Inhibition of Pyk2 blocks lung inflammation and injury in a mouse model of acute lung injury

Yingli Duan,Angelo Y Meliton,Alan R Leff,Jonathan Learoyd,Xiangdong Zhu

doi:10.1186/1465-9921-13-4

Abstract

BackgroundProline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo.MethodsC57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically.ResultsIntratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment.ConclusionsThese results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.

Highlights

Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro
We found that TAT-Pyk2-CT blocked LPS-induced neutrophilic lung inflammation and vascular leakage without blocking macrophage inflammatory protein-2 (MIP-2) and keratinocyte- derived chemokine (KC) production in LPS challenged lungs
Levels of phosphorylated Pyk2 in lungs obtained from LPS challenged mice pretreated with TAT-GFP control were comparable to mice receiving LPS treatment alone

Summary

Introduction

Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. Our laboratory recently has reported that TAT-Pyk2-CT, a fusion protein in which Pyk C-terminal domain (amino acid 6801009) is fused to a cell-permeable TAT peptide, blocks eosinophilic airway inflammation and airway hyperresponsiveness in an ovalbumin- induced mouse model of asthma [17]. From these observations we have hypothesized that the Pyk signaling pathway may play an important role in LPS-mediated lung inflammation and that inhibition of Pyk may reduce neutrophil infiltration in the lung and reduce lung injury in vivo

Objectives

Methods

Results

Conclusion