A CONSORT-guided, randomized controlled clinical trial of nebulized administration of dexamethasone and saline on lower airway cytokine mRNA expression in horses with moderate asthma

Abstract

Background: Nebulized administration of dexamethasone on cytokine regulation in horses with moderate asthma has not been investigated.

Objective: To investigate the changes in expression of inflammatory cytokine mRNA after nebulized administration of dexamethasone treatment of horses with moderate asthma.

Animals: Horses with naturally occurring moderate asthma (n = 16) and healthy control horses (n = 4). All horses were kept in a dusty environment during the study.

Methods: Prospective, parallel, randomized, controlled, blinded clinical trial. Blood endogenous cortisol, tracheal mucus, and bronchoalveolar lavage (BAL) were sampled before and after 13 days treatment with either nebulized administration of dexamethasone (15 mg once daily) or 0.9% saline (3 mL). Treatment groups were randomly allocated via randomization function (Microsoft Excel). Amplification of target mRNA in BAL fluid (IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, IFN-γ, Eotaxin-2, and TNF-α) was achieved by qPCR, and the relative expression software tool was used to analyze BAL inflammatory cytokine mRNA.

Results: Horses treated with nebulized administration of dexamethasone had increased relative expression of IL-5 (1.70-fold), IL-6 (1.71-fold), IL-17 (3.25-fold), IL-12 (1.66-fold), and TNF-α (1.94-fold), and decreased relative expression of IL-23 (1.76-fold; P = .04) in samples collected on Day 14, in comparison to samples collected on Day 0 (all P < .05). Horses treated with nebulized administration of saline had no significant difference in the relative expression of any gene (all P > .05).

Conclusions and clinical importance: Nebulized administration of dexamethasone was associated with increased expression of inflammatory cytokine mRNA. There was no improvement in inflammatory airway cytology associated with either dexamethasone or saline treatment.

Keywords: REST analysis; cytokine expression; mRNA; mild equine asthma; one health; qPCR; respirable dust.

FIGURE 1

Bronchoalveolar lavage fluid differential cell counts in 16 horses with moderate asthma before (Pre: Day 0) and after (Post: Day 14) treatment with nebulized dexamethasone (15 mg q24h, n = 8), or 0.9% nebulized saline (3 mL q24h, n = 8). Bronchoalveolar lavage fluid differential cell counts for no treatment controls (n = 4) are also provided. (A) Differential neutrophils' counts. (B) Differential mast cells' count. (C) Differential eosinophils' count.

FIGURE 2

Effect of 13 days of treatment with nebulized dexamethasone (15 mg q24h; Day 14 vs Day 0) on relative expression of inflammatory cytokine mRNA from bronchoalveolar lavage‐derived cells obtained from 8 horses with moderate asthma. An expression ratio of 1 indicates that there was no change in the relative expression of that gene over time (>1 indicates up‐regulation; <1 indicates down‐regulation). Asterisk (*) indicates that the change in relative expression was significant (P < .05).

FIGURE 3

Relative expression of inflammatory cytokine mRNA from bronchoalveolar lavage‐derived cells obtained on Day 14, in horses with moderate asthma treated with nebulized dexamethasone (15 mg q24h), in comparison to no‐treatment control horses. An expression ratio of 1 indicates that there was no difference in the relative expression of that gene between groups (>1 indicates up‐regulation; <1 indicates down‐regulation). Asterisk (*) indicates that the difference in relative expression was significant (P < .05).

FIGURE 4

Relative expression of inflammatory cytokine mRNA from bronchoalveolar lavage‐derived cells obtained on Day 14, in horses with moderate asthma treated with nebulized 0.9% saline (3 mL q24h), vs treated with nebulized dexamethasone (15 mg q24h). An expression ratio of 1 indicates that there was no difference in the relative expression of that gene between groups (>1 indicates up‐regulation; <1 indicates down‐regulation). Asterisk (*) indicates that the difference in relative expression was significant (P < .05).

FIGURE 5

Effect of 13 days of treatment with nebulized 0.9% saline (3 mL, SID; Day 14 vs Day 0) on relative expression of inflammatory cytokine mRNA from bronchoalveolar lavage‐derived cells obtained from 8 horses with moderate asthma. An expression ratio of 1 indicates that there was no change in the relative expression of that gene over time (>1 indicates up‐regulation; <1 indicates down‐regulation). Asterisk (*) indicates that the change in relative expression was significant (P < .05).

FIGURE 6

Relative expression of inflammatory cytokine mRNA from bronchoalveolar lavage‐derived cells obtained on Day 14, in horses with moderate asthma treated with nebulized 0.9% saline (3 mL q24h), in comparison to no‐treatment control horses. An expression ratio of 1 indicates that there was no difference in the relative expression of that gene between groups (>1 indicates up‐regulation; <1 indicates down‐regulation). Asterisk (*) indicates that the difference in relative expression was significant (P < .05).

FIGURE 7

Effect of 13 days in a dusty environment (Day 14 vs Day 0) on relative expression of inflammatory cytokine mRNA from bronchoalveolar lavage‐derived cells obtained from no‐treatment control horses (n = 4). An expression ratio of 1 indicates that there was no change in the relative expression of that gene over time (>1 indicates up‐regulation; <1 indicates down‐regulation). Asterisk (*) indicates that the change in relative expression was significant (P < .05).