Heaves affects mature horses and is characterized by episodes of labored breathing at rest caused by bronchoconstriction, neutrophilic inflammation of the airways, mucus accumulation, and airway remodeling. Neutrophilic airway inflammation in horses with heaves can be reversed by antigen withdrawal. However, evidence suggests that the inflammatory processes are not completely shut down as residual peripheral airway bronchoconstriction, elevated smooth muscle cell turnover surrounding the airways, and higher nuclear factor κB (NF-κB) activity are observable in asymptomatic horses with heaves. Although heaves is a disease of the airways, peripheral blood leukocyte activation[5-7] and increased concentration of circulatory inflammatory mediators[8-10] have been observed in affected horses during disease exacerbation suggesting that the inflammatory process might not be limited to the lungs.
There is an increasing interest for the systemic component of chronic inflammatory diseases in human and veterinary medicine. It is thought that systemic inflammation magnifies the local response and leads to immunological reactions distant from the lungs. Therefore, it is hypothesized that therapies targeting bronchospasm and local inflammation in chronic airway diseases using inhaled bronchodilators and corticosteroids, respectively, might not be optimal for long-term treatment and health improvement. Asthma, a disease that shares many pathophysiological features with equine heaves, is considered to be a systemic disease, as an increase in several inflammatory markers has been observed in the blood of affected patients. These include immunity-related mediators (eg, cytokines, eicosanoids, and cyclooxygenase products, IgEs) and the acute phase markers CRP,[13, 14] haptoglobin, fibrinogen, and SAA. Acute phase proteins (APP) are typically induced by proinflammatory cytokines and are produced by the liver after trauma or infection, but are also detected in a number of noninfectious conditions. Systemic inflammation in patients with chronic airway diseases is thought to contribute to comorbidities.[11, 16-19] In equine medicine, APPs are increasingly used to determine prognosis and monitor response to treatment in infectious and noninfectious diseases or traumatic injuries (reviewed in Ref. ).
The aim of this study was to better characterize the systemic inflammation present in horses affected with heaves. Selected APPs as well as a panel of cytokines were quantified in the serum of horses with heaves in the asymptomatic and symptomatic clinical phases as well as healthy controls kept in a similar environment.
- Top of page
- Materials and Methods
This study shows that proteins of the acute phase are increased in the peripheral blood of horses with heaves. More specifically, serum haptoglobin concentrations were found to be above those of healthy horses in all clinical phases of the disease, and were further increased by antigenic challenge. SAA was also transiently increased by antigen challenge only in heaves-affected horses; however, these markers did not correlate with lung function parameters or BALF neutrophil percentages. In addition, this study reveals that CRP is not a good marker of chronic inflammatory airway disease in horses and that none of the cytokines detected in serum samples in this study were upregulated in heaves. In addition, these results suggest that the presence of subclinical airway inflammation in heaves-susceptible horses even with strict environmental dust control may lead to sustained systemic inflammation (ie, haptoglobin synthesis) or inversely, residual airway inflammation may derive from persistent systemic inflammation if it reveals to be an intrinsic feature of heaves pathology.
The results of this study support and further extend previous reports suggesting that systemic inflammation is a component of clinical exacerbation of heaves (reviewed by Leclere et al). Herein, both SAA and haptoglobin, but not CRP, were induced with antigenic challenge in heaves-affected horses. The results also indicate that systemic inflammation remains present during clinical remission of the disease as high haptoglobin concentrations were found in asymptomatic heaves-susceptible horses. This is in agreement with a recent study that observed high serum TNF concentrations in heaves-susceptible horses kept in a low dust environment and free of clinical signs. Further support for a persistent systemic inflammation in asymptomatic horses is the increased adhesion of peripheral blood neutrophils to fibronectin-coated plastic and priming for proinflammatory cytokine expression in response to bacterial-derived products.
Haptoglobin has been shown to be increased 5–10 fold in horse serum as a result of inflammation and infection. Its primary recognized role is to scavenge hemoglobin released into the circulation because of hemolysis or normal red blood cell turnover. Equine haptoglobin is a glycoprotein with an alpha and beta chain subunit structure, resembling human haptoglobin type 1–1. Normal concentrations in adult horses are ~1,000 μg/mL, whereas higher concentrations are reported in foals,[24, 35] suggesting that haptoglobin concentrations decrease with age. In this study, haptoglobin concentrations were lower than reported previously, which may be attributable to the fact that the horses studied were aged horses. Haptoglobin concentrations in asymptomatic heaves-susceptible horses were ~2.5 fold higher than control horses at T0, and rose up to ~3 fold after exposure to natural antigenic challenge for 30 days. This is similar to findings in human patients with asthma or chronic obstructive pulmonary disease.[15, 36] Importantly, serum haptoglobin concentrations in heaves-affected horses did not overlap those of controls kept under identical environmental conditions, suggesting that haptoglobin may be used as a biological marker for heaves. Interestingly, control horses also showed an increase in haptoglobin serum concentrations after hay exposure. This suggests that the magnitude, but not the uniqueness of the systemic inflammatory response distinguishes heaves-affected from healthy horses. Of note, however, haptoglobin concentrations returned to baseline in controls at T30, reinforcing the hypothesis that heaves underlies a defect in negative feedback mechanisms of inflammation. Monitoring of haptoglobin in horses with lower inflammatory airway diseases may possibly be of help in selecting therapies, as haptoglobin concentrations are higher in serum of asthmatic patients refractory to β2-agonist treatment and requiring systemic corticosteroids.
