The effect of hyperoxia on inflammation and platelet responses in an ex vivo extracorporeal membrane oxygenation circuit.

Use of extracorporeal membrane oxygenation (ECMO) is expanding, however, it is still associated with significant morbidity and mortality. Activation of inflammatory and innate immune responses and hemostatic alterations contribute to complications. Hyperoxia may play a role in exacerbating these responses. Nine ex vivo ECMO circuits were tested using fresh healthy human whole blood, with two oxygen levels: 21% inspired fraction of oxygen (FiO2 ; mild hyperoxia; n = 5) and 100% FiO2 (severe hyperoxia; n = 4). Serial blood samples were taken for analysis of platelet aggregometry, leukocyte activation, inflammatory, and oxidative stress markers. ECMO resulted in reduced adenosine diphosphate- (P < .05) and thrombin receptor activating peptide-induced (P < .05) platelet aggregation, as well as increasing levels of the neutrophil activation marker, neutrophil elastase (P = .013). Additionally, levels of the inflammatory chemokine interleukin-8 were elevated (P < .05) and the activity of superoxide dismutase, a marker of oxidative stress, was increased (P = .002). Hyperoxia did not augment these responses, with no significant differences detected between mild and severe hyperoxia. Our ex vivo model of ECMO revealed that the circuit itself triggers a pro-inflammatory and oxidative stress response, however, exposure to supra-physiologic oxygen does not amplify that response. Extended-duration studies and inclusion of an endothelial component could be beneficial in characterizing longer term changes.

Combined Mesenchymal Stromal Cell Therapy and Extracorporeal Membrane Oxygenation in Acute Respiratory Distress Syndrome. A Randomized Controlled Trial in Sheep.

Rationale: Mesenchymal stromal cell (MSC) therapy is a promising intervention for acute respiratory distress syndrome (ARDS), although trials to date have not investigated its use alongside extracorporeal membrane oxygenation (ECMO). Recent preclinical studies have suggested that combining these interventions may attenuate the efficacy of ECMO.Objectives: To determine the safety and efficacy of MSC therapy in a model of ARDS and ECMO.Methods: ARDS was induced in 14 sheep, after which they were established on venovenous ECMO. Subsequently, they received either endobronchial induced pluripotent stem cell-derived human MSCs (hMSCs) (n = 7) or cell-free carrier vehicle (vehicle control; n = 7). During ECMO, a low Vt ventilation strategy was employed in addition to protocolized hemodynamic support. Animals were monitored and supported for 24 hours. Lung tissue, bronchoalveolar fluid, and plasma were analyzed, in addition to continuous respiratory and hemodynamic monitoring.Measurements and Main Results: The administration of hMSCs did not improve oxygenation (PaO2/FiO2 mean difference = -146 mm Hg; P = 0.076) or pulmonary function. However, histological evidence of lung injury (lung injury score mean difference = -0.07; P = 0.04) and BAL IL-8 were reduced. In addition, hMSC-treated animals had a significantly lower cumulative requirement for vasopressor. Despite endobronchial administration, animals treated with hMSCs had a significant elevation in transmembrane oxygenator pressure gradients. This was accompanied by more pulmonary artery thromboses and adherent hMSCs found on explanted oxygenator fibers.Conclusions: Endobronchial hMSC therapy in an ovine model of ARDS and ECMO can impair membrane oxygenator function and does not improve oxygenation. These data do not recommend the safe use of hMSCs during venovenous ECMO.

Viability of Mesenchymal Stem Cells in an Ex Vivo Circulation System.

Extracorporeal membrane oxygenation (ECMO) is a well-known therapy for refractory cardiac and respiratory failure. Stem cell therapy has been investigated as an adjunctive treatment for use during ECMO, but little is known about the viability of stem cells during ECMO support. We evaluated the viability and activity of mesenchymal stem cells (MSCs) in ex vivo circulation (EVC) conditions. The experimental groups were divided into two subgroups: EVC with oxygenator (OXY group) and EVC without oxygenator (Non-OXY group). Mesenchymal stem cells (1.0 × 10) were injected into the EVC system. Cell counting, a lactate dehydrogenase (LDH) cytotoxicity assay, and the mitochondrial functions of viable MSCs were analyzed. The post-EVC oxygen consumption rate (OCR) was significantly lower than the pre-EVC OCR, regardless of whether the oxygenator was used. The LDH levels were significantly higher in the OXY group than in the Non-OXY group. The cellular loss was mainly due to lysis of the cells whereas the loss of cellular activity was attributed to the nonphysiologic condition itself, as well as the oxygenator. We concluded that direct infusion of MSCs during ECMO support did not serve as adjunctive therapy. Further studies are needed to improve the viability in an ECMO setting.

