Neutrophil-Derived Cytosolic PLA2α Contributes to Bacterial-Induced Neutrophil Transepithelial Migration

Abstract

Eicosanoids are a group of bioactive lipids, shown to be important mediators of neutrophilic inflammation, and selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 (HxA3) is a pathogen-elicited, epithelial-produced neutrophil chemoattractant that directs trans-epithelial migration in response to infection. Following HxA3-driven trans-epithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2 (PLA2), and the cytosolic PLA2α (cPLA2α) isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil trans-epithelial migration has not been explored. Human and mouse neutrophil-epithelial co-cultures were employed to evaluate the role of neutrophil-derived cPLA2α in infection-induced trans-epithelial signaling using pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-Optical Coherence Tomography (μOCT), a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil trans-epithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil trans-epithelial migration by mediating leukotriene B4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection.

A1 adenosine receptor signaling reducesStreptococcus pneumoniaeadherence to pulmonary epithelial cells by targeting expression of platelet‐activating factor receptor

“…To test that, we used H292 cells, a human lung epithelial cell line extensively used in modeling pulmonary bacterial-host interaction (Bhowmick et al, 2013;Choi, Lee, Lee, Park, & Lee, 2008;R. T. Clark, Hope, Lopez-Fraga, Schiller, & Lo, 2009;Marks, Parameswaran, & Hakansson, 2012;Wagner et al, 2007;Yonker et al, 2017). H292 cells were treated with extracellular adenosine or vehicle control, infected with S. pneumoniae TIGR4 strain and the amounts of bacteria that adhered to the host cells measured.…”

Expansion of Airway Basal Cells and Generation of Polarized Epithelium

“…With this newly discovered ability to expand airway basal stem cells, patient-specific cells can be isolated and preserved from small biopsies of the airway, generating a virtually limitless supply of patient-specific airway epithelium in vitro (Mou et al , 2016). When differentiated on air-liquid interface, airway basal stem cell-derived epithelium forms a polarized mucociliary layer that exhibits proper epithelial architecture, physiology and response to clinically relevant pharmacologic agents (Mou et al , 2016; Yonker et al , 2017a and 2017b). Further, airway disease can be studied by generating basal stem cell-derived epithelium from individuals with airway disease, such as cystic fibrosis (CF), a genetic disease caused by a mutation in the CFTR gene, resulting in progressive respiratory failure.…”

“…A better understanding of cellular and molecular mechanisms mediating neutrophil trans-epithelial migration has the potential to inform and improve the efficacy of anti-inflammatory therapeutic strategies critically needed to treat inflammation mediated lung damage associated with CF. Recently, we have applied our advanced culture method in a co-culture system with neutrophils to investigate neutrophil-airway epithelium interaction and P. aeruginosa -induced neutrophil trans-epithelial migration (Yonker et al , 2017a and 2017b), Our findings from cultured human airway basal stem cells differentiated on ALI are consistent with prior molecular mechanistic studies implicating epithelial-derived neutrophil chemoattractant hepoxilin A3, an arachidonic acid metabolite synthesized by 12-lipoxygenase, as key in driving P. aeruginosa -induced trans-epithelial migration (Yonker et al. , 2017a and 2017b).…”

“…Recently, we have applied our advanced culture method in a co-culture system with neutrophils to investigate neutrophil-airway epithelium interaction and P. aeruginosa -induced neutrophil trans-epithelial migration (Yonker et al , 2017a and 2017b), Our findings from cultured human airway basal stem cells differentiated on ALI are consistent with prior molecular mechanistic studies implicating epithelial-derived neutrophil chemoattractant hepoxilin A3, an arachidonic acid metabolite synthesized by 12-lipoxygenase, as key in driving P. aeruginosa -induced trans-epithelial migration (Yonker et al. , 2017a and 2017b). Additionally, these studies revealed previously unknown cellular mechanisms associated with neutrophilic breach of the airway mucosal barrier.…”

Cytosolic phospholipase A2-α participates in lipid body formation and PGE2 release in human neutrophils stimulated with an l-amino acid oxidase from Calloselasma rhodostoma venom

“…cPLA 2 -α is phosphorylated by MAPKs, which increases its catalytic activity 39,41 . Once activated, cPLA 2 -α releases arachidonic acid from the membrane phospholipids, which triggers the activity of enzymes that oxygenate arachidonic acid (AA), generating various eicosanoids 42 . During this process, AA is formed and subsequently metabolised by COXs, which depending on the stimulus, can be driven by COX-1 (constitutively active) or COX-2 (induced), resulting in PGE 2 as the end product 9,10 .…”