Keywords
ARDS, Acute respiratory distress syndrome, Mesenchymal stromal/stem cells, MSCs,
ARDS, Acute respiratory distress syndrome, Mesenchymal stromal/stem cells, MSCs,
Acute respiratory distress syndrome (ARDS) constitutes a spectrum of increasingly severe acute respiratory failure that may account for 10% of intensive care unit admissions worldwide and continues to have a mortality rate of over 40%1. ARDS can develop in response to multiple predisposing factors, including pneumonia, systemic infection, and major surgery or multiple traumas1. According to the Berlin Definition, the disease can be classified as mild, moderate, or severe, depending on the severity of hypoxemia2. Patients with ARDS have an acute onset of symptoms and signs that include severe dyspnea, chest pain, and cyanosis and bilateral pulmonary infiltrates indicative of edema on chest radiography; these symptoms usually appear within a one-week period from a known clinical insult2.
Inflammation is one of the main drivers of ARDS pathogenesis. Damage to the lung endothelial and epithelial layers is caused by inflammation of the lung parenchyma which is mediated by a predominantly neutrophilic influx into the alveolar space3. This leads to the release of pro-inflammatory cytokines, including interleukin-6 (IL-6), IL-1β, and IL-8 and tumor necrosis factor-alpha (TNF-α)3. This in turn leads to the generation of reactive oxygen species that can impair lung barrier function and increase vascular permeability and finally, where ARDS persists, can cause end-stage fibrosis3. Currently, there are no therapies for this condition, and protective mechanical ventilation and fluid-restrictive strategies are the only supportive treatments that can improve outcome. Mesenchymal stromal/stem cells (MSCs) have attracted considerable interest as a potential therapy for ARDS. In this review, we will discuss early key studies that first demonstrated the potential of MSCs for ARDS and subsequently focus on recent findings and advances published within the last 2 years.
MSCs are multipotent adult progenitor cells that can be isolated from numerous sources, including bone marrow, umbilical cord, and adipose tissue, and can differentiate into mesenchymal lineage cells such as fat or cartilage tissue4. The interest in MSCs as a possible therapy for ARDS stems largely from their potential to modulate the host immune response to injury and infection and to promote repair following tissue injury. A number of seminal studies showed the therapeutic potential of MSCs for ARDS. Gupta et al. demonstrated that MSCs significantly improve survival and reduced lung injury in mice following Escherichia coli endotoxin lung injury5. Importantly, MSCs modulated the immune response by inhibiting TNF-α release and by enhancing anti-inflammatory IL-10 secretion5. Mei et al. demonstrated that MSCs genetically modified to overexpress angiopoietin 1, an important protein involved in vascular growth and angiogenesis, were more effective than naïve MSC treatment in a mouse model of lipopolysaccharide (LPS)-induced lung injury6. Chang et al. demonstrated that human umbilical cord blood-derived MSCs attenuated hyperoxia-induced lung injury in the neonatal rat, a key demonstration of the efficacy of MSCs from other, potentially more plentiful, tissue sources7. Nemeth et al. demonstrated the therapeutic potential of MSCs in a murine model of systemic sepsis, and efficacy was mediated via reprogramming of macrophage function8. Of translational significance, Lee et al. demonstrated that human MSCs and MSC conditioned medium (MSC-CM) decreased endotoxin-induced injury to the human ex vivo perfused lung via mechanisms involving improved alveolar fluid clearance9. Krasnodembskaya et al. highlighted the role of MSC-secreted antimicrobial peptides in augmenting the host response to bacterial infection10. Curley et al. demonstrated the potential for MSC therapy to repair the lung following ventilation-induced acute lung injury (ALI)11.
The immune-modulating effect of MSCs is increasingly well understood. MSCs promote T regulatory cell expansion, which causes the suppression of the proliferation of effector T cells and dampens the immune response12. Furthermore, MSCs can modify T cells, dendritic cells, and natural killer cells and decrease their release of pro-inflammatory cytokines or increase their release of anti-inflammatory molecules12. MSCs can also cause the release of soluble potent anti-inflammatory mediators such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), and IL-1012,13. In terms of their reparative function, MSCs secrete growth factors such as keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF)14,15. Importantly for an acute inflammatory disorder such as ARDS, MSCs do not elicit a significant immune response in the host and thus can be used for allogeneic transplantation12.
