A VILI rabbit model will be used to test the in vivo relevance of the abovementioned in vitro findings. In particular, alarmins will be detected in lung lavage fluids from rabbit submitted to prolonged mechanical ventilation, and alarmin inhibitors will also be tested in this model with the aim of neutrophil-dependent preventing lung injury. Finally alarmins levels will be measured in human BAL fluid from patients mechanically ventilated and in the plasma from patients with sepsis and ARDS.
A series of in vitro experiments using the cyclic stretch model aim at studying the signaling of alarmins (inflammasome), and test various inhibitors of alarmin-dependent activation of cells. These experiments will focus on the activation of lung cells by mtDNA (TLR9 pathway), ATP (caspase-1 activation) and fMLP (neutrophil activation and chemotaxis). Co-cultures with neutrophils will also be performed to investigate their relative role in epithelial cell injury during cyclic stretch.
Preliminary results using an in vitro model show that human alveolar cells submitted to cyclic stretch release such alarmins. These results serve as a proof-of-concept to further investigate the role of alarmins in the generation of aseptic lung inflammation during mechanical ventilation. We hypothesize that alarmins released from cells injured during unusual mechanical strains are responsible for the alveolar production of IL-1ß creating a local inflammatory reaction (mtDNA and ATCec). Released alarmins (mitochondrial fMLP) also attract, home to the airways, and activate neutrophils that will produce additional lung injury.
Mechanical ventilation of critically ill patients, particularly when high pressures and volumes are delivered to the lung, produces plasma membrane breaks in lung cells and recruitment of activated neutrophils to the airways. This phenomenon is now called “ventilator-induced lung injury, or VILI”. Although some of the (secondary) mediators have been recognized, such as IL-1ß and IL-8, the proximal mediators responsible for generating the local inflammation are not known. It is the aim of this proposal to test the relevance of mitochondrial alarmins and extracellular ATP in the mediation of VILI.
Mediators from human tissues responsible for generating local and systemic inflammation are poorly known. Recently, the concept of alarmins (or danger signals) released from injured (necrotic) tissues was proposed to explain the situation of inflammatory responses in the absence of an infection. Experimental models have recently identified alarming of mitochondrial origin, released from necrotic tissues that signal the presence of danger and activate the inflammatory and immune systems. Among the molecules that are released from tissues that play a local (and possibly systemic) role are: mitochondrial DNA, the formyl peptide fMLP, and adenosine triphosphate, ATP. When present extracellularly, these mediators induce an inflammatory reaction via the production of the pro-inflammatory IL-1ß cytokine by neighboring cells, via the intracytoplasmic assembly of the inflammasome. fMLP plays also an important role as a chemotactic and activating factor for neutrophils. Activated neutrophils recruited to the site of tissue injury in turn participate in further injury.
This project carries the exciting potential to unravel the role of novel and proximal endogenous mediators of lung inflammation and injury, and also to identify critical pathways that could be modulated with pharmacological inhibitors.