Dorothy M. Ainsworth

Recurrent airway obstruction (RAO) in horses is characterized by IFN-γ and IL-8 production in bronchoalveolar lavage cells

In horses prone to developing recurrent airway obstruction (RAO), we tested the hypotheses that the cytokine profile in the bronchoalveolar lavage (BAL) cells of affected horses would reflect a polarized Th-2 response; that cytokine and chemokine alterations would occur within 24 h of allergen exposure; and that allergen exposure would induce alterations in the expression of the transcription factor t-bet (t-box-expressed in T-cells). The expression levels of interleukin-4 (IL-4), IL-13, Interferon-gamma (IFN-gamma), t-bet, IL-8 and granulocyte-macrophage colony stimulating factor (GM-CSF) were measured in BAL cells obtained from control and RAO-susceptible horses during an asymptomatic phase and at 24 h and 5 weeks post-stabling and hay exposure. At each sampling time, BAL neutrophil percentages in the RAO-group exceeded controls. In the RAO-group, only IL-13 expression was decreased 2-fold during the asymptomatic phase. No differences in cytokine or chemokine expression were detected during the acute exposure phase. During the chronic phase, IFN-gamma and IL-8 expression levels were 2.5- and 3-fold greater, respectively, in the RAO-group. No other differences in gene expression were detected. We conclude that the cytokine profile of the airway cells does not reflect a polarized Th-2 response; that increases in IFN-gamma result from a t-bet independent pathway and that chemokines from epithelial or interstitial cells may contribute to early neutrophil influx.

Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals

Cytokines produced by T helper (Th) cells are important in orchestrating the immune response during health and disease. Recent reports indicated that cytokine mRNA expression in foals is often quantitatively lower than that of adult horses suggesting that foal T cells are not fully mature. Here, peripheral blood mononuclear cells from foals and adult horses were stimulated with phorbol 12-myristate 13-acetate and analyzed for intracellular interferon-gamma (IFN-γ), interleukin-4 (IL-4) and IL-10 production, representing the Th1, Th2 and regulatory TR1 cell phenotypes respectively, by flow cytometry. In agreement with previous reports, all three cytokines were quantitatively reduced in foals compared to adults. However, the balance between Th1 and Th2 cytokines (IFN-γ/IL-4 ratio) showed a clear Th1-biased response in foals by 6 and 12 weeks of life, while similar IFN-γ/IL-10 ratios were found in foals and adult horses. By day 5 after birth, intracellular IFN-γ production by foal CD4+ and CD8+ T cells resembled that in adults. Overall, IL-4 production was low in foals. IL-4+ cells peaked at day 5 of age when IL-4 was mainly produced by IgE+ cells. Relative percentages of IL-4+ Th2 cells were significantly lower in foals at all time points. The data suggested that equine neonates and young foals have an impaired Th2 response, that the immune response of foals is Th1 biased, that IFN-γ production by Th and cytotoxic T cells is qualitatively similar to adult horses, and regulatory IL-10 production by T cells is developmentally mature in foals during the first three months of life.

Respiratory muscle perfusion and energetics during exercise

The oxygen cost of breathing and blood flow requirements of the respiratory muscles during exercise are discussed along with the implications for limitation of locomotor muscle and exercise performance. Findings show that the oxygen cost of the hyperpnea achieved during very heavy exercise may approach 15% or more of VO2max under conditions that require extraordinary levels of ventilatory work. These conditions include those in the highly trained endurance athlete (at VE > 150 l.min-1), the older athlete at VE of 110-120 l.min-1), and athletic cursorial mammals at VO2max--all of whom experience significant expiratory flow limitation and sometimes even complete ventilatory limitation during heavy or maximum exercise. Rates of blood flow to the respiratory muscles under these peak exercise conditions may equal or exceed those to the limb locomotor muscles. The hypothesis is advanced that excessive requirements of ventilatory work (and therefore VO2 and blood flow) during heavy exercise may cause reflex vasoconstriction of locomotor muscles resulting in curtailment of endurance exercise performance.

The effect of strenuous exercise on mRNA concentrations of interleukin-12, interferon-gamma and interleukin-4 in equine pulmonary and peripheral blood mononuclear cells.

The effect of strenuous exercise on the mRNA concentrations of interleukin-12p35 subunit (IL-12p35), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in equine pulmonary and peripheral blood mononuclear cells (PBMCs) was investigated. We hypothesized that strenuous exercise would suppress the expression of IL-12p35, IFN-gamma and augment the expression of IL-4. Eleven horses were randomly divided into two groups, a stall-confined control group (n=5) and an exercise-conditioned treatment group (n=6). Bronchoalveolar and PBMCs were obtained from horses in the treatment group prior to the commencement of a 9-week conditioning program and 24h after the completion of a maximum exercise test conducted in week 12. Samples were obtained simultaneously from control horses. Differential counts were performed on the bronchoalveolar lavage cells. Real-time PCR was performed on the pulmonary and PBMCs to quantitate cytokine expression using equine-specific primers and Taqman probes. Target gene expression was normalized to 18s rRNA expression. With the exception of IL-4 in the BALF cells, mRNA for the three cytokines was detected in the mononuclear cells from all horses at both sampling times. There were no significant differences in the cytokine mRNA concentrations between the two groups of horses at either of the sampling times. These findings demonstrate that strenuous treadmill exercise does not exert a deleterious effect on gene expression for IL-12p35, IFN-gamma or IL-4 when assessed in horses 24h following the intense physical activity.

