Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: In this project we investigated a clinically-relevant lower respiratory tract disease,heaves or recurrent airway obstruction (RAO), which occurs in certain mature horses fed hay. This disease, which is estimated to affect at least 25% of horses living in the Northeastern and Midwestern US, is characterized by the development of airway neutrophilia, bronchospasm and excessive mucus production. The lung inflammatory reaction negatively impacts on athletic performance and may contribute to increased horse wastage. In a small percentage of cases, right heart failure develops and may contribute to death. This disease is typically diagnosed and treated by equine general practitioners or by equine internists at tertiary care veterinary hospitals. Astute horse owners and trainers are also capable of recognizing the clinical signs exhibited by affected horses. Thus, when disseminating our research results, we have targeted practicing veterinarians, veterinary researchers, horse owners and trainers by: 1. Presenting results at local, regional, national and international conferences attended by these stakeholders. Specific examples include: a. Ainsworth DM. "The airway epithelium is not an innocent bystander in horses with recurrent airway obstruction (RAO)." Third International World Equine Airways Symposium, Ithaca, NY, July 19-22, 2005. b. Ainsworth DM. "Update on the diagnosis and treatment of equine respiratory disorders." Hudson Valley Chapter of the NYS Veterinary Medical Association, Millbrook, NY, October 5, 2005. c. Ainsworth DM. "Recurrent airway obstruction in horses-equivalent to farmer's lung" John Rankin Laboratory of Medicine Symposium, University of Wisconsin, Madison, WI, May 26, 2007. d. Ainsworth DM, et al. "The IL-23/IL-17 axis in RAO: just how important are alveolar macrophages in propagating the inflammation Third Equine Allergy Workshop, Holar, Iceland, June 17-21, 2007." e. Ainsworth DM. "Heaves current concepts" Equine Practitioners Conference, Cornell University, Ithaca, NY, November 1, 2007. f. Ainsworth DM, et al. "Primary bronchial epithelial cell cultures from heavey horses exposed to hay dust exhibit early up-regulation of CXCL2." Twenty-sixth Veterinary Internal Medicine Forum, San Antonio, TX, June 3-6, 2008. 2. Publishing results in relevant veterinary journals and textbooks including the American Journal of Veterinary Research, the Journal of Veterinary Internal Medicine, Equine Internal Medicine (textbook) and Large Animal Internal Medicine (textbook). 3. Involving pre-vet and veterinary students in research projects so that they exposed to this disease early in their careers and know state of the art information regarding its pathophysiology and treatment regimens. PARTICIPANTS: 1. Dorothy M. Ainsworth, Professor of Medicine in the Department of Clinical Sciences, College of Veterinary Medicine Cornell University, Ithaca, NY was the Project Director. She participated in all aspects of this study including sample collection, conducting molecular biology assays, performing data analysis (in collaboration with Professor Hollis N. Erb), presenting results and preparing or overseeing the preparation of the manuscripts. As an Equine Internist at the Cornell University Hospital of Animals, Dr. Ainsworth has a vested interest in understanding the pathophysiology of this common disease as well as designing therapeutic and prophylactic interventions. She has direct contact with many of the stakeholders of the equine industry. 2. Douglas F. Antczak is the Dorothy Havemeyer McConville Professor of Equine Medicine in the Department of Microbiology & Immunology, College of Veterinary Medicine Cornell University, Ithaca, NY. Dr. Antczak is an equine immunogeneticist who studies immunological interactions at another equine mucosal surface the uterus and brings a strong genomics perspective to this project. As Director of the J.A. Baker Institute of Animal Health (CVM-Cornell University), Dr. Antczak also made available to the research team state of the art equipment housed at the Institute. He also provided critical input on the experimental design and interpretation of results. 3. Hollis N. Erb is a Professor of Epidemiology in the Department of Population Medicine & Diagnostic Sciences, College of Veterinary Medicine Cornell University, Ithaca, NY. Dr. Erb contributed her expertise in the area of experimental design, statistical analysis and in critical review of the manuscripts. 4. Gabriele Grunig is a Research Scientist in the Department of Pathology, St. Lukes Hospital, Columbia University, NY, NY. Dr. Grunig is a veterinarian with extensive research training in the area of human asthma. She provided additional immunological insights into relevant chemokines or cytokines to be investigated. 