Syk Regulates Neutrophilic Airway Hyper-Responsiveness in a Chronic Mouse Model of Allergic Airways Inflammation

Abstract

BackgroundAsthma is a chronic inflammatory disease characterized by airways hyper-responsiveness (AHR), reversible airway obstruction, and airway inflammation and remodeling. We previously showed that Syk modulates methacholine-induced airways contractility in naïve mice and in mice with allergic airways inflammation. We hypothesize that Syk plays a role in the pathogenesis of AHR; this was evaluated in a chronic 8-week mouse model of house dust mite (HDM)-induced allergic airways inflammation.MethodsWe used the Sykflox/flox//rosa26CreERT2 conditional Syk knock-out mice to assess the role of Syk prior to HDM exposure, and treated HDM-sensitized mice with the Syk inhibitor, GSK143, to evaluate its role in established allergic airways inflammation. Respiratory mechanics and methacholine (MCh)-responsiveness were assessed using the flexiVent® system. Lungs underwent bronchoalveolar lavage to isolate inflammatory cells or were frozen for determination of gene expression in tissues.ResultsMCh-induced AHR was observed following HDM sensitization in the Syk-intact (Sykflox/flox) and vehicle-treated BALB/c mice. MCh responsiveness was reduced to control levels in HDM-sensitized Sykdel/del mice and in BALB/c and Sykflox/flox mice treated with GSK143. Both Sykdel/del and GSK143-treated mice mounted appropriate immune responses to HDM, with HDM-specific IgE levels that were comparable to Sykflox/flox and vehicle-treated BALB/c mice. HDM-induced increases in bronchoalveolar lavage cell counts were attenuated in both Sykdel/del and GSK143-treated mice, due primarily to decreased neutrophil recruitment. Gene expression analysis of lung tissues revealed that HDM-induced expression of IL-17 and CXCL-1 was significantly attenuated in both Sykdel/del and GSK143-treated mice.ConclusionSyk inhibitors may play a role in the management of neutrophilic asthma.

A 4-Week Model of House Dust Mite (HDM) Induced Allergic Airways Inflammation with Airway Remodeling

“…Rocuronium (1.8–4.8 mg/kg i.p., Sandoz Canada Inc., Boucherville, QC, Canada) was administered prior to pulmonary function testing to prevent respiratory drive artifacts. Baseline measurements of respiratory mechanics (including quasistatic compliance; C st ) were assessed prior to challenge with increasing concentrations of nebulized methacholine (MCh; Sigma-Aldrich, Mississauga ON), and determination of total respiratory resistance (R rs ) and central airways Newtonian resistance (R N ), as described previously 21 , 22 , 26 .…”

“…After completion of the pulmonary function testing, mice were euthanized with an overdose of ketamine/xylazine, and blood was collected via cardiac puncture 21 , 22 . In a subset of mice, bronchoalveolar lavage (BAL) was performed, and lungs were harvested and placed in RNAlater® (Life Technologies, Burlington, ON, Canada) for storage at −20 °C, or inflation-fixed to 25 cm H 2 O in 4% paraformaldehyde for subsequent histologic analysis 27 .…”

“…Serum HDM-specific IgE was assessed using the antigen-capture ELISA method 22 . Briefly, 96 well plates (NUNC MaxiSorp1 plates, Sigma Aldrich Co., Mississauga, ON) were coated with 5 µg HDM in 100 µl coating buffer and incubated overnight at 4 °C.…”

“…In this paper, we describe two 4-week models of HDM-induced allergic airways inflammation using a lower (10 µg) and higher (25 µg) dose of Dermatophagoides pteronyssinus , and compare the phenotypic features to 2- and 8-week HDM models 22 . We found that 4 weeks of intranasal exposure to 25 µg HDM daily during the week, resulted in AHR, airways inflammation and remodeling with collagen deposition, which were comparable to the 8-week HDM-sensitization and -challenge model.…”

LC3-Associated Phagocytosis Is Required for Dendritic Cell Inflammatory Cytokine Response to Gut Commensal Yeast Saccharomyces cerevisiae

“…The inducible Syk flox/flox /rosa26CreERT2 mouse strain was maintained at Toronto University (Toronto, ON, Canada) ( 21 ). The tamoxifen induction protocol and characterization of the Syk knockout phenotype have been described ( 22 , 23 ). Control littermates were treated with 10% v/v ethanol in sunflower oil, the diluent for tamoxifen.…”