The main source of haptoglobin in acute phase responses is the liver. Interestingly, a heavily glycosylated form of haptoglobin was found to be synthesized and stored by human neutrophils in specific granules during their maturation and released after their activation. It may be hypothesized that peripheral blood, airway neutrophil degranulation, or both after activation by inflammatory factors present in the blood (eg, TNF[10, 31]) may constitute a source of haptoglobin in heaves-affected horses. Studies have shown that haptoglobin is largely induced in the lungs of mice after LPS challenge and produced by human alveolar macrophages, alveolar epithelial cells type II, and bronchiolar cells. As plasmatic haptoglobin decreases neutrophil functions through binding to specific membrane receptors and counteracting calcium signalization and respiratory burst activation, it may be hypothesized that this APP plays a protective role and prevents overwhelming inflammation.
Serum amyloid A is the most commonly APP evaluated in the horses, as it is induced 10–1,000 fold during inflammation. It has previously been used as a blood marker of lower airway inflammation caused by viral or bacterial infections. In humans, both serum and sputum SAA is increased in asthma. The production of local SAA by the airway tissues was also suggested. Herein, all heaves-affected horses had detectable serum SAA after 7 days of challenge with increases in concentrations up to at least 50 times those of baseline, whereas it was detected in only 1 control horse serum at this time point. These results are in agreement with previous reports indicating that blood concentrations of SAA are very low or undetectable in healthy individuals. A limitation of this study is the assignment of arbitrary values to samples with very high or low concentrations, decreasing the power of statistical analysis. Despite uncertainty about the absolute protein concentrations, differences in biological significance between groups at T7 were clearly highlighted. Interestingly, it has recently been shown that SAA can promote airway neutrophilia, activate neutrophils, and retroactively, be degraded by their granule content. SAA is also rapidly degraded by the liver.
The evaluation of the cytokine profile associated with heaves has been the focus of intense research for the last decade and has generated conflicting results.[49-51] This is the first study to our knowledge that characterizes multiple serum cytokines at the protein level in heaves-affected horses. We found that only IL-10, CCL2, and IFN-γ were detectable in a majority of horses, whereas IL-4, IFN-α, and IL-2 remained under detection levels. The latter cytokines are unlikely to contribute to the disease through systemic circulation, as the ELISA assay detects cytokines in the physiological range (low pg/mL). Correlation analysis suggested that CCL2 (also named MCP-1), a chemokine expressed by stimulated human and murine neutrophils promoting the development of Th17-type or Th2-type adaptive immunity, respectively, was positively associated with lung resistance (rs = 0.89) at T30 in heaves-affected horses. Similarly, INFγ showed a positive association with BALF neutrophil percentages in heaves-affected horses in clinical remission (rs = 0.93). Indeed, this Th1-associated cytokine is known for its role in promoting neutrophilic inflammation and the activation of neutrophils. As multiple statistical tests were performed in a relatively small cohort of animals and because cytokine concentration did not vary according to lung obstruction or inflammation, one must be careful in interpreting these results. Further exploration is required to identify the source of these cytokines and their significance in the disease.
Whether systemic inflammation represents an “overspill” of lung inflammation into the bloodstream or an intrinsic feature of heaves remains to be elucidated. In this study, as neither the lung function parameters nor the BALF cytology were correlated with the concentration of APPs, the causal relationship between those events could not be ascertained. Nevertheless, it has been shown in human patients that systemic inflammation in young adults is associated with decreased lung function independently of the presence of asthma, smoking or obesity. Hence, systemic inflammation has been proposed to predate the development of chronic lung diseases, and this hypothesis was recently verified in a 15-year prospective study. From these, it may be hypothesized that the presence of systemic inflammation earlier in life may have contributed to the development of heaves in these horses. Long-term prospective studies investigating systemic inflammatory markers in young horses from heaves-prone families and the development of the disease would also benefit in resolving this issue. In addition, studies where serum APPs are quantified in symptomatic horses treated with inhaled or systemic corticosteroids may give valuable information.
In conclusion, results of this study indicate that heaves is associated with increased serum concentrations of the acute phase proteins, SAA, and haptoglobin. These findings confirm previous assumption that heaves involves systemic inflammatory changes, in both the remission and exacerbation phases. Further investigation is required to assess the usefulness of these markers for the evaluation of response to treatments and the consequences of systemic inflammation on the development of collateral pathologies in affected horses.