Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening forms of acute respiratory failure that are among the leading causes of morbidity and mortality worldwide. In the U.S., they affect approximately 200,000 patients annually with mortality rates around 20-40%. However, there are currently no approved pharmacologic therapies for ALI/ARDS. Acute lung injury (ALI) involves complement activation, as indicated by attenuated severity in C5-deficient mice following formation of immune-complexes. The cellular and molecular interactions between the coagulation pathways and the immune system are believed to represent an important pillar of the pathophysiology of ALI/ARDS.
Protease-activated receptors (PARs) are a family of four G-protein-coupled receptors (GPCRs) for proteases from the circulation, inflammatory cells and epithelial tissues. Unlike other GPCRs, which are activated by ligand binding, PARs are irreversibly activated by proteolytic cleavage. The general mechanism of proteolytic activation is similar for all PARs. After proteolytic activation of PARs, a novel tethered ligand is exposed that binds to the receptor for triggering several downstream signaling pathways. These events mediate diverse biological functions including the degradations of proteins and the control of the cell cycle. The proteases that activate PARs include coagulation factors [e.g. thrombin, FVIIIa (factor VIIa), FXa], proteases from inflammatory cells (mast cell tryptase, neutrophil cathepsin G) and enzymes from epithelial tissues (trypsins). Each of these proteases can activate individual PARs with different affinity and trigger specific responses via biased agonist signaling.
PARs are expressed on a variety of immune and non-immune cells in different tissues. For example, PAR2 expression is observed in kidney, pancreas, stomach, intestine, airway, skin, bladder and brain. In the lung, PARs are expressed in almost all cell types including alveolar epithelial and endothelial cells, fibroblasts and macrophages. PAR1 and PAR2 are expressed on lung epithelial cells which are frequently exposed to various pathogenic microbes. In healthy homeostasis, the alveolar lumen contains mainly alveolar macrophages (AM) and low levels of active proteases. During inflammatory conditions, polymorphonuclear neutrophils (PMN) infiltrate the alveoli and release abundant amounts of proteases. These proteases can either activate or inhibit members of the PAR family, as well as the coagulation activating factors present in inflammation. The roles of PARs in different settings of pathogen-induced or sterile lung injury is not fully characterized and controversially discussed.
In this proposal, PAR2 mutant and cell type-specific PAR2 knockout mice will be subjected to acute lung injury as induced by intra-tracheal rmC5a administration. Analyses based on inflammatory mediators such as cytokines and chemokines as well as on the cellular level (e.g. cell phenotyping by multicolor FACS, lung histology) will provide insights into the immunology and coagulation networks modulated by PAR2 signaling. The formation of neutrophil extracellular traps (NET), which mainly contain extracellular histones, will be evaluated through the quantification of citrullinated histones (e.g. citH3) by ELISA and by FACS on BALF samples. Furthermore, I envision to utilize an in situ hybridization assay (e.g. PrimeFlow™ RNA assay) that combines the power of branched-DNA technology with the single-cell resolution of flow cytometry, enabling the simultaneous detection of PAR2 RNA target in combination with immune-phenotyping for cell surface to evaluate the expression of PAR2 in distinct cell populations including neutrophils. In addition, quantification of PAR2 protein in BALF sample by Western Blot will be considered to confirm the results obtained from PrimeFlow™ RNA assay at protein level. To obtain further insights into the PAR2-initiated intracellular events, RNA-sequencing will be performed on highly purified FACS sorted cell populations obtained from inflamed lungs. Pathways analysis may be precisely performed to establish accurate conclusions regarding the complex interactions of the coagulatory and inflammatory signaling networks.
In future, the data obtained during the proposed working program could have relevance for translational biomedicine to deepen our understanding of the lung patho-physiologies of patients suffering from ALI/ARDS and other inflammatory airway diseases.