The goal of this study was to investigate the MARCKS (Myristoylated Alanine Rich C Kinase Substrate) protein as a potential novel therapeutic target for Equine Asthma Syndrome (EAS). Our lab, and others, previously established a role for MARCKS in both neutrophil and macrophage inflammatory functions. In the current study, we hypothesized that MARCKS protein is upregulated in BAL cells in horses with asthma; and that inhibition of the MARCKS protein with a MARCKS-specific inhibitor peptide known as MANS, will diminish the zymosan-induced respiratory burst of equine alveolar macrophages, and peripheral blood neutrophils, ex vivo. Bronchoalveolar lavage (BAL) was performed as previously described using a 3 m cuffed Bivona ® tube and 300 mLs sterile saline. BAL cytology (400 cell count differential), physical, and rebreathing exam, were used to classify horses as normal, mild/moderate, or severe EAS. Total MARCKS protein expression in BAL cell lysates was quantified using an equine specific MARCKS ELISA (MyBioSource, San Diego, CA, USA) and normalized to total protein (BCA assay) in cell lysate. Equine alveolar macrophages were isolated by adhesion. Peripheral blood neutrophils were isolated by ficol density gradient centrifugation. Zymosan-induced respiratory burst was measured as luminol-enhanced chemiluminescence. Cells were pretreated with 0, 10, 25, 50, or 100 uM MANS (MARCKS-specific inhibitor peptide), or 100 uM RNS (control peptide). Normalized MARCKS protein expression was significantly higher in BAL cell lysates from horses with mild/moderate (n=14) and severe asthma (n=5), compared to BAL cell lysates from horses with normal lower airway cytology (n=6) (One-way ANOVA, p < 0.05). MANS peptide pretreatment, but not RNS, significantly attenuated zymosan-induced respiratory burst of equine alveolar macrophages and peripheral blood neutrophils (One-way ANOVA, p < 0.05). These findings determine that MARCKS protein expression is altered in BAL cells from horses with EAS, indicating a possible role for MARCKS in the pathophysiology of EAS, and support MARCKS inhibition as a potential therapeutic strategy to alter the inflammatory response of airway cells including alveolar macrophages and neuteophils.
Learning Objectives:
Upon completion, the participant will be able to explain the potential role of neutrophils and macrophages in the pathophysiology of severe Equine Asthma.
Upon completion, the participant will be able to recall at least two reasons that the MARCKS protein was chosen as a potential therapeutic target for severe Equine Asthma.
Upon completion, the participant will be able to able to describe the effects of MANS peptide mediated MARCKS inhibition on the respiratory burst of equine neutrophils and alveolar macrophages.
