Abstract
Abstract 18
Eosinophils are increasingly recognized as important myeloid effector cells in the inflammatory environment of many human diseases. Although eosinophils critically contribute to chronic asthmatic inflammation, few therapies directly target these cells. Eosinophils rapidly migrate to eotaxin elicited by allergic sensitization and challenge, a chemokine that ligates the CCR3 receptor. Eotaxin:CCR3 signaling critically regulates allergen-induced eosinophil infiltration in murine models by activating the Rho-family proteins. In several cell systems, the Rho proteins Rac and CDC42 activate p21-activated kinase 1 (PAK1), which we have previously shown to regulate F-actin dynamics and histamine release in the degranulating mast cell. In these studies, we examined eotaxin-induced eosinophil migration using genetic and hematopoietic ablation of Pak1 (Pak1−/−) in a murine asthma model.
Using an in vitro transwell migration assay system, we evaluated the migration of bone marrow derived eosinophils of both genotypes to eotaxin (N=10). Pak1−/− eosinophils exhibited profoundly diminished eotaxin-induced chemotaxis in vitro relative to wild-type (Pak1+/+) eosinophils (p < 0.0001) with a 30% overall decrease in migrating Pak1−/− compared to Pak1+/+ eosinophils. Furthermore, we compared the eotaxin-induced localization and arrangement of F-actin in eosinophils of both genotypes by fluorescence cytometry and deconvolution confocal microscopy of fluorescently-tagged phalloidin in seeking to explain this migration defect. Preliminary findings suggest decreased F-actin polymerization in eotaxin-treated Pak1−/− eosinophils. In an independent line of experiments designed to compare eotaxin-mediated eosinophil recruitment in vivo we injected mice of both genotypes with an intraperitoneal dose of eotaxin or saline. Pak1+/+ mice showed an 8 fold eotaxin-mediated increase in eosinophil recruitment over control whereas Pak1−/− mice demonstrated only a modest 3–4 fold increase (p< 0.05). Finally we pursued PAK1's function in an experimental disease model in which the eosinophil's key role in pathogenesis is well documented. In 3 cohorts of 7 age, gender and strain matched Pak1+/+ and Pak1−/− ova albumin (OVA)-sensitized and challenged mice, we scored lung eosinophilic inflammation by histology and compared eosinophil counts and eotaxin concentrations in broncho-alveolar lavage fluid (BALF) by fluorescence cytometry and ELISA respectively. We also assessed OVA-specific T-cell subset cytokine secretion in our asthma mice by ELISA. Lung-parenchymal eosinophilic inflammation was diminished in Pak1−/− ova-sensitized mice versus Pak1+/+'s (p<0.01) with neither differences in BALF eotaxin content nor OVA-specific in vitro T-helper cell secretion of asthma-induced cytokines between the 2 genotypes. Based on our findings in this model, we assessed PAK1's hematopoietic role using two complementary chimeric mouse models. In a cohort of matched recipient Pak1+/+ mice we transplanted Pak1+/+ and Pak1−/− bone marrow and after hematopoietic reconstitution we incited asthmatic inflammation in these mice. Significantly, hosts transplanted with Pak1−/− bone marrow developed decreased eosinophilic inflammation scores compared to Pak1+/+ bone marrow recipients (p<0.05). To complement the bone marrow experiments, we transplanted left-lung grafts from Pak1+/+ and Pak1−/− mice into matched Pak1+/+ and Pak1−/− recipient mice and after surgical recuperation we elicited asthmatic inflammation as above. Similar to our bone marrow transplant experiments, irrespective of the genotype of the lung graft, hosts with Pak1−/− bone marrow developed decreased lung eosinophil infiltrate.
Our data suggest that genetic PAK1 disruption hinders the in vitro and in vivo eotaxin-mediated migration of eosinophils by altering polymerization of F-actin. In an OVA murine model of asthma, we show that the genetic ablation of PAK1 attenuates the eosinophilic inflammation without affecting T-cell function. We similarly demonstrate that hematopoietic expression of PAK1 is critical to the development of eosinophil inflammation in two complementary transplant murine models. Pharmacologically targeting PAK1 may thus provide a specific way to impede eosinophil tissue infiltration, alleviate chronic eosinophil inflammation, and hamper long-term tissue remodeling in diseases like asthma.
Disclosures:
No relevant conflicts of interest to declare.
Leukotriene Receptor Antagonists in the Treatment of Asthma: Implications for Eosinophilic Inflammation
