Emma Scholefield

Monocytes Control Second-Phase Neutrophil Emigration in Established Lipopolysaccharide-induced Murine Lung Injury

RATIONALE Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood. OBJECTIVES To determine the influence of peripheral blood monocytes (PBMs) in established ALI. METHODS In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2(hi) monocytes. MEASUREMENTS AND MAIN RESULTS PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1(hi) and Gr1(lo) PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes. CONCLUSIONS These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI.

Novel role for endogenous mitochondrial formylated peptide-driven formyl peptide receptor 1 signalling in acute respiratory distress syndrome

Background Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. Methods Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography–tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. Results Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1−/− mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. Conclusions We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS.

Two-color widefield fluorescence microendoscopy enables multiplexed molecular imaging in the alveolar space of human lung tissue

Abstract. We demonstrate a fast two-color widefield fluorescence microendoscopy system capable of simultaneously detecting several disease targets in intact human ex vivo lung tissue. We characterize the system for light throughput from the excitation light emitting diodes, fluorescence collection efficiency, and chromatic focal shifts. We demonstrate the effectiveness of the instrument by imaging bacteria (Pseudomonas aeruginosa) in ex vivo human lung tissue. We describe a mechanism of bacterial detection through the fiber bundle that uses blinking effects of bacteria as they move in front of the fiber core providing detection of objects smaller than the fiber core and cladding (∼3  μm). This effectively increases the measured spatial resolution of 4  μm. We show simultaneous imaging of neutrophils, monocytes, and fungus (Aspergillus fumigatus) in ex vivo human lung tissue. The instrument has 10 nM and 50 nM sensitivity for fluorescein and Cy5 solutions, respectively. Lung tissue autofluorescence remains visible at up to 200 fps camera acquisition rate. The optical system lends itself to clinical translation due to high-fluorescence sensitivity, simplicity, and the ability to multiplex several pathological molecular imaging targets simultaneously.

Timed feeding of mice modulates light-entrained circadian rhythms of reticulated platelet abundance and plasma thrombopoietin and affects gene expression in megakaryocytes

Circadian (c. 24 h) rhythms of physiology are entrained to either the environmental light‐dark cycle or the timing of food intake. In the current work the hypothesis that rhythms of platelet turnover in mammals are circadian and entrained by food intake was explored in mice. Mice were entrained to 12 h light‐dark cycles and given either ad libitum (AL) or restricted access (RF) to food during the light phase. Blood and megakaryocytes were then collected from mice every 4 h for 24 h. It was found that total and reticulated platelet numbers, plasma thrombopoietin (TPO) concentration and the mean size of mature megakaryocytes were circadian but not entrained by food intake. In contrast, a circadian rhythm in the expression of Arnt1 in megakaryocytes was entrained by food. Although not circadian, the expression in megakaryocytes of Nfe2, Gata1, Itga2b and Tubb1 expression was downregulated by RF, whereas Ccnd1 was not significantly affected by the feeding protocol. It is concluded that circadian rhythms of total platelet number, reticulated platelet number and plasma TPO concentration are entrained by the light‐dark cycle rather than the timing of food intake. These findings imply that circadian clock gene expression regulates platelet turnover in mammals.

T4 Optically detectable antimicrobial peptides enable the immediate detection of bacteria and fungi in the lung

Introduction The immediate detection of pathogens in the lungs of patients with unexplained pulmonary opacities in the intensive care unit would represent a significant advance in their management. An optical imaging strategy, including the endobronchial administration of bacterial specific Smartprobes, would confer a number of advantages over conventional techniques such as bronchoalveolar lavage, principally real-time detection to immediately inform antimicrobial therapy. The aims of this study were to fluorescently label and iteratively develop anti-microbial peptides to image bacteria in situ in the lung using fibered confocal fluorescence microscopy (FCFM). Methods Antimicrobial peptides (AMP) have been synthesised on a dendrimeric scaffold (AMP-1) and conjugated to an environmentally sensitive fluorophore called NBD, following the continuous development a linear counterpart. A further construct consists of an AMP with gram-selectivity conjugated to the NBD fluorophore (AMP-2). These are combined with FCFM to allow distal alveolar imaging at micron resolution in an ex vivo ovine model of bacterial infection. Results AMP-1 demonstrates bacterial binding affinity in a concentration dependent manner and labels a diverse panel of bacteria, including a panel consisting of >70% of ventilator-associated pneumonia causing organisms and the pathogenic fungi Aspergillus fumigatus. AMP-1 demonstrates significantly higher fluorescence over isomolar linear equivalents for E. coli, K. pneumoniae, P. aeruginosa, MSSA, A. baumannii and S. pneumoniae (all p < 0.01), is selective for bacteria over mammalian cells and has improved chemical stability over the linear equivalent when incubated with bronchoaoveolar lavage from patients with acute respiratory distress syndrome. Furthermore, AMP-1 can label E. coli, K. pneumoniae, P. aeruginosa and MSSA in situ in an ex vivo ovine model when instilled endobronchially and imaged with FCFM (pin vitro and remains selective for gram-negative bacteria over mammalian cells. In the ex-vivo model AMP-2 selectively labels the gram-negative bacterial segments (P. aeruginosa, K. pneumonia and E. coli) over the gram-positive (MSSA, MRSA and S. pneumoniae) or control pulmonary segments (all p < 0.05). Conclusions A Smartprobe/FCFM strategy to immediately detect bacteria with gram selectivity in size relevant pre-clinical models is described, and are undergoing first-in-man translation.

