of these cells may pave the way for MSC modifications, which might enable more effective translation into clinical practice.