Effect of ex vivo extracorporeal membrane oxygenation flow dynamics on immune response.

BACKGROUND: Extracorporeal membrane oxygenation is a life-saving support for heart and/or lung failure patients. Despite technological advancement, abnormal physiology persists and has been associated with subsequent adverse events. These include thrombosis, bleeding, systemic inflammatory response syndrome and infection. However, the underlying mechanisms are yet to be elucidated. We aimed to investigate whether the different flow dynamics of extracorporeal membrane oxygenation would alter immune responses, specifically the overall inflammatory response, leukocyte numbers and activation/adhesion surface antigen expression. METHODS: An ex vivo model was used with human whole blood circulating at 37°C for 6 hours at high (4 L/minute) or low (1.5 L/minute) flow dynamics, with serial blood samples taken for analysis. RESULTS: During high flow, production of interleukin-1ß (p < 0.0001), interleukin-6 (p = 0.0075), tumour necrosis factor-α (p = 0.0013), myeloperoxidase (p < 0.0001) and neutrophil elastase (p < 0.0001) were significantly elevated over time compared to low flow, in particular at 6 hours. While the remaining assessments exhibited minute changes between flow dynamics, a consistent trend of modulation in leukocyte subset numbers and phenotype was observed at 6 hours. CONCLUSION: We conclude that prolonged circulation at high flow triggers a prominent pro-inflammatory cytokine response and activates neutrophil granule release, but further research is needed to better characterize the effect of flow during extracorporeal membrane oxygenation.

Mesenchymal stem cells may ameliorate inflammation in an ex vivo model of extracorporeal membrane oxygenation.

INTRODUCTION: Mesenchymal stem cells exhibit immunomodulatory properties which are currently being investigated as a novel treatment option for Acute Respiratory Distress Syndrome. However, the feasibility and efficacy of mesenchymal stem cell therapy in the setting of extracorporeal membrane oxygenation is poorly understood. This study aimed to characterise markers of innate immune activation in response to mesenchymal stem cells during an ex vivo simulation of extracorporeal membrane oxygenation. METHODS: Ex vivo extracorporeal membrane oxygenation simulations (n = 10) were conducted using a commercial extracorporeal circuit with a CO2-enhanced fresh gas supply and donor human whole blood. Heparinised circuits (n = 4) were injected with 40 × 106-induced pluripotent stem cell-derived human mesenchymal stem cells, while the remainder (n = 6) acted as controls. Simulations were maintained, under physiological conditions, for 240 minutes. Circuits were sampled at 15, 30, 60, 120 and 240 minutes and assessed for levels of interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, tumour necrosis factor-α, transforming growth factor-ß1, myeloperoxidase and α-Defensin-1. In addition, haemoglobin, platelet and leukocyte counts were performed. RESULTS: There was a trend towards reduced levels of pro-inflammatory cytokines in mesenchymal stem cell-treated circuits and a significant increase in transforming growth factor-ß1. Blood cells and markers of neutrophil activation were reduced in mesenchymal stem cell circuits during the length of the simulation. As previously reported, the addition of mesenchymal stem cells resulted in a reduction of flow and increased trans-oxygenator pressures in comparison to controls. CONCLUSIONS: The addition of mesenchymal stem cells during extracorporeal membrane oxygenation may cause an increase in transforming growth factor-ß1. This is despite their ability to adhere to the membrane oxygenator. Further studies are required to confirm these findings.

Administration of mesenchymal stem cells during ECMO results in a rapid decline in oxygenator performance.

Mesenchymal stem cells (MSCs) have attracted attention as a potential therapy for Acute Respiratory Distress Syndrome (ARDS). At the same time, the use of extracorporeal membrane oxygenation (ECMO) has increased among patients with severe ARDS. To date, early clinical trials of MSCs in ARDS have excluded patients supported by ECMO. Here we provide evidence from an ex-vivo model of ECMO to suggest that the intravascular administration of MSCs during ECMO may adversely impact the function of a membrane oxygenator. The addition of clinical grade MSCs resulted in a reduction of flow through the circuit in comparison to controls (0.6 ±0.35 L min-1vs 4.12 ± 0.03 L min-1, at 240 minutes) and an increase in the transoygenator pressure gradient (101±9 mmHg vs 21±4 mmHg, at 240 minutes). Subsequent immunohistochemistry analysis demonstrated quantities of MSCs highly adherent to membrane oxygenator fibres. This study highlights the potential harm associated with MSC therapy during ECMO and suggests further areas of research required to advance the translation of cell therapy in this population.

Packed Red Blood Cell Transfusion Modulates Myeloid Dendritic Cell Activation and Inflammatory Response In Vitro.