Structural modifications of the antimicrobial peptide ubiquicidin for pulmonary imaging of bacteria in the alveolar space

Abstract Background The direct visualisation of bacteria in the distal lung would increase the spatiotemporal understanding of pulmonary infection and be a powerful tool to stratify patients with suspected pneumonia. In critically ill patients, the diagnostic dilemma of pulmonary opacities leads to overprescribing of antimicrobial agents while waiting for culture results from bronchoalveolar lavage. Ubiquicidin (UBI) is an innate cytosolic antimicrobial peptide with a twelve aminoacid portion (UBI 29–41 ) that specifically binds bacteria. We aimed to modify the chemical structure of UBI 29–41 so that in-situ bacterial imaging with optical endomicroscopy (OEM) could be achieved. Methods UBI 29-41 compounds were labelled with the environmentally sensitive fluorophore NBD (NBD-UBI), with incorporation of synthetic aminoacids (NBD-UBI nma ) and alteration of the secondary structure of the native peptide on a dendrimeric scaffold (NBD-UBI dend ). These compounds were assessed in vitro and delivered endobronchially in an ex-vivo sheep lung model and then OEM applied to allow alveolar imaging. The NBD-UBI dend –OEM platform was also evaluated in explanted whole cystic fibrosis lungs. Findings NBD-UBI selectively labelled bacteria over mammalian cells but remained susceptible to proteolytic degradation and poor affinity. NBD-UBI nma improved stability but not affinity. NBD-UBI dend remained structurally stable and exhibited high affinity for bacteria in vitro. It retained bacterial selectivity over mononuclear cells (p=0·0015), neutrophils (p=0·0034), bronchoalveolar lavage macrophages (p=0·0169), and labelled Escherichia coli (p=0·0035), Klebsiella pneumoniae (p=0·0003), Pseudomonas aeruginosa (p=0·0009), and meticillin-sensitive Staphylococcus aureus (p 5 colony-forming units per mL on lavage. NBD-UBI dend also detected bacteria in situ in ex-vivo explanted human cystic fibrosis lungs (p=0·0027 compared with peptide and fluorophore control segments). Interpretation We describe an OEM strategy that can immediately detect bacteria in size-relevant preclinical models, with crucial requirements for pulmonary molecular imaging of peptide stability and affinity. This method has the potential to stratify pulmonary opacities in the intensive care unit when pneumonia is suspected and offers the substantial advantage of real-time detection, therefore allowing immediate decision making about antimicrobial treatment. This imaging strategy is now undergoing first-in-man translation. Funding Wellcome Trust, Department of Health, Engineering and Physical Sciences Research Council.

Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils

Serine proteases are released by neutrophils to act primarily as antimicrobial proteins but excessive and unbalanced serine protease activity results in serious host tissue damage. Here the synthesis of a novel chemical sensor based on a multi-branched fluorescence quencher is reported. It is super-silent, exhibiting no fluorescence until de-quenched by the exemplar serine protease human neutrophil elastase, rapidly enters human neutrophils, and is inhibited by serine protease inhibitors. This sensor allows live imaging of intracellular serine protease activity within human neutrophils and demonstrates that the unique combination of a multivalent scaffold combined with a FRET peptide represents a novel and efficient strategy to generate super-silent sensors that permit the visualisation of intracellular proteases and may enable point of care whole blood profiling of neutrophils.