Serum interleukin-6 (IL-6) and IL-10 concentrations in normal and septic neonatal foals

Previously it was reported that compared to surviving septic foals, non-surviving foals had a 35-fold increase in interleukin-10 (IL-10) and 15-fold increase in IL-6 gene expression in their peripheral blood mononuclear cells (PBMC). As gene expression profiles can be time-consuming, we sought to determine if serum IL-6 and IL-10 in foals would aid in the diagnosis and prognosis of septicemia. A prospective study of septic neonatal foals admitted to the Cornell University Equine Hospital during 2007 and 2008 was performed. Septicemia was confirmed in 15 foals using blood culture results and sepsis scores. Blood samples for measurement of serum IL-6 and IL-10 concentrations were collected at the time of admission (T0) and again 24 (T24) and 48 (T48) hours later. Blood samples from age-matched control foals (n=15) born at the Cornell Equine Park were obtained from foals 12-72h after birth (T0) and again 24 (T24) and 48 (T48) hours later. IL-6 and IL-10 concentrations were determined in the serum from dams of septic foals and serum and colostrum from dams of control foals. Serum IL-6 was also measured in healthy foals prior to ingestion of colostrum. Interleukin-6 was detected using an ELISA and IL-10 was detected using a bead-based fluorescent immunoassay. Group differences were detected using a Wilcoxon rank sum test with a Bonferroni correction applied to the p value. There were no significant differences in serum IL-10 concentration between the two groups of foals. Relative to control foals, septic foals had significantly lower serum IL-6 concentrations at all 3 time points. Relative to septic foals, control foals had significantly higher serum IL-6:IL-10 ratios. Serum IL-6 was undetectable in foals prior to ingestion of colostrum. However, colostral IL-6 concentration measured in the control mares was high (> or =215ng/mL) in all samples suggesting passive transfer of maternal IL-6 to the equine neonate. Colostral IL-10 was undetectable in 11/12 samples. Failure of passive transfer may directly influence the serum IL-6 concentration in septic foals. Neither serum IL-6 nor IL-10 alone, were useful diagnostic indices of sepsis in equine neonates. Although the number of animals involved in this study was too small for the identification of a concrete value, the serum IL-6:IL-10 ratio is likely to provide a valuable prognosticator for neonatal septicemia.

The Effect of Adding Oral Dexamethasone to Feed Alterations on the Airway Cell Inflammatory Gene Expression in Stabled Horses Affected with Recurrent Airway Obstruction

BACKGROUND Chemokine expression in airway epithelium and bronchoalveolar lavage fluid (BALF) cells of horses with recurrent airway obstruction (RAO) is increased. HYPOTHESIS For RAO-affected horses that are stabled and fed a pelleted ration, the addition of oral dexamethasone further improves pulmonary function and reduces inflammatory gene expression in pulmonary cells. ANIMALS Twelve RAO-affected horses. METHODS In a randomized cross-over experiment, the effect of feeding pellets in lieu of hay to stabled, RAO-affected horses was compared with the effect of feeding pellets and administering a 21-day decreasing dose regimen of oral dexamethasone on the expression (by kinetic polymerase chain reaction) of interleukin-8 (IL-8), chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1beta, IL-6, and beta-actin in the BALF cells and of IL-8, CXCL2, 2 IL-1 receptor (IL-1R2), Toll-like receptor 4 (TLR4), and glyceraldehyde 3-phosphate dehydrogenase in the bronchial epithelium 2 days after the final dose. RESULTS Both treatments reduced airway neutrophilia and breathing efforts but the addition of dexamethasone was associated with fewer treatment failures. Compared with feed changes alone, dexamethasone administration further reduced the expression of IL-8, CXCL2, and IL-1beta in the BALF cells 3.3-, 2.5-, and 4.7-fold, respectively. In the airway epithelium, both treatments were equally efficacious in reducing the expression of IL-8 and CXCL2 expression relative to pretreatment values, but either treatment failed to alter the expression of IL-1R2 and TLR4. CONCLUSIONS AND CLINICAL IMPORTANCE For a rapid and consistent improvement in pulmonary function and a reduction in inflammatory gene expression of the BALF cells, a decreasing dose of oral dexamethasone in combination with feed alterations is more efficacious for horses that must remain stabled.

