Objectives: The pathophysiology of inflammatory airway disease (IAD) is unknown, but in some cases involves the accumulation of mast cells, neutrophils, or both in the bronchoalveolar lavage fluid (BALF). The objective of this study was to characterize cytokine gene expression in the BALF cells of horses with IAD, including a comparison of cytokine gene expression between IAD horses with increased BALF mast cells (IAD-Mast) or neutrophils (IAD-Neutro).Methods: The mRNA expression of IL-4, IFN-c, IL-17, IL-8, IL-1b, IL-5, IL-6, IL-10, IL-12p35, and eotaxin-2 was studied by quantitative polymerase chain reaction (QPCR) with efficiency correction in BALF samples of 17 horses with IAD (IAD-total), also subcategorized as 8 IAD-Mast and 9 IAD-Neutro, and 10 controls. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. Relative expression software tool (REST) analysis provided ratios of expression, statistical analysis, and confidence intervals for the results.Results: Compared with the control group, IL-5, IL-1b, IL-6, IL-8, and IL-10 mRNA expression was upregulated 3.5-, 3.4-, 2.8-, 2.2-, and 1.9-fold, respectively, in the IAD-total group. The IAD-Neutro group showed increased expression of IL-17, IL-8, and IL-5 (4.7-, 2.5-, and 2.9-fold, respectively) and a decreased expression of IL-4 (3.4-fold) compared with the IAD-Mast group.Conclusion: Cytokines from the Th2 family plays a key role in IAD and a different pathophysiology may be involved in mast cell versus neutrophil BALF accumulation in IAD horses.

BackgroundThe stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify reference genes with the most stable mRNA expression in the BAL cells of horses with IAD irrespective of corticosteroids treatment.ResultsThe expression stability of seven candidate reference genes (B2M, HPRT, GAPDH, ACTB, UBB, RPL32, SDHA) was determined by qRT-PCR in BAL samples taken pre- and post- treatment with dexamethasone and fluticasone propionate for two weeks in 7 horses with IAD. Primers' efficiencies were calculated using LinRegPCR. NormFinder, GeNorm and qBasePlus softwares were used to rank the genes according to their stability. GeNorm was also used to determine both the ideal number and the best combination of reference genes. GAPDH was found to be the most stably expressed gene with the three softwares. GeNorm ranked B2M as the least stable gene. Based on the pair-wise variation cut-off value determined with GeNorm, the number of genes required for optimal normalization was four and included GAPDH, SDHA, HPRT and RPL32.ConclusionThe geometric mean of GAPDH, HPRT, SDHA and RPL32 is recommended for accurate normalization of quantitative PCR data in BAL cells of horses with IAD treated with corticosteroids. If only one reference gene can be used, then GAPDH is recommended.