Microvesicles as crucial mediators of cytokine-dependent perioperative lung injury induced by mechanical ventilation

Each year, over 310 million major operations are performed worldwide. In most cases, patients are kept alive using a breathing machine or 'ventilator', ensuring oxygen is delivered to the lungs. Lung complications are common after an operation which in severe cases can lead to disability or death. These complications can be caused by the ventilator stretching the lung cells making them become inflamed but our current understanding of the mechanisms behind this is poor leading to no effective treatments.

Lung inflammation is controlled by a wide variety of substances such as "cytokines". One cytokine shown to be crucial in lung injury related to being on a ventilator is tumour necrosis factor (TNF). This can be found in various forms, but we do not know which type of TNF is most important in this disease. When cells are activated or injured, they release tiny particles known as microvesicles (MV) which can carry cargo such as TNF within them, delivering information between cells. There is increasing evidence that these MVs play a vital role in several lung diseases, specifically the cytokines associated with them.

We performed preliminary experiments that show if a ventilator over-stretches the lungs of mice, a substance known to stimulate MV production (ATP) is released and there is an increase in MV numbers that contain TNF; this also happens if you stretch lung cells in the laboratory. Also, when lung cells are inflamed due to cell stretch or infection, the interaction between MVs and the cells increases, causing the cells to become even more injured. Finally, we studied patients having lung transplant surgery and found more lung MVs in those patients that suffered from low oxygen levels after the operation, highlighting the importance of lung MVs.

Therefore, we propose that over-stretch of the lungs caused by a ventilator initiates the release of substances that leads to the production of cytokine-containing MVs. These MVs then interact with stretched lung cells causing lung inflammation, possibly leading to lung complications after surgery.

Our primary objective is to identify the role of cytokines associated with MVs that cause lung inflammation when on a ventilator. To do this, we will perform detailed laboratory experiments (using samples taken from patients) to achieve the following aims: 1) Identify the mechanisms by which cytokines are packaged within MVs following lung overstretch caused by a ventilator, 2) Investigate the interaction between MVs and resting or stretched lung cells, 3) Understand if the biological effect of MVs on lung cells is enhanced if the cells are stretched.

We will replicate the lung environment in the laboratory using different types of cells which will then be stretched using a machine. We will thoroughly study the production of MVs and the distribution of cytokines in these conditions. Additionally, we will assess the biological effects of these MVs on lung cells and see if their effects are enhanced by cell stretch. To see if our laboratory findings are replicated in humans, we will study patients undergoing 'oesophagectomy', a major operation to remove cancer of the gullet. These patients are exposed to lung overstretch due to a special type of ventilation necessary for this complex operation. We will dynamically assess MV production, the distribution of cytokines in various forms and lung inflammation/ injury using lung fluid and blood samples. We will also assess if MV numbers or the cytokines associated with them can predict which patient will have a lung complication after surgery. Finally, we will combine MVs taken from patients, with stretched/non-stretched lung cells in the laboratory to assess the MVs biological effects and interactions.

We hope this work enables future attempts to prevent lung inflammation and subsequent complications for patients on a ventilator during an operation or needing intensive care, which has the potential to vastly improve outcomes.

Postoperative pulmonary complications (PPCs) are a common yet potentially fatal consequence of major surgery. Perioperative mechanical ventilation often produces unphysiological lung stretch instigating inflammation, which predisposes the lungs to PPCs and may evolve into ventilator-induced lung injury (VILI). Intra-alveolar cytokines such as TNF have been implicated in the pathophysiology of VILI, but its precise mechanism is still unclear. Microvesicles (MV) may serve as a hidden compartment of such cytokines within the alveolus, inducing cytokine-mediated lung inflammation during VILI.

In this proposal, we aim to investigate the role of intra-alveolar MV-mediated inflammation in VILI, specifically focusing on MV-associated cytokine signalling, using a combined in vitro and in vivo approach. For in vitro, we will use a cell culture model of VILI, in which human alveolar epithelial cells (AECs) are mechanically stretched in mono-culture or co-culture with monocyte-derived macrophages. We will identify the mechanisms of stretch-induced MV production, cytokine packaging within MVs/MV subtypes, and MV-mediated AEC activation through their cargo cytokines. For in vivo, we will use a human model of VILI, in which serial bronchoalveolar lavage samples will be obtained from patients undergoing oesophagectomy and one lung ventilation (OLV). We will investigate MV profiles/cargo cytokines in lavage samples, define if pathological lung stretch during OLV diverts cytokine signalling from a soluble to MV-mediated pathway, and assess the bioactivity of patient-derived MVs using the in vitro culture model.

The results will provide fundamental yet clinically-translatable knowledge to understand the in vivo roles/mechanisms of MV-mediated cytokine signalling during VILI. Our findings will offer important insights towards the development of novel MV-targeting strategies for ventilator-associated lung inflammation/injury in both perioperative and critical care patients.