Comparison of genomic and proteomic data in recurrent airway obstruction affected horses using ingenuity pathway analysis\u3csup\u3e®\u3c/sup\u3e

Background: Recurrent airway obstruction (RAO) is a severe chronic respiratory disease affecting horses worldwide, though mostly in the Northern hemisphere. Environmental as well as genetic factors strongly influence the course and prognosis of the disease. Research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. The goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage study and proteins expressed in bronchoalveolar lavage fluid (BALF) collected from healthy and RAO-affected horses. The analysis was carried out with Ingenuity Pathway Analysis® bioinformatics software. Results: The gene with the greatest number of indirect interactions with the set of proteins identified is Interleukin 4 Receptor (IL-4R), whose protein has also been detected in BALF. Interleukin 21 receptor and chemokine (C-C motif) ligand 24 also showed a large number of interactions with the group of detected proteins. Protein products of nother genes like that of SOCS5, revealed direct interactions with the IL-4R protein. The interacting proteins NOD2, RPS6KA5 and FOXP3 found in several pathways are reported regulators of the NFkB pathway. Conclusions: The pathways generated with IL-4R highlight possible important intracellular signaling cascades implicating, for instance, NFkB. Furthermore, the proposed interaction between SOCS5 and IL-4R could explain how different genes can lead to identical clinical RAO phenotypes, as observed in two Swiss Warmblood half sibling families because these proteins interact upstream of an important cascade where they may act as a functional unit

Proteomic analysis of bronchoalveolar lavage fluid in an equine model of asthma during a natural antigen exposure trial

Background Heaves is a complex, asthma-like respiratory disease that affects many older horses. While environmental and genetic components to the disease have been proposed, the specific pathophysiology of heaves is still poorly understood. Using proteomic techniques, we compared the protein profile of bronchoalveolar lavage fluid (BALF) in the lungs of healthy horses and horses affected with heaves. Methods Clinical signs of the disease were induced in heaves-affected horses using an experimental hay exposure model.  Samples of BALF were collected from all horses before and after the hay exposure trial. Mass spectrometry (LC-MS) was used to evaluate the differences in the global BALF peptide profile between the control and heaves-affected horses. Tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins in the two groups of horses. The identification of two proteins was validated with Western blot assays. Results One hundred peptides were differentially expressed between healthy controls and heaves-affected horses; 76 peptides were over-expressed in controls and 24 were over-expressed in heaves-affected horses. The identifications of transferrin and secretoglobin were confirmed with Western blot. Conclusions This study demonstrates that proteomics can be used to compare the protein profiles of BALF from healthy and diseased horses. These techniques may prove helpful in determining the pathophysiology of complex diseases

DNA binding activity of transcription factors in bronchial cells of horses with recurrent airway obstruction.

Horses with recurrent airway obstruction (RAO) present many similarities with human asthmatics including airway inflammation, hyperresponsiveness, reversible obstruction, and increased NF-kappaB expression. Studies in experimental asthma models have shown that transcriptions factors such as activator protein-1 (AP-1), GATA-3, cyclic AMP response element binding protein (CREB) and CAAT/enhancer binding protein (C/EBP) may also play an important role in airway inflammation. The purpose of this study was to measure DNA binding activity of these transcription factors in the airways of horses with RAO and to compare it to pulmonary function and bronchoalveolar lavage fluid (BALF) cytology. Seven horses with RAO and six control animals were studied during a moldy hay challenge and after 2 months at pasture. Pulmonary function, BALF cytology and transcription factors' activities in bronchial brushings were measured during hay and pasture exposures. During moldy hay challenge, RAO-affected horses developed severe airway obstruction and inflammation and a significantly higher airway AP-1 binding activity than in controls. After 2 months on pasture, pulmonary function and airway AP-1 binding activity were not different between RAO and control horses. The DNA binding activity of CREB in airways of RAO-affected horses increased significantly after 2 months at pasture and became higher than in controls. A significant positive correlation was detected between AP-1 binding activity and indicators of airway obstruction and inflammation. Airway GATA-3, CEBP and CREB binding activities were negatively correlated with indices of airway obstruction. However, contrarily to CREB binding activity, GATA-3 and CEBP binding activities were not different between RAO and control horses and were unaffected by changes in environment. These data support the view that AP-1 and CREB play a role in modulating airway inflammation in horses with RA