Understanding the Mechanisms of Action of Hypercapnic Acidosis and the Therapeutic Potential of Human Mesenchymal Stromal Cells in Diminishing Inflammation and Enhancing Repair in Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a term used to describe severe lung injury characterised by uncontrolled inflammatory response and resultant damage to endothelial and epithelial layers leading to eventual loss of pulmonary function. ARDS can be aggravated by the only therapy currently available to prolong survival - mechanical ventilation. To date many attempts have been made to alter ventilation protocols to reduce the over-distension and cycle of atelectasis and shear stresses associated with artificial gas delivery to the lung. Thus far the most effective therapeutic strategy overall has been to reduce the tidal volumes used which can lead to the build-up of CO2, this is termed "permissive hypercapnia". Experimental studies recommend avoiding buffering the resultant acidosis as there is no evidence of benefit. In fact, both the decrease in pH and the elevated CO2 may confer their own beneficial effects during lung inflammation and injury, but may also have adverse effects such as slowing repair and inhibiting the host response to infection. The anti-inflammatory effects of hypercapnic acidosis (HCA), appears to be mediated, at least in part, by the suppression of NF-kB, a key transcription factor in inflammation, injury and repair. however the exact mechanisms by which HCA suppresses activation of the NF-kB pathway remain to be elucidated. A greater understanding of these mechanisms may provide opportunities to develop strategies to harness the benefits of hypercapnia while minimising any potential for harm. The investigation of the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is a rapidly escalating, including recent application to the area of lung disease and injury. The safety, and in some cases, efficacy of hMSCs has been established in disease states such as skeletal muscle injury, myocardial infarction, stroke, and graft versus host disease. In fact, an initial phase 1 dose escalation efficacy and safety study of MSCs has recently commenced in patients with ARDS. However, much remains to be understood in regard to the efficacy and mechanisms of action of MSCs before we can move forward to definitive clinical testing. Therefore these studies, in a continuation of previous studies from our laboratory are aimed at determining the precise mechanism of action of HCA on the pulmonary NF-kB pathway and following this, to provide critical pre-clinical data that will enable the safe and effective use of human MSCs in ARDS. Methods: In vitro models of lung injury were used to determine the effects of HCA on the NF-kB pathway. Pulmonary cell lines were transfected or transduced with an NF-kB luciferase reporter and subjected to TNF-a, IL-1b or endotoxin injury. Therapeutic HC was administered by increasing CO2 levels in cell culture environments for 24 hours. NF-kB activation was assessed based on luciferase production in cell lysates and IL-8 concentration in cell culture media. Effects of HCA on intracellular proteins were analysed in cellular fractions which were prepared using nuclear extraction kits and the proteins were analysed using Western blotting, ELISA, binding assays, kinase assays and transcription, translation assays. In vivo experiments were conducted to determine the optimal route, therapeutic window and the benefits of using purified sub-populations of MSCs in a rodent model of recovery following VILI. VILI was induced using high pressure ventilation until a severe lung injury, as evidenced by a 50% drop in lung compliance, was induced. Following establishment of the injury, the animals were then treated with BM-MSCs (4x106cells/kg; IV, IT or IP), either immediately post injury or 0, 6 or 24 hours post injury, depending on the specific experimental protocol. The ORB-1+ and ORB-1- sub-populations of MSCs were compared to heterogeneous "parental" MSCs. At 24 or 48 hours following VILI, animals were harvested and analysis performed to determine the extent of repair of the lung injury. Key indices of injury and recovery assessed included arterial oxygenation, lung compliance, lung inflammation, cytokine response and histologic morphology. Results: HCA was demonstrated to reduced NF-kB activation in pulmonary and systemic cell lines as indicated by both luciferase assay and IL-8 ELISA. HCA also suppressed NF-kB pathway activation following over-expression of key pathway proteins. HCA was shown to affect protein activation by inhibiting phosphorylation and kinase activity as demonstrated using ELISA and in vitro kinase assays. Further to this HCA was shown to decrease NF-kB dimer activity and the binding of NF-kB to its consensus sequence, but not the formation of these dimers in vitro. HCA was shown to inhibit transcription factor to DNA binding and subsequent transcription and translation. In vivo, it was determined that an optimal dose of 4x106cells/kg was effective in the attenuation of VILI when administered IT or IV up to 24 hours post injury. This was demonstrated by improvements in arterial blood oxygenation, static lung compliance, cytokine profiles and histological morphology compared to controls. In addition to this the use of a particular, more rigorously defined, sub-population of cells was almost as effective as mixed, parental populations of MSCs. Conclusions: The application of therapeutic HCA to injured pulmonary and systemic cell lines suppress NF-kB activation by acting at multiple points in the pathway. Further to this, HCA reduces the ability of NF-kB complexes to translocate to the nucleus, bind to DNA and potentially decreases their ability to initiate transcription. The administration of hMSCs IV or IT demonstrates superior efficacy to IP administration to an animal model of VILI. In addition, these cells can be administered up to 24 hours post injury without significant loss in efficacy toward repair and recovery. The use of ORB-1 hMSC sub-populations confers no additional benefit in the repair and recovery following VILI