Transfusion of packed red blood cells (PRBCs) modulates patients' immune responses and clinical outcomes; however, the underpinning mechanism(s) remain unknown. The potential for PRBC to modulate myeloid dendritic cells (mDC) and blood DC antigen 3 was assessed using an in vitro transfusion model. In parallel, to model processes activated by viral or bacterial infection, toll-like receptor agonists polyinosinic:polycytidylic acid or lipopolysaccharide were added. Exposure to PRBC upregulated expression of CD83 and downregulated CD40 and CD80 on both DC subsets, and it suppressed production of interleukin (IL)-6, IL-8, IL-12, tumor necrosis factor-α, and interferon-gamma-inducible protein-10 by these cells. Similar effects were observed when modeling processes activated by concurrent infection. Furthermore, exposure to PRBC at date of expiry was associated with more pronounced effects in all assays. Our study suggests PRBC have an impact on recipient DC function, which may result in failure to establish an appropriate immune response, particularly in patients with underlying infection.

Platelet concentrates modulate myeloid dendritic cell immune responses.

Platelet transfusion has been reported to modulate the recipients' immune system. To date, the precise mechanism(s) driving poor patient outcomes (e.g., increased rate of mortality, morbidity, infectious complications and prolonged hospital stays) following platelet transfusion are largely undefined. To determine the potential for platelet concentrates (PC) to modulate responses of crucial immune regulatory cells, a human in vitro whole blood model of transfusion was established. Maturation and activation of human myeloid dendritic cells (mDC) and the specialized subset blood DC antigen (BDCA)3+ DC were assessed following exposure to buffy-coat derived PC at day (D)2 (fresh) and D5 (date-of-expiry). In parallel, to model recipients with underlying viral or bacterial infection, polyinosinic:polycytidylic acid or lipopolysaccharide was added. Exposure to PC had less of an impact on mDC responses than BDCA3+ DC responses. PC alone downregulated BDCA3+ DC expression of co-stimulatory molecules CD40 and CD80. In the model of viral infection, PC downregulated expression of CD83, and in the bacterial model of infection, PC downregulated CD80, CD83, and CD86. PC alone suppressed mDC production of interleukin (IL)-8, IL-12 and tumor necrosis factor (TNF)-α and BDCA3+ DC production of IL-8, IL-12, and IL-6. In the model of viral infection, production of IL-12 and interferon-gamma inducible protein (IP)-10 was reduced in both DC subsets, and IL-8 was reduced in BDCA3+ DC following PC exposure. When modeling bacterial infection, PC suppressed mDC and BDCA3+ DC production of IL-6 and IL-10 with a reduction in TNF-α evident in mDC. This study assessed the impact of PC "transfusion" on DC surface antigen expression and inflammatory mediator production and provided the first evidence that PC transfusion modulates blood mDC and BDCA3+ DC maturation and activation, particularly in the models of infection. Results of this study suggest that patients who receive PC, particularly those with underlying infectious complications, may fail to establish an appropriate immune response precipitating poor patient outcomes.

Immunomodulatory effect of cryopreserved platelets: altered BDCA3+ dendritic cell maturation and activation in vitro.

BACKGROUND: Cryopreservation of platelets (PLTs) is useful in remote areas to overcome logistic problems associated with supply and can extend the shelf life to 2 years. During cryopreservation, properties of PLTs are modified. Whether changes in the cryopreserved PLT (CPP) product are associated with modulation of recipients' immune function is unknown. We aimed to characterize the immune profile of myeloid dendritic cells (mDCs) and the specialized blood DC antigen (BDCA)3+ subset after exposure to CPPs. STUDY DESIGN AND METHODS: Using an in vitro whole blood model of transfusion, the effect of CPPs on mDC and BDCA3+ DC surface antigen expression and inflammatory mediator production was examined using flow cytometry. In parallel, polyinosinic:polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) was utilized to model processes activated in viral or bacterial infection, respectively. RESULTS: Cryopreserved PLTs had minimal impact on mDC responses but significantly modulated BDCA3+ DC responses in vitro. Exposure to CPPs alone up regulated BDCA3+ DC CD86 expression and suppressed interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interferon-γ inducible protein (IP)-10 production. In both models of infection-related processes, exposure to CPPs down regulated BDCA3+ DC expression of CD40, CD80, and CD83 and suppressed BDCA3+ DC production of IL-8, IL-12, and TNF-α. CPPs suppressed CD86 expression in the presence of LPS and IP-10 and IL-6 production with poly(I:C). CONCLUSION: Cryopreserved PLTs may be immunosuppressive, and this effect is more evident when processes associated with infection are concurrently activated, especially for BDCA3+ DCs. This suggests that transfusion of CPPs in patients with infection may result in impaired BDCA3+ DC responses.