MSCs have previously shown promising efficacy in numerous preclinical ARDS models, including pneumonia, ventilator-induced lung injury (VILI), and sepsis. A recent study showed that mouse MSCs significantly improved survival, reduced histologic lung injury, and reduced pulmonary edema in a mouse model of E. coli-induced ALI via a mechanism involving reduced oxidant stress (Table 1)16. Devaney et al. also recently demonstrated that human bone marrow (hBM)-derived MSCs reduce injury severity and increase survival in a rat model of E. coli pneumonia (Table 1)17. hBM-MSCs reduced the lung bacterial load following intra-tracheal E. coli administration via a mechanism involving enhanced antimicrobial peptide secretion and increased macrophage phagocytosis17. hBM-MSCs decreased alveolar concentrations of IL-6 while increasing concentrations of the anti-inflammatory cytokine IL-10 and increasing KGF17. Other findings of translational significance included delineation of the human MSC dose response curve and the demonstration that cryopreserved cells retain efficacy, but the secretome alone was less effective in the setting of E. coli pneumonia17. In a key study, Asmussen et al. showed that clinical-grade human MSCs significantly enhanced oxygenation while reducing pulmonary edema without any adverse effects in a large animal (sheep) model of combined smoke inhalation and Pseudomonas aeruginosa pneumonia (Table 1)18. The translational importance of this study is underlined by the fact that these cells are now in early phase clinical trials for ARDS.
Injury setting | Cell therapy | Effects |
---|---|---|
Unmodified MSC therapy | ||
Escherichia coli-induced ALI in mice16 | Mouse bone marrow MSCs 5 × 105 cells/mL/22g IV | - Increased survival - Decreased lung edema and lung histology score - Decreased oxidative stress injury (decreased myeloperoxidase activity and malondialdehyde levels) |
E. coli pneumonia in rats17 | hBM MSCs 10 and 20 million per kilogram IV | - Increased oxygenation and survival - Decreased lung histology score and bacterial load - Frozen delivery of cells is effective - Increased phagocytosis and LL-37 release in vitro |
Pseudomonas aeruginosa administration and smoke inhalation in sheep18 | hBM MSCs 5 and 10 million per kilogram IV | - Increased oxygenation - Decreased lung edema - No adverse effects observed |
Repair after ventilator- induced lung injury in rats21 | hBM MSCs 1–10 million per kilogram intra-tracheally and IV | - Increased blood oxygenation and lung compliance - Decreased lung edema and histologic injury - Decreased bronchoalveolar lavage CINC-1 and IL-6 - Lowest efficacious dose is 2 million per kilogram |
Ex vivo lungs under prolonged ischemic conditions23 | hBM MSCs 5 million per kilogram IV | Decreased edema in human lungs rejected for transplantation |
Modified MSC therapy | ||
Lipopolysaccharide- induced endothelial injury in vitro24 | MSCs overexpressing ACE2 | Enhanced decrease in endothelial damage, edema, and TNF-α and IL-6 concentrations when compared with naïve MSCs |
Bleomycin-induced ALI in mice25 | Human umbilical cord MSCs overexpressing ACE2 | Enhanced decrease in TNF-α, interferon-gamma, transforming growth factor-beta, and IL-1β, IL-2, and IL-6 and enhanced increase of superoxide dismutase and IL-10 when compared with naïve MSCs |
Previous studies observed MSCs to enhance lung repair following VILI. Chimenti et al. demonstrated the potential for MSC pretreatment to prevent the development of VILI following high-volume ventilation. MSC pretreatment reduced the lung water content and improved the lung histology score19. Bronchoalveolar lavage (BAL) levels of protein, neutrophils, macrophage inflammatory protein-2, and IL-1β were also considerably reduced7. Curley et al. observed that intra-tracheal and intravenously delivered rat MSCs and their secretome comparably enhanced restoration of lung oxygenation and compliance and restored lung structure after high-pressure VILI20. Inflammation was also significantly attenuated by MSC delivery as indicated by lower concentrations of IL-6 and TNF-α in the BAL20. More recently, Hayes et al. showed that MSCs retained their therapeutic efficacy following high-pressure VILI at doses as low as 2 million cells per kilogram (Table 1)21. They also observed that efficacy with MSC treatment was maintained with delayed delivery (that is, 6 hours after injury)21.
MSCs enhance alveolar fluid clearance, an important process in the recovery following ARDS. Bacteria-induced inflammation was also significantly inhibited and this appeared to be due to the enhanced phagocytic ability of macrophages and to antibacterial effects of secreted KGF in an ex vivo perfused human lung pneumonia model22. Of importance in transplant medicine, MSCs also restored alveolar fluid clearance in another study which used ex vivo lungs that were ischemic and rejected for transplantation (Table 1)23.
