Determining the contribution of formylated peptides and formyl peptide receptor 1 to the pathogenesis of acute lung injury
Dorward, David Andrew
Neutrophils as key effector cells of the innate immune system migrate from the circulation into sites of inflammation and are essential for the containment, killing and clearance of invading pathogens through a variety of highly regulated cell functions. Despite this beneficial role their involvement can also be detrimental. In a number of diseases dysregulated neutrophil influx and activation results in significant tissue damage and worsening of the acute inflammatory event as well as long term tissue injury, scarring and fibrosis. One such pulmonary condition is acute respiratory distress syndrome (ARDS) which, despite decades of intensive research and multiple clinical trials, remains without a cure and has an associated mortality rate of approximately 40%. Delineating and understanding the key pathogenic mediators that drive neutrophil recruitment into the lung in the context of both bacterial and sterile injury is therefore vital in the development of novel therapies. Neutrophils migrate towards a variety of agents but amongst such factors a hierarchy exists with bacterial-derived products, including formylated peptides, dominant in this process. In sterile tissue injury where no bacterial factors are present the mediators involved change but a hierarchy still exists. Mitochondrial formylated peptides are released following necrotic cell death and bind to formyl peptide receptor 1 (FPR1) on the neutrophil surface inducing migration and activation. Like bacterial formylated peptides they are powerful chemoattractants and are therefore likely to be important in recruiting neutrophils to sites of injury and inflammation. Hypothesis: The central hypothesis of this thesis is that mitochondrial formylated peptides, as end-target chemoattractants, are elevated in patients with ARDS and drive neutrophil recruitment through binding to FPR1. Inhibition of FPR1 in models of acute lung injury will therefore result in attenuation of this inflammatory response through multiple FPR1-mediated effects implicating both formylated peptides and their cognate receptor in the pathogenesis of sterile ARDS. Results: Free mitochondrial DNA and formylated peptides were elevated in the circulation of patients with ARDS or severe paracetamol-induced hepatic failure relative to healthy controls. In addition, FPR1 receptor number was increased on the surface of neutrophils isolated from critically ill septic patients. Isolated mitochondrial formylated peptides induced FPR1- dependent chemotaxis in primary human neutrophils in vitro. Alongside this, FPR1 ligand binding resulted in increased cell surface β2-integrin expression [integrin alpha M beta 2 (ITGB2); also called CD11b/CD18, Mac-1 or CR3] through intracellular activation of PI-3Kand MAPK-dependent signalling pathways. Indeed, blockade of neutrophil cell-surface integrin alpha M (ITGAM; also known as CD11b)) resulted in a reduction in mitochondrial formylated peptide-induced chemotaxis. To determine the production of human neutrophil IL-1β, a pivotal chemokine within a sterile inflammatory environment, a novel method for the in vitro isolation of ultrapure neutrophils was developed. Neutrophils were isolated by autofluorescence-based flow sorting as determined by intrinsic differences in neutrophil and eosinophil autofluorescence and their size and granularity relative to circulating mononuclear cells. Analysis of this approach demonstrated the ability to rapidly collect a highly pure neutrophil population (99.95 ± 0.03%). Flow sorting did not alter the activation state or functional capacity of these cells relative to unsorted neutrophils with regards to several measures of neutrophil behaviour/function. Cells also remained fully responsive to a variety of neutrophil agonists with no evidence of neutrophil priming. The capacity of highly pure neutrophils to secrete IL-1β was determined to be approximately 160-fold lower than equivalent numbers of circulating peripheral blood mononuclear cells. In the context of an inflammatory environment however this is likely to be of biological significance given the large number of infiltrating neutrophils. In sterile hydrochloric acid-induced acute lung injury pharmacological inhibition of FPR1 with cyclosporin H (CsH), or use of transgenic FPR1-/- mice, resulted in inhibition of neutrophil migration into the alveolar space 24 hours after injury. This was associated with a reduction in pulmonary haemorrhage, extravascular protein leak and pro-inflammatory cytokine expression with improved histological appearances. Furthermore, the HCl acid-induced reduction in alveolar macrophage numbers was inhibited by CsH with interstitial macrophages displaying an alternatively activated phenotype. Importantly, delivery of CsH 12 hours after the onset of injury also reduced acute lung inflammation demonstrating its potential therapeutic relevance in the treatment of human disease. In non-sterile E. coli-mediated acute lung injury partial antagonism of FPR1 with CsH resulted in a reduction in neutrophil migration and vascular leak with no effect on pulmonary bacterial load. A narrow therapeutic window existed however as increased concentrations of CsH, or infection in FPR1-/- mice, resulted in a reduction in alveolar neutrophil number and increase in E. coli at 24 hours. Alongside effects on myeloid cells within the lung FPR1 was found to be expressed on mouse lung epithelial cells. A technique to isolate and culture mouse type 1 alveolar epithelial (AT1) cells was therefore developed. Flow sorting of anti-type 1 alpha (anti-T1α) stained single cell lung homogenates with subsequent culture on transwell membranes resulted in the development of confluent AT1 cell monolayers after 10 days. Formylated peptides appear to induce a reduction in transepithelial resistance and increase in permeability across a monolayer in vitro alongside an increase in release of the neutrophil chemo-attractant mouse CXCL8 (KC). Conclusions: Taken together, mitochondrial formylated peptides released following cell necrosis and FPR1 play a significant role in the pathogenesis of sterile acute lung injury. This is likely to be predominantly through neutrophil-dependent means but data presented here also suggests that their role in macrophage function and alveolar epithelial cell permeability may be important. Inhibition of FPR1 may therefore represent a novel and multi-cellular therapeutic target in the treatment of ARDS.