5. Students provided the backbone of this investigation both with sample collection, care of horses, development of tissue culture techniques, performing molecular biology assays, and writing of manuscripts. Ms. Jean-Yin Tan, who helped develop tissue culture assays as a veterinary student, has since graduated and become a boarded equine internist. Ms.Danielle Retallick and Ms. Lauren DeLuca were both undergraduate students in Animal Science when they assisted with various aspects of the project. Their efforts resulted in publications. Ms. Retallick is currently training as a flight emergency nurse and Ms. DeLuca is in law school with an interest in science. Ms. Claudia Reyner, a pre-veterinary student in the College of Agriculture and Life Sciences (Cornell) has been involved with the whole animal aspects as well as laboratory portions of this study. She is a co-author of several papers with the PD and has recently submitted a first-author manuscript to the Am J Vet Res. She has applied for admission to veterinary school and hopes to specialize in equine medicine. TARGET AUDIENCES: Multiple groups were targeted including horse owners and trainers, general equine practitioners, equine specialists (boarded internists) as well as researchers who focus on airway disease. PROJECT MODIFICATIONS: No major changes in the objectives were implemented during this investigation. A no-cost extension was requested so that the PD could provide didactic and clinical coverage for 2 colleagues in LA Medicine who took maternity or sabbatical leaves during the project funding period.
Impacts
Real-time PCR and immunohistochemistry were used to test the hypothesis that the bronchial epithelium incites and propagates neutrophil influx in RAO. Our 2 objectives were to measure time-dependent changes in epithelial and bronchoalveolar cell (BC) chemokine expression during disease development and to examine bronchial epithelial cell culture responses of RAO-affected horses to hay dust. Objective one, part one. Bronchial biopsies and BCs were isolated from 8 RAO-prone and 9 control horses after horses were pastured (disease free) and after stabling and dusty hay exposure (DHE) for 1, 14, 35 and 49 days. Gene expressions of IL-8, CXCL1, GM-CSF and G-CSF (epithelium) and of IL-8 and IL-17 (BCs) were measured. At baseline, no differences between the 2 groups in epithelial or BC target gene expressions were found. After 24 hrs of DHE, epithelial IL-8 expression in RAO horses had increased 3-fold but did not exceed control responses (P=0.053). With continuous DHE, epithelial IL-8 gene and protein expression in RAO horses exceeded control values. In either group, epithelial CXCL1, GM-CSF or G-CSF gene expressions remained unchanged from baseline. Conclusions: epithelial IL-8 is involved in the chronic phases of RAO and may be instrumental in initiating early neutrophil influx but additional horses need to be studied at this earlier time point. Objective one, part two. Airway mononuclear cells were isolated from 8 diseased and 7 control horses after 1 or 14 days of DHE. Cells were treated with PBS, hay dust and LPS for 24 hrs before gene expressions of IL-8, CXCL2, IL-1beta, IL-23 (p19, p40 subunits) and IL-17 were measured. For cells isolated 1 or 14 d after DHE and treated with PBS, no differences between the 2 groups in target gene expression were found. Hay dust or LPS treatment of cells isolated at either time point up-regulated all cytokines equally in both groups. Conclusions: Airway mononuclear cells from RAO horses (acute or chronic stages) are not more reactive to hay dust than are cells from controls; increased IL-8 expression in BCs in RAO horses is due to neutrophils; the inflammation of RAO is not a result of a skewed IL-23/IL-17 axis. Objective two. Chemokine (IL-8, CXCL2, IL-1beta) and cell-surface receptor (TLR2, TLR4, IL-1R1) gene expressions in primary bronchial epithelial cell cultures (BECCs) established from 6 RAO-affected and 6 control horses (after 14 days of DHE) were examined. Gene expressions were measured in BECCs treated with PBS, hay dust and LPS for 6 or 24 hrs. No inherent differences in chemokine or cell surface receptor expressions were detected between the 2 groups at either time point following PBS treatment. Hay dust or LPS treatment for 6 or 24 hrs increased chemokine gene expression in both groups but early CXCL2 expression in RAO horses was significantlygreater than that of controls. Conclusion: Epithelial CXCL2 may incite early neutrophil influx in RAO-prone horses and deserves further investigation (in vivo). These studies provide evidence that the epithelial-derived IL-8 and CXCL2 may initiate and propagate the neutrophil influx in RAO.
