Activated neutrophil fluorescent imaging technique for human lungs

Tashfeen Walton,Thomas H Craven,Chris Haslett,Duncan C Humphries,Thane A Campbell,Emma Scholefield,Neil Mcdonald,Adam D.L Marshall,Adam T Hill,Annya Bruce,Kevin Dhaliwal,James W Dear,Ahsan R Akram,Bethany Mills,Joanne Mair,David E Newby,Timothy S Walsh

doi:10.1038/s41598-020-80083-w

Tashfeen Walton, Thomas H Craven + Show 15 more

Open Access

https://doi.org/10.1038/s41598-020-80083-w

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Abstract

Neutrophil activation is an integral process to acute inflammation and is associated with adverse clinical sequelae. Identification of neutrophil activation in real time in the lungs of patients may permit biological stratification of patients in otherwise heterogenous cohorts typically defined by clinical criteria. No methods for identifying neutrophil activation in real time in the lungs of patients currently exist. We developed a bespoke molecular imaging probe targeting three characteristic signatures of neutrophil activation: pinocytosis, phagosomal alkalinisation, and human neutrophil elastase (HNE) activity. The probe functioned as designed in vitro and ex vivo. We evaluated optical endomicroscopy imaging of neutrophil activity using the probe in real-time at the bedside of healthy volunteers, patients with bronchiectasis, and critically unwell mechanically ventilated patients. We detected a range of imaging responses in vivo reflecting heterogeneity of condition and severity. We corroborated optical signal was due to probe function and neutrophil activation.

Highlights

Neutrophil activation is an integral process to acute inflammation and is associated with adverse clinical sequelae
In airway and parenchymal diseases characterised by neutrophilic inflammation and damage, we hypothesized that the combination of a specific optical SmartProbe and bedside optical endomicroscopy (OEM) would provide an in situ indicator of alveolar neutrophil activation
Neutrophil Activation Probe (NAP) acted as a pH sensor, in keeping with a multitude of fluorescein protolytic forms, but with a serendipitous steepening of the fluorescent response as pH increases over the physiologically relevant range compared to the emission spectra of carboxyfluorescein (Fig. 1D), with the pH profile modulated by the tri-branched nature of the construct, thereby altering the pKa values expected for carboxyfluorescein

Summary

Introduction

Neutrophil activation is an integral process to acute inflammation and is associated with adverse clinical sequelae. Detecting and monitoring real-time neutrophil activity in vivo in situ may offer useful insights into disease pathology. In airway and parenchymal diseases characterised by neutrophilic inflammation and damage, we hypothesized that the combination of a specific optical SmartProbe and bedside optical endomicroscopy (OEM) would provide an in situ indicator of alveolar neutrophil activation. We previously reported a multi-branched compound for fluorescent detection of neutrophil e lastase[23] and have modified that structure to generate a bespoke chemical SmartProbe for the detection of activated neutrophils that would be suitable for clinical deployment. This Neutrophil Activation Probe (NAP) consists of three internally quenched fluorescein moieties, each conjugated to an optimized peptide sequence, and all attached to a tri-branched multivalent scaffold. The targeting of these augmented metabolic processes makes the generated cellular fluorescent signal specific to activated neutrophils

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