A key early study demonstrated the potential to enhance MSC efficacy by using gene overexpression strategies, specifically overexpression of Ang-16. More recently, MSCs overexpressing angiotensin-converting enzyme 2 (ACE2) showed enhanced endothelial repair and enhanced reduction in the expression of inflammatory molecules (including TNF-α and IL-6) following in vitro LPS injury when compared with naïve MSCs24 (Table 1). ACE2 plays a major role in negating the pro-inflammatory and pro-apoptotic effects of angiotensin II on the endothelium24. In vivo, Min et al. showed that MSCs overexpressing ACE2 caused an enhanced reduction in inflammation and reduced lung edema, collagen deposition, and fibrosis after bleomycin-induced lung injury in mice25. Furthermore, these cells decreased oxidative stress damage to a greater degree than naïve MSCs25. Finally, human MSCs overexpressing soluble IL-1 receptor-like-1, which plays an immune-modulatory role in the lung, decreased inflammation, BAL protein and neutrophil content to a greater extent than naïve MSCs in a mouse model of LPS-induced ALI26.
MSCs exert their therapeutic efficacy in part via paracrine mechanisms (Table 2). MSC-CM—termed the ‘MSC secretome’—attenuates injury following endotoxin-induced ALI in the mouse27, restoring lung structure and decreasing alveolar concentrations of IL-6 and macrophage inflammatory protein-2. These effects were partly mediated via the attenuation of the expression of nuclear factor-kappa B (NF-κB) p65 and phospho-NF-κB p65 in the mouse lung27. MSC-CM also induced neutrophil apoptosis as indicated by a reduction of the expression of Bcl-xl and Mcl-1 on said neutrophils27. In a similar model, MSC-CM enhanced the activation of the M2 macrophage phenotype, thus promoting an anti-inflammatory and pro-healing environment28. Interestingly, these effects were correlated with increased levels of insulin-like growth factor 1 (IGF-1)18.
Injury setting | Therapy | Effects |
---|---|---|
Entire MSC secretome | ||
Endotoxin-induced ALI in mice27 | Mouse MSC conditioned medium | - Decreased histology score and lung neutrophils - Decreased lung interleukin-6, macrophage inflammatory protein-2, and NF-κB p65 and phospho-NF-κB p65 expression - Increased apoptosis of neutrophils |
MSC-derived microvesicles | ||
Endotoxin-induced ALI in mice30 | Human MSC microvesicles 30 μL | - Decreased lung edema - Decreased protein, neutrophil, and macrophage inflammatory protein-2 levels - In part via enhanced KGF expression |
Escherichia coli pneumonia in mice32 | Human MSC microvesicles | - Increased survival - Decreased bacterial load - Decreased BAL protein - Increased phagocytosis by monocytes in vitro |
E. coli pneumonia in mice33 | Human bone marrow MSCs | MSCs through mitochondrial transfer in microvesicles enhanced the phagocytic action of macrophages in vitro and in vivo |
The potential for the MSC secretome to enhance lung repair following VILI has also been demonstrated. Intra-tracheal delivery of the MSC secretome products enhanced lung oxygenation and compliance and reduced the lung histology score and pulmonary edema at 48 hours after high-pressure VILI20. Another study examined the effects of MSCs and their secretome in the earlier phases of the repair process following VILI29. The MSCs were effective and significantly improved blood oxygenation and respiratory compliance while reducing lung edema, BAL neutrophil counts, and IL-6 concentrations29. In contrast, the MSC secretome failed to exert therapeutic effects at this earlier stage in the recovery process, suggesting that not all of the effects of the MSC are recapitulated by the secretome alone29.
A relatively new discovery is that some of the effects of MSCs appear to be mediated via the release of particles, specifically microvesicles and exosomes that can transfer genetic material, cellular organelles such as mitochondria, and biologically active molecules between cells (Table 2). One study observed that microvesicles from hBM-MSCs significantly decreased lung edema and BAL protein and neutrophil counts in an LPS lung injury model and this was facilitated partly by the increased expression of KGF30. Islam et al. demonstrated that MSCs delivered to LPS-injured mice transferred mitochondria-containing microvesicles through gap junctions to the mouse alveolar epithelia and that the therapeutic effect was abrogated with the use of MSCs with non-functional mitochondria or gap junction-incompetent MSCs31. Another study observed that human MSC-derived microvesicles enhanced survival in a mouse model of E. coli pneumonia32. The microvesicles were comparable to MSC cell treatment in their ability to reduce bacterial load and BAL protein and cytokine concentrations, and their effects were mediated in part by KGF secretion32. Furthermore, the phagocytic capability of monocytes in vitro was also increased by the microvesicles32. Jackson et al. also observed that MSCs enhanced macrophage phagocytosis in vitro and produced anti-bacterial effects in an E. coli pneumonia mouse model in part via microvesicle transfer of mitochondria33.