Publications
- Ainsworth DM, Wagner B, Franchini M, Grunig G, Erb HN, Tan J-Y. Time-dependent alterations in IL-8 gene expression in the bronchial epithelium of horses with recurrent airway obstruction. Am J Vet Res 67:669-677, 2006.
- Ainsworth DM, Wagner B, Erb HN, Young JC, Retallick D. Effects of in vitro exposure to hay dust on expression of interleukin-17, -23, -8, -1β and chemokine (C-X-C motif) ligand 2 (CXCL2) by pulmonary mononuclear cells isolated from horses chronically affected with recurrent airway disease. Am J Vet Res 68: 1361-1369, 2007.
- DeLuca L, Erb HN, Young JC, Perkins GA, Ainsworth DM. The effect of adding oral dexamethasone to feed alterations on the airway cell inflammatory gene expression in stabled horses affected with recurrent airway obstruction (RAO). J Vet Intern Med 22: 427-435, 2008.
- Ainsworth DM, Matychak MB, Reyner CL, Erb HN, Young JC. Effects of in vitro exposure to hay dust on the expression of three chemokines and three cell surface receptors in primary bronchial epithelial cell cultures established from horses with chronic recurrent airway obstruction. In press, Am J Vet Res 2008.
- Ainsworth DM and Cheetham J. The Respiratory System. In: Bayly W.M., Reed S., Sellon D., eds.: Equine Internal Medicine, W.B. Saunders, third edition. In press, 2008.
- Ainsworth DM. Recurrent airway obstruction. In: Smith B.P., ed.: Large Animal Internal Medicine, Mosby, 4th edition, 2008, pp 556-563.
- Reyner CL, Wagner B, Young JC, Ainsworth DM. Pulmonary mononuclear cells isolated from horses acutely affected with RAO do not exhibit a greater expression of interleukin-17, -23, -8, -1β and chemokine (C-X-C motif) ligand 2 (CXCL2) when exposed in vitro to hay dust. Under review, Am J Vet Res 2008.
