The Harry M. Zweig Memorial Fund for Equine Research


Deciphering the Mechanism of Equine Inflammatory Airway Disease in vitro

Dr. Dorothy M. Ainsworth

AinsworthRespiratory disorders of the equine athlete are one of the major causes of reduced athleticism. In particular, inflammatory airway disease (IAD) affects between 20-80% of racehorses and performance horses. Horses with IAD exhibit a cough and exhibit excessive mucus and inflammatory cell accumulations in the tracheobronchial secretions. The net effect of the pulmonary inflammation is to reduce lung function, to make the lung stiffer and more susceptible to further injury during exercise. The cause of IAD is currently unknown. Some investigators hypothesize that inhalation of hay dust which contains environmental particulates, endotoxin and fungal cell wall components, is responsible for the development of IAD. That the racehorse or performance horse typically spends the majority of its time stall-confined and that the risk of IAD increases with stabling makes this hypothesis likely. Attenuating or preventing inflammatory airway disease would likely improve athleticism and the overall comfort of the elite athlete.

We recently have developed a bronchial epithelial cell culture system in order to model pulmonary disorders in vitro. In the cell culture system, hay dust exposure up-regulates a multitude of pro-inflammatory proteins which we believe would incite white blood cell migration into the airways and excessive mucus production by the epithelium in vivo. The mechanism by which the hay dust interacts with the epithelial cells as well as the intracellular signaling pathways which eventually promote up-regulation of pro-inflammatory gene expression are unknown.

In the proposed project, we seek to identify the cell surface receptors and intracellular signaling pathways involved in hay dust induced inflammation by pre-incubating the epithelial cell cultures (prior to hay dust exposure) with specific inhibitors or blockers of these cell surface receptors and intracellular signaling pathways. Depicted in the figure below are the 4 major components that would be investigated in this proposal. These include the NF-KB intracellular pathway (pink torpedoes that interact with the cellular DNA or helix within the nucleus) and the three cell surface receptors which ultimately activate the NF-KB pathway. The cell surface receptors include the toll-like receptor 4 (TLR4, depicted in green); the TNF-a receptor (TNFR, shown in yellow) and the interleukin-1 receptor (IL-1R, diagramed in pink).

diagram
Inhalation of hay dust components activates the intracellular signaling pathway, NF-?B, which promotes pro-inflammatory cytokine production both at the gene and protein expression levels. (Our most likely candidate is TLR4 induced NF-KB activation but we will investigate the contribution of two other cell surface receptors, TNFR and IL-1R).

We will test our hypothesis in vitro, using bronchial epithelial cell cultures established from young athletic horses that have been humanely euthanized because of musculoskeletal, neurological or behavioral problems not amenable to treatment.

We will test the hypothesis by pre-treating the bronchial epithelial cell cultures with 4 different agents, prior to the cell cultures being challenged with hay dust. The specific agents include:

  1. Quercetin, a plant-derived bioflavonoid that directly inhibits intracellular NF-KB pathway, preventing its “activation” and translocation into the nucleus where it would normally enhance expression of selected pro-inflammatory genes on the DNA (helix in blue oval).
  2. E5564, a synthetic inhibitor of the toll-like receptor 4 (TLR4). This drug structurally resembles LPS (endotoxin) which normally combines with TLR4. However, E5564 structurally prevents any hay dust components from interacting with TLR4 and secondarily activating the NF-KB pathway.
  3. Infliximab, a monoclonal antibody that is able to interact with all forms of the pro-inflammatory cytokine TNF-α(free, membrane-bound or receptor-bound) blocking TNF-α induced activation of the receptor (and secondary activation of NF-KB pathway).
  4. Equine interleukin-1 receptor antagonist, a recombinant protein that binds to the IL-1 receptor and prevents the pro-inflammatory cytokine, IL-1ß, from signaling through the NF-KB pathway.

The outcome parameters that would be measured in this study include the activation of NF-?B and the expression of pro-inflammatory cytokines both at the gene level (mRNA concentrations of IL-8, TNF-α and IL-1ß) and protein level (IL-8, TNF-α).

We are very excited about the potential “investment return” that these studies offer. Furthermore, we are confident that completion of these studies will not only enhance our understanding of the pathophysiology of IAD, but enable evidence-based therapeutic interventions for horses with IAD to be designed and implemented.