Based on their therapeutic promise in preclinical studies, MSCs have entered early phase clinical testing in patients with ARDS (Table 3). The recently reported phase 1b START trial (safety proof-of-concept trial) enrolled nine patients to receive 1, 5, or 10 million cells per kilogram of a single intravenous dose of allogeneic hBM MSCs using a three-by-three dose escalation design and showed no adverse effects at any of the doses used34. These investigators are now enrolling patients in a phase 2 study using the dose of 10 million cells per kilogram (NCT01775774). In another phase 1 trial, adipose-derived allogeneic MSCs were administered to 12 patients in an intravenous dose of 1 million cells per kilogram or placebo in a 1:1 ratio and again the cells were well tolerated in the patients35. This study also measured IL-6, IL-8, and surfactant protein D levels and found that surfactant protein D concentrations were lower after MSC delivery at day 5 but that MSC administration had no effects on IL-6 or IL-8 levels35. The study concluded that higher doses may be needed at the phase 2 stage35. Importantly, given the relationship between ARDS and sepsis, MSCs are also in early phase clinical studies in patients with septic shock in Canada (Cellular Immunotherapy in Septic Shock study, NCT02421484).
Although MSCs demonstrate considerable promise and early phase clinical trials are encouraging, several challenges still impede the clinical translation of MSCs for patients with ARDS. Cell batch variability between donors remains a significant hurdle that raises efficacy and possible safety concerns. Robust assays of MSC batch potency for ARDS are lacking. However, one recent study observed that cell motility can predict the differentiation potential of MSCs, which may provide a bioassay for characterizing MSC efficacy for certain disease conditions36. Another study also recently outlined that MSCs from young mouse donors were more effective than those from aging donors in reducing pulmonary fibrosis37. Of relevance to ARDS, MSCs from aging mice were less effective in reducing endotoxemia-induced lung injury38. Importantly, the efficacy of these cells in an in vitro cell motility assay paralleled their effectiveness in subsequent in vivo studies, suggesting that this may be a useful assay of potency38.
Furthermore, the generation of large quantities of clinical-grade MSCs for dose delivery to humans is both time consuming and costly. Doses of up to 1 billion MSCs per patient are currently being studied in clinical trials. Should MSCs prove an effective therapy at these doses, alternative, more easily accessible and abundant sources, such as adipose tissue or the umbilical cord, are likely to prove more feasible than bone marrow, which is the current source of cells used in most clinical studies. The demonstration that MSCs retain their efficacy when cryopreserved is important in making bulk production and storage of MSCs for clinical use feasible17 and will facilitate MSC translation to the clinic.
In conclusion, MSCs may provide the immunomodulatory and regenerative tool needed to target ARDS pathogenesis. Preclinical studies have highlighted their therapeutic potential and mechanisms of action. These preclinical studies may also be of relevance to other clinical situations, such as smoke inhalation injury, lung transplantation, and chemotherapy-induced lung injury. Although there are considerable grounds for optimism regarding the therapeutic potential of MSCs for ARDS, hurdles to translation still exist, and only time will tell whether MSCs realize their therapeutic potential in later-phase clinical trials.
ACE2, angiotensin-converting enzyme 2; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; BAL, bronchoalveolar lavage; hBM, human bone marrow; IL, interleukin; KGF, keratinocyte growth factor; LPS, lipopolysaccharide; MSC, mesenchymal stromal/stem cell; MSC-CM, mesenchymal stromal/stem cell conditioned medium; NF-κB, nuclear factor-kappa B; TNF-α, tumor necrosis factor-alpha; VILI, ventilator-induced lung injury.
Shahd Horie is supported by funding from the Health Research Board, Ireland. John G. Laffey is funded by the Canadian Institute of Health Research and Physicians Services Incorporated, Ontario, and holds a Merit Award from the University of Toronto, Department of Anesthesia.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
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