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Progress 01/01/07 to 12/31/07
Outputs
Year 3 (Specific aim 2): To characterize the specificity of particulate-airway epithelial cell interactions in cell cultures and to determine if the epithelium of diseased (RAO-affected) horses is hyper-responsive to hay dust. Primary cell cultures were established from the second to fifteenth generation airways isolated from 6 healthy and 6 RAO-affected horses that had been stabled and fed dusty hay for 2 weeks. This time point was selected because prior studies (Ainsworth et al., 2006) had demonstrated that epithelial chemokines are up-regulated in RAO-susceptible but not in healthy horses two weeks after natural challenge exposure (stabling, exposure to dusty hay). Cell cultures were grown in BEGM on collagen-fibronectin coated wells for 3-5 days until 80% confluency was achieved. Following a media change, cells were challenged for 6 or 24 hours with one of 6 treatment solutions consisting of PBS, hay dust (3 x 106, 3 x 105 particulates), LPS (10 μg/ml, 20
ng/ml) or beta-glucan (12.5 ng/ml). RNA was isolated, genomic DNA destroyed and cDNA was synthesized. The gene expressions of epithelial-derived chemokines (IL-8, CXCL2, IL-1beta) and of epithelial cell surface receptors (TLR4, TLR2, IL-1R1, IL-1R2) were measured using real-time PCR. Target gene expression was normalized to GAPDH. Between group treatment differences were analyzed using a Wilcoxon rank sum test and within group treatment effects (relative to PBS) were examined using a Wilcoxon signed rank test. In primary epithelial cell cultures that were incubated for 24 hours with one of the six different ligands we found no significant differences between the RAO-affected horses and the control horses in the expression of IL-8, CXCL2 or in IL-1beta. In either group, relative to PBS treatment exposure to hay dust or LPS increased IL-8 or CXCL2 expression from 2- to 4-fold and increased IL-1beta expression from 3 to 20-fold. Treatment with beta-glucan failed to enhance chemokine
expression in either group. Gene expression of cell surface receptors following 24 hr ligand treatment is currently being measured. Within the next two months we expect to complete our analysis of chemokine and cell surface receptor expression following the 6 hour ligand treatment. Based upon our preliminary data thus far, primary epithelial cells from RAO-affected horses are not more hyper-responsive to a 24 hour challenge with hay dust, LPS or beta-glucan than are cells from control horses. This finding, along with previous results from our laboratory, suggests that the characteristic up-regulation of epithelial-derived chemokines in RAO-affected horses requires additional promoting signals (cytokines, chemokines) possibly derived from airway luminal or interstitial cells that are not present in cell cultures
Impacts
Studies from our laboratory have sought to determine the pathways involved in inducing pulmonary inflammation in horses with heaves (COPD, RAO). Temporal changes in gene and protein expression in the airway lining and airway luminal cells provide clues to the mechanism of the disease following inhalation of hay dust. By themselves, either the airway luminal cells or airway epithelial cells are not hyper-reactive to the hay dust but require in vivo signals for the exaggerated inflammatory responses.
Publications
- Ainsworth DM, Wagner B, Franchini M, Grunig G, Erb HN, Tan J-Y. Time-dependent alterations in IL-8 gene expression in the bronchial epithelium of horses with recurrent airway obstruction. Am J Vet Res 67:669-677, 2006.
- Ainsworth DM. Recurrent airway obstruction. In: Smith BP ed.: Large Animal Internal Medicine, Mosby, 4th edition, (in press, 2007).
- Ainsworth DM, Wagner B, Erb HN, Young JC, Retallick D. Pulmonary mononuclear cells isolated from horses chronically affected with RAO do not exhibit a greater expression of interleukin-17, IL-23, IL-8, IL-1beta and chemokine (C-X-C motif) ligand 2 (CXCL2) when exposed in vitro to hay dust. (in press, Am J Vet Res, 2007).
- DeLuca L, Erb HN, Young JC, Perkins GA, Ainsworth DM. The effect of oral dexamethasone and feed alterations compared to feed alterations alone on the inflammatory gene expression of the bronchoalveolar lavage cells and the airway epithelium in stabled horses affected with recurrent airway obstruction (RAO). (Submitted, J Vet Intern Med, 2007).
- Ainsworth DM. The role of the airway epithelium in the pulmonary inflammatory responses of horses. Proc World Equine Airways Symposium 3: 37-39, 2005.
- Ainsworth DM. Recurrent airway obstruction in horses. John Rankin Laboratory of Medicine Symposium, University of Wisconsin, May 26, 2007, Madison, WI.
- Ainsworth DM, Wagner B, Erb HN, Retallick D. The IL-23/IL-17 axis in RAO: just how important are alveolar macrophages in propagating the inflammation? 3rd Equine Allergy Workshop at Holar, Iceland June 17-21, 2007.
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