Therapies for acute respiratory distress syndrome: determination of the mechanisms of action of hypercapnia and the therapeutic efficacy of mesenchymal stromal cells

Rationale: Acute Respiratory Distress Syndrome (ARDS) causes severe respiratory failure and is associated with high mortality. Pharmacological therapies are non-existent and supportive ventilation treatments are limited. High ventilator induced stretch, termed Ventilator Induced Lung Injury (VILI), is a common propagator of ARDS development and causes severe inflammation and death of the lung parenchyma. Hypercapnia with its associated acidosis (HCA) appears to play a role in ARDS modulation by regulating inflammation but the mechanisms of this are not fully understood in the context of high stretch injury. Furthermore, Mesenchymal Stem/Stromal cells (MSCs) are promising contenders for ARDS therapy but issues with source, batch and donor variability, culture conditions and delivery to the clinic have not been fully resolved. Aims: (1): Determine the mechanisms by which HCA attenuates NF-κB activation and epithelial injury following high mechanical stretch in vitro. (2): Determine the mechanisms by which the MSC “secretome” attenuates lung injury in vitro and the potential for enhancement by MSC pre-activation. (3): Re-establish a relevant VILI animal model of ARDS and elucidate the potential for different MSC populations to enhance recovery. (4): Determine the efficacy of umbilical cord (UC) versus bone marrow (BM) derived MSCs. (5): Determine the impact of cryo-preservation and (6) culture in xeno-free (XF) conditions on the efficacy of MSC repair; and (7) the potential to enhance efficacy of XF and/or cryo-preserved MSCs by pre-stimulation. Methods: (1): Pulmonary alveolar epithelial cells were subjected injurious mechanical stretch injury and the potential for HCA to attenuate stretch induced cell inflammation and death was assessed. The interaction of the NF-κB pathway in HCA mediated effects was also assessed, as was the role of acidosis versus CO2. (2): The pulmonary alveolar epithelium was subjected to oxidative stress injury, inflammatory activation, wound injury and mechanical stretch injury and the efficacy of the MSC secretome to attenuate damage and promote repair was assessed. The potential to enhance the MSC secretome efficacy by pre-activation with inflammatory cytokines was also determined. (3): Animals were subjected to high pressure injurious ventilation followed by intravenous MSC administration. The efficacy of a defined S2+ MSC subpopulation to enhance recovery and repair following VILI was assessed. (4): The efficacy of UC derived MSCs to promote recovery post VILI was compared to standard BM derived MSC therapy and the effect of cryo-preservation on this efficacy was also determined. (5-7): The efficacy of cryo-preserved XF MSCs to promote recovery and repair at a time point of maximal VILI was determined, as was the potential for enhancement of efficacy by pre-activation. Results: (1) HCA attenuates high stretch induced inflammation and injury in the pulmonary epithelium through inhibition of the canonical NF-κB pathway and by way of generating an acidosis. (2): Pre-activation of MSCs enhances the protective effects the secretome in the injured pulmonary epithelium. (3): An S2+ subpopulation of MSCs enhances resolution and repair post VILI. (4): UC derived MSCs promote recovery post VILI with similar efficacy to BM MSCs and (5) this efficacy is retained after cryo-preservation. (6) XF cultured MSCs retain efficacy, while (7) pre-activation enhances the efficacy of cryo-preserved, XF expanded MSCs in lung recovery and repair following ventilator injury. Conclusion: (1): HCA modulates inflammation and may contribute to the benefit seen with protective ventilation in ARDS patients. (2): MSCs possess protective paracrine mechanisms of action and the secretome may be a viable alternative to the cell treatment. (3): S2+ MSCs may represent a more defined and less variable therapy for ARDS, and needs further investigation in other pre-clinical ARDS models. (4): UC derived MSCs are efficaciously comparable to standard BM MSCs, (5) even after cryo-preservation and may be a more readily available and less variable MSC therapy for the ARDS patient. (6): XF MSCs are also therapeutic, while (7) pre-activation enhances the protective and immunomodulatory functions of cryo-preserved XF MSCs and may provide a more efficacious treatment in the clinical setting of ARDS.2019-09-2