Background: Neutrophil activation is a key pathological hallmark of acute lung injury yet bedside methods to identify the presence of activated neutrophils in the distal lung parenchyma remain elusive. Elucidating the cytokine, proteomic and lipidomic signatures of alveolar neutrophil transmigration in response to pulmonary injury has the potential to facilitate diagnostic and therapeutic advances.

Aims and objectives: Develop a signature library of neutrophilic influx in the distal human lung utilising an optical Neutrophil Activation Probe (NAP) fluorescent reporter coupled with fibre based fluorescence lifetime imaging (FLIM) and alveolar fluid cytokine, proteomic and lipidomic data.

Methods: Ex vivo human lungs declined for transplantation were ventilated and perfused. Lipopolysaccharide (LPS) was administered into a single lobe via the working channel of a flexible bronchoscope to induce lung injury. A bespoke delivery and imaging fibre (Panoptes, <2mm diameter) was deployed into the distal lung and fluorescent intensity/FLIM images were obtained pre- and post-delivery of NAP. Alveolar and bronchoalveolar lavage (AL, BAL) for cytokines, omics and tissue digest for flow cytometry were obtained.

Results: We observed a distinct change in optical signal in the LPS-treated lobe following NAP delivery, with a bespoke detection algorithm identifying a 7-fold increase in activated neutrophils. A distinct flow cytometry, cytokine and omics signature were also derived.

Conclusion: FLIM, Panoptes and NAP can identify neutrophil influx in the distal lung and AL can generate distinct omic and cytokine signatures. This platform could be used to identify inflammatory causes of pulmonary infiltrates as to guide stratified treatment.