Pulmonary-locomotory interactions in exercising dogs and horses

In exercising quadrupeds, limb movement is often coupled with breathing frequency. This finding has lead some investigators to conclude that locomotory forces, associated with foot plant, abdominal visceral displacements or lumbo-sacral flexion, are the primary determinants of airflow generation. Analysis of respiratory muscle electrical activation (EMG) and contraction profiles in chronically instrumented dogs and horses, along with measurements of esophageal pressure (Pes) changes and limb movements, provide evidence that each breath during the exercise hyperpnea is determined by respiratory neuromuscular events. Specifically: (1) Phasic diaphragmatic EMG and tidal shortening are always synchronous with decreases in Pes; (2) decrements in Pes are always associated with inspiratory flow generation; and (3) strict phase coupling between breathing and stride frequency is not obligatory. Thus, although locomotory-associated forces may minimally assist with flow generation, they are not the primary determinants of breathing during exercise.

The effect of exercise on diaphragmatic activation in horses

Horses chronically-instrumented with costal diaphragmatic electromyographic electrodes were studied during exercise while unencumbered by a breathing mask. Exercise-associated changes in esophageal (Pes), gastric (Pga) and transdiaphragmatic (Pdi) pressures were measured and related to diaphragmatic electromyographic activity (CS EMG) and to left forelimb impact. In all breaths examined, CS EMG always coincided with decrements in Pes. For all exercise trials, linear increases in CS EMG, Pga and Pdi and linear decreases in Pes, as a function of exercise intensity, always occurred. During all gaits, breathing frequency (fR) was entrained with stride frequency (fS) one for one. However, a constant phase-coupling relationship between fR and fS, observed when horses cantered and galloped, was absent when horses walked or trotted. We conclude that biomechanical forces contribute minimally to ventilation in exercising horses, that the diaphragm is always phasically active during each breath and its total electrical activity and mechanical output are proportional to the exercise hyperpnea.

Genomic analysis establishes correlation between growth and laryngeal neuropathy in Thoroughbreds

BackgroundEquine recurrent laryngeal neuropathy (RLN) is a bilateral mononeuropathy with an unknown pathogenesis that significantly affects performance in Thoroughbreds. A genetic contribution to the pathogenesis of RLN is suggested by the higher prevalence of the condition in offspring of RLN-affected than unaffected stallions. To better understand RLN pathogenesis and its genetic basis, we performed a genome-wide association (GWAS) of 282 RLN-affected and 268 control Thoroughbreds.ResultsWe found a significant association of RLN with the LCORL/NCAPG locus on ECA3 previously shown to affect body size in horses. Using height at the withers of 505 of these horses, we confirmed the strong association of this locus with body size, and demonstrated a significant phenotypic and genetic correlation between height and RLN grade in this cohort. Secondary genetic associations for RLN on ECA18 and X did not correlate with withers height in our cohort, but did contain candidate genes likely influencing muscle physiology and growth: myostatin (MSTN) and integral membrane protein 2A (ITM2A).ConclusionsThis linkage between body size and RLN suggests that selective breeding to reduce RLN prevalence would likely reduce adult size in this population. However, our results do not preclude the possibility of modifier loci that attenuate RLN risk without reducing size or performance, or that the RLN risk allele is distinct but tightly linked to the body size locus on ECA3. This study is both the largest body size GWAS and the largest RLN GWAS within Thoroughbred horses to date, and suggests that improved understanding of the relationship between genetics, equine growth rate, and RLN prevalence may significantly advance our understanding and management of this disease.

Effects of in vitro exposure to hay dust on the gene expression of chemokines and cell-surface receptors in primary bronchial epithelial cell cultures established from horses with chronic recurrent airway obstruction

OBJECTIVE To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or beta-glucan on chemokine and cell-surface receptor (CSR) gene expression in primary bronchial epithelial cell cultures (BECCs) established from healthy horses and horses with recurrent airway obstruction (RAO). SAMPLE POPULATION BECCs established from bronchial biopsy specimens of 6 RAO-affected horses and 6 healthy horses. PROCEDURES 5-day-old BECCs were treated with PBS solution, hay dust solutions, LPS, or beta-glucan for 6 or 24 hours. Gene expression of interleukin (IL)-8, chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1beta, toll-like receptor 2, toll-like receptor 4, IL-1 receptor 1, and glyceraldehyde 3-phosphate dehydrogenase was measured with a kinetic PCR assay. RESULTS Treatment with PBS solution for 6 or 24 hours was not associated with a significant difference in chemokine or CSR expression between BECCs from either group of horses. In all BECCs, treatment with hay dust or LPS for 6 hours increased IL-8, CXCL2, and IL-1beta gene expression > 3-fold; at 24 hours, only IL-1beta expression was upregulated by > 3-fold. In all BECCs, CSR gene expression was not increased following any treatment. With the exception of a 3.7-fold upregulation of CXCL2 in BECCs from RAO-affected horses (following 6-hour hay dust treatment), no differences in chemokine or CSR gene expression were detected between the 2 groups. At 24 hours, CXCL2 gene expression in all BECCs was downregulated. CONCLUSIONS AND CLINICAL RELEVANCE Epithelial CXCL2 upregulation in response to hay dust particulates may incite early airway neutrophilia in horses with RAO.