Modulation of neutrophil dysfunction in tracheostomised children to prevent respiratory infections

Many young children who have serious medical problems require help with their breathing long-term. In this setting some children need a procedure called a "tracheostomy'', which involves a surgeon making a small hole in the windpipe, and inserting a small tube, allowing them to breathe more easily. The tracheostomy can sometimes be removed at a later date. However, many children have the tracheostomy for a number of years. Living with a tracheostomy carries a significant burden as carers have to ensure the tube does not block, which would result in death.

Children with tracheostomies are much more prone to chest infections than other children. These infections can result in multiple admissions to hospital and can themselves be life-threatening. Little is known about why children with tracheostomies get so many chest infections and therefore we do not know how to prevent them.

A major problem with research in this area has been getting samples from children undergoing a tracheostomy. My previous research has overcome this by working closely with those involved in the care of these children, including doctors, nurses and parents. This work has shown that children's windpipes "overreact" to the tracheostomy procedure, and that this might actually reduce the body's ability to fight infection. During infection, cells called neutrophils normally recognise, "eat" and kill bacteria and we believe this essential process does not happen properly after a tracheostomy. In children with tracheostomies, neutrophils seem to cause damage to the lining of the windpipe, allowing harmful bacteria to enter and cause chest infections.

I plan to use samples of sputum (taken directly from the windpipe) and blood taken from children with and without a tracheostomy to: 1) understand why neutrophils might not be fighting bacteria effectively - this will include using innovative technologies to look at and measure the way neutrophils, taken out of the body, respond to bacteria; 2) assess how this defective function might be causing the lungs to become damaged; and 3) look at ways of "repairing" these neutrophils using drugs that restore their ability to kill bacteria and stop neutrophils from releasing chemicals that harm the windpipe. This, we anticipate, will reduce damage in the airway, allowing neutrophils to kill bacteria in the way they are intended to.

By using live cells taken out of the body (from children's sputum or blood samples) and testing them in the laboratory I can check if these drugs work, without any risk of harm to the child, and without using animal testing. The drugs that I identify to be most efficient at restoring neutrophil function can subsequently be tested on children in a clinical trial. The ultimate aim of the work is to find new drugs to reduce the number of chest infections that children with tracheostomies children have to endure, and the associated complications of these infections.

Findings of the study will be shared with other researchers, and much of this work will help researchers studying other lung conditions in which neutrophils play a damaging role.

I will collaborate with researchers in other centres, including Durham, Belfast, Berlin and Vancouver. As part of my vision for this project I have assembled a team of people with relevant experiences and skills from different backgrounds, such as clinicians, physicists and laboratory scientists all bringing specific skills together to solve an important problem. The research will provide me with a unique skillset to become a world-leading researcher seeking new solutions to improve the quality of life and survival of children requiring tracheostomies.

Tracheostomy in children is associated with ongoing respiratory infections resulting in significant morbidity, poor quality of life, healthcare costs, and excess mortality. The mechanisms underlying these adverse outcomes are poorly understood resulting in a lack of management options.

I previously demonstrated that paediatric tracheostomy is associated with the ongoing presence of pathogenic bacteria and neutrophilic inflammation. Neutrophils are critical to bacterial clearance and previous studies have demonstrated phagocytic dysfunction in extravasated and blood-derived neutrophils in severe illness via a failure of RhoA-dependent cytoskeletal re-arrangement. I have developed skills in functional evaluation of extravasated, tissue neutrophils. I subsequently demonstrated elevated neutrophil proteases, reactive oxygen species and proteolysis in the trachea of tracheostomised children. This strongly suggests that dysfunctional tissue neutrophils drive inflammation secondary to tracheostomy, perpetuating further neutrophil recruitment.

In this fellowship I will use extravasated airway and blood-derived neutrophils to characterise mechanisms of tissue neutrophil dysfunction, combining established techniques and novel real-time imaging modalities to assess microrheological changes during phagocytosis. I will characterise protease activity at the cell surface of extravasated neutrophils in response to stimulation. I will also develop an air-liquid interface co-culture model using primary tracheal cells to study the effects of dysfunctional neutrophils on the epithelium, and interrogate bacterial susceptibility in this model. Finally, I will investigate potential therapeutic agents anticipated to (a) restore neutrophil function (e.g. PI3Kd inhibitors) or (b) inhibit neutrophil proteases mediating epithelial damage. This data will lay the foundations for a career investigating mechanisms of airway injury after tracheostomy and suggest compounds for future trials.