Time-resolved spectroscopy at 19,000 lines per second using a CMOS SPAD line array enables advanced biophotonics applications

A SPAD-based line sensor fabricated in 130 nm CMOS technology capable of acquiring time-resolved fluorescence spectra (TRFS) in 8.3 milliseconds is presented. To the best of our knowledge, this is the fastest time correlated single photon counting (TCSPC) TRFS acquisition reported to date. The line sensor is an upgrade to our prior work and incorporates: i) parallelized interface from sensor to surrounding circuitry enabling high line rate to the PC (19,000 lines/s) and ii) novel time-gating architecture where detected photons in the OFF region are rejected digitally after the output stage of the SPAD. The time-gating architecture was chosen to avoid electrical transients on the SPAD high voltage supplies when gating is achieved by excess bias modulation. The time-gate has an adjustable location and time window width allowing the user to focus on time-events of interest. On-chip integrated center-of-mass (CMM) calculations provide efficient acquisition of photon arrivals and direct lifetime estimation of fluorescence decays. Furthermore, any of the SPC, TCSPC and on-chip CMM modes can be used in conjunction with the time-gating. The higher readout rate and versatile architecture greatly empower the user and will allow widespread applications across many techniques and disciplines. Here we focused on 3 examples of TRFS and time-gated Raman spectroscopy: i) kinetics of chlorophyll A fluorescence from an intact leaf; ii) kinetics of a thrombin biosensor FRET probe from quenched to fluorescence states; iii) ex vivo mouse lung tissue autofluorescence TRFS; iv) time-gated Raman spectroscopy of toluene at 3056 cm-1 peak. To the best of our knowledge, we detect spectrally for the first time the fast rise in fluorescence lifetime of chlorophyll A in a measurement over single fluorescent transient.

In-situ imaging of neutrophil activation in the human alveolar space with neutrophil activation probe and pulmonary optical endomicroscopy

Abstract Background Acute respiratory distress syndrome is a severe and heterogeneous condition. Clinical diagnostic criteria lack specificity and fail to differentiate the various inflammatory phenotypes. Diagnosis and stratification can be improved by profiling the neutrophil and one of its enzymes, neutrophil elastase (NE), within the alveolar space. We aimed to combine fibre-based optical endomicroscopy (OEM) with a bespoke imaging probe to deliver a new strategy to elucidate pathobiological processes in the lung. Methods We designed and synthesised an optical imaging probe termed neutrophil activation probe to fluoresce as it enters the alkaline neutrophilic vacuole and through NE-specific cleavage. Validation was performed with spectrophotometry, confocal microscopy, and flow cytometry. The probe was tested in a perfused large sheep lung model in conjunction with bronchoscopic OEM. Neutrophil activation probe was synthesised to GMP, and a phase 1 study is underway in healthy volunteers and patients in the intensive care unit. The phase 1 study is registered with EudraCT, number 2011-066167-17, and with ClinicalTrials.gov, number NCT01532024. Findings Neutrophil activation probe was neutrophil specific, specific to NE among other serine proteinases, and demonstrated dequenching in alkaline pH. Intracellular fluorescence signal determined by flow cytometry increased in chemically activated neutrophils (mean fluorescence index signal increased from 24 144 (SE 6175) to 1·218 × 10 6 (59 325), p Interpretation We have demonstrated the potential utility of this bedside molecular imaging strategy, from bench to patient: the probe functions in a predictable manner, in keeping with its design. A two-site international phase 2 clinical study is planned, which will assess the test characteristics of this novel technique and its ability to predict and stratify clinically important outcomes in a heterogeneous ventilated population in the intensive care unit. Funding Medical Research Council.

In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A

A topically administered fluorescently labeled peptide targeting lipid A permits rapid, real-time visualization of bacteria in the distal human lung. Lung infection in real time Lung infections are frequent causes of complications in mechanically ventilated and immunosuppressed patients. However, the diagnosis is challenging, requires risky procedures, and is time consuming. Now, Akram et al. have developed an imaging method that is able to detect Gram-negative bacteria in real time in the distal part of the human lung. Using a fluorescent probe binding to lipid A, a molecule expressed on Gram-negative bacterial membranes, in combination with an optical endomicroscope, the researchers rapidly detected Gram-negative infections in distal airways in hospitalized individuals. The results suggest that the approach could accelerate the diagnosis of bacterial lung infection and facilitate the evaluation of antibiotic treatment efficacy. Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.