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.


Clinical samples
Informed consent was received from participants, or next of kin in cases of incapacity, prior to inclusion in the study in accordance with the approved study protocol with the nature and possible consequences of the study explained. For collection of bronchoalveolar lavage fluid the bronchoscope was wedged in a subsegment corresponding to the area of radiological involvement and sterile saline (20ml) was instilled and the aspirate discarded, then 200ml of sterile saline was instilled in aliquots and the aspirate (representing an alveolar sample) retained. BALF was centrifuged at 700g for 10 min and the supernatant retained. Whole blood was collected into 3.8% sodium citrate and following centrifugation (350g for 20 min) serum was made by addition of 1M calcium chloride to the aspirated plasma layer. Samples were stored at -80 o C prior to analysis. ARDS was defined at initial recruitment according to the American-European Consensus Conference definition and satisfy the current Berlin criteria with PaO2/FiO2 ≤ 300mmHg, bilateral opacification seen on chest x-ray, positive end expiratory pressure (PEEP) ≥ 5cm H2O and non-cardiogenic pulmonary oedema. [1,2]

Quantification of N-terminal formylated peptides
Biological and clinical samples (mitochondria (MTD), BALF and serum) were subjected to acetone precipitation. The acetone fraction, containing peptides, was dried under vacuum and reconstituted in 0.5% acetic acid. The peptides were then analysed by liquid chromatographytandem mass spectrometry (LC-MS/MS) in positive ion mode using a Thermo LTQ-Orbitrap XL mass spectrometer coupled to a Waters nanoAcquity UPLC system with a linear gradient over 39 min (mobile phase A: 0.5% acetic acid in water; mobile phase B: 0.5% acetic acid in acetonitrile). N-formylated peptides were identified based on their accurate mass, retention times and characteristic fragmentation patterns compared to custom synthesised standards

Flow cytometry
Neutrophils were resuspended at 5 x 10 6 /ml in PBS with divalent cations (

Neutrophil chemotaxis
Neutrophil chemotaxis was conducted using a 96 well chemotaxis chamber fitted with a 3μm filter (Neuro Probe, Gaithersburg, MD, USA) as described. [6] Neutrophils, 3x10 6 /ml (IMDM with 10% autologous serum) were pre-treated with CsH, anti-CD11b antibody (20μg/ml, ICRF44, BioLegend) or vehicle or istoype control for 30 min at 37°C and loaded in the upper well of the chamber. Chemoattractants MTD (50μg/ml), fMIT (100nM) or vehicle control were added in the lower well. Transmigrated neutrophils in the lower chamber were counted with a haemocytometer following 90 min incubation at 37 o C 5% CO2.

Western blotting
Neutrophils, 7x10 5 cells per sample, were suspended in PBS with divalent cations and pretreated with or without cyclosporin H (CsH, 2.5μM) for 30 min. MTD were added and cells incubated for 1 min at 37°C prior to cell lysis and protein extraction as described. [7] Briefly, following protein quantification by bicinchoninic acid assay samples were mixed with 4x NuPAGE sample buffer (ThermoScientific) and denatured at 96°C for 5 min. Gel electrophoresis in NuPAGE 4-12% Bis-Tris protein gels (Invitrogen) was performed and transferred to PVDF membrane prior to 1 h incubation with 5% dried milk in TBS-Tween Tissue harvesting and processing Mice were euthanased with an overdose of terminal anaesthesia followed by exsanguination by cardiac puncture. Harvested blood was mixed 1:1 with 3.8% sodium citrate prior to antibody staining for flow cytometry as described. [9] Lungs were lavaged with three boluses of 800μl sterile 0.9% NaCl and centrifuged at 850g for 5 min. Supernatants were stored at - was instilled as outlined previously with subsequent tissue harvest after 24 h.