Effects of Macrolide and Corticosteroid in Neutrophilic Asthma Mouse Model

Abstract

Background: Asthma is a disease of chronic airway inflammation with heterogeneous features. Neutrophilic asthma is corticosteroid-insensitive asthma related to absence or suppression of TH2 process and increased TH1 and/or TH17 process. Macrolides are immunomodulatory drug that reduce airway inflammation, but their role in asthma is not fully known. The purpose of this study was to evaluate the role of macrolides in neutrophilic asthma and compare their effects with those of corticosteroids.

Methods: C57BL/6 female mice were sensitized with ovalbumin (OVA) and lipopolysaccharides (LPS). Clarithromycin (CAM) and/or dexamethasone (DXM) were administered at days 14, 15, 21, 22, and 23. At day 24, the mice were sacrificed.

Results: Airway resistance in the OVA+LPS exposed mice was elevated but was more attenuated after treatment with CAM+DXM compared with the monotherapy group (p<0.05 and p<0.01). In bronchoalveolar lavage fluid study, total cells and neutrophil counts in OVA+LPS mice were elevated but decreased after CAM+DXM treatment. In hematoxylin and eosin stain, the CAM+DXM-treated group showed less inflammation additively than the monotherapy group. There was less total protein, interleukin 17 (IL-17), interferon γ, and tumor necrosis factor α in the CAM+DXM group than in the monotherapy group (p<0.001, p<0.05, and p<0.001). More histone deacetylase 2 (HDAC2) activity was recovered in the DXM and CAM+DXM challenged groups than in the control group (p<0.05).

Conclusion: Decreased IL-17 and recovered relative HDAC2 activity correlated with airway resistance and inflammation in a neutrophilic asthma mouse model. This result suggests macrolides as a potential corticosteroid-sparing agent in neutrophilic asthma.

Keywords: Adrenal Cortex Hormones; Asthma; Histone Deacetylases; Inflammation; Macrolides; Neutrophils; Th17 Cells.

Figure 1. Schematic outline of the neutrophilic asthma mouse model. Mice were sensitized via OVA on days 0 and 7 by i.p. injection and challenged through inhalation of OVA on days 14, 15, 21, 22, and 23. For neutrophilic asthma, mice were additionally challenged with LPS on days 21, 22, and 23. DXM by i.p. injection and CAM via oral gavage were challenged every 24 hours on days 14, 15, 16, 17, 18, 21, and 23. OVA: ovalbumin; i.p.: intraperitoneally; LPS: lipopolysaccharide; DXM: dexamethasone; CAM: clarithromycin; i.n: intranasally.

Figure 2. Airway responsiveness to methacholine by forced oscillation technique. Airway responsiveness was expressed in terms of respiratory systemic Rrs. Baseline Rrs value was assessed after 3 minutes of nebulized PBS. Rrs was measured in response to increasing challenges of methacholine dose (6.25 mg/mL, 12.5 mg/mL, 25 mg/mL, and 50 mg/mL) in control, O+L, O+L+C, O+L+D, and O+L+D+C group. Rrs was increased after OVA+LPS challenge. Rrs of combination of CAM+DXM group was significantly changed compared to the CAM or DXM group especially at 50 mg/mL of methacholine challenge. O+L vs. all (^*p<0.05, ^**p<0.01, ^***p<0.001), O+L+C vs. O+L+D+C (^†p<0.05), O+L+D vs. O+L+D+C (^‡‡p<0.01). Rrs: resistance; PBS: phosphate buffered saline; O: ovalbumin; L: lipopolysaccharide; C: clarithromycin; D: dexamethasone.

Figure 3. Combination therapy of macrolide and corticosteroid inhibited lung inflammation in murine model of neutrophilic asthma. (A) BALF analysis was performed to evaluate lung inflammation. The number of total cell counts, eosinophil, and neutrophil was significantly increased after OVA+LPS challenge. The number of total cell counts was significantly more decreased in CAM+DXM group compared to decrease in CAM or DXM group. Neutrophils in combination of CAM+DXM treatment group was decreased and it was not seen in CAM or DXM group. The number of eosinophils was not decreased in CAM, DXM, and CAM+DXM group. (B) Accumulation of inflammatory cells improved significantly more in CAM+DXM group compared to DXM group in H&E staining. O+L vs. all (^*p<0.05, ^**p<0.01, ^***p<0.001), O+L+C vs. O+L+D+C (^†p<0.05, ^††p<0.01, ^†††p<0.001), O+L+D vs. O+L+D+C (^‡‡p<0.01, ^‡‡‡p<0.001). BALF: bronchoalveolar lavage fluid; O: ovalbumin (OVA); L: lipopolysaccharide (LPS); C: clarithromycin (CAM); D: dexamethasone (DXM).

Figure 4. T_H17 inflammation was attenuated by combination of macrolide and corticosteroid. Total protein, T_H2 inflammation, T_H17 inflammation, and proinflammatory cytokine in BALF analysis was increased after OVA+LPS challenge. (A) The number of total protein in BALF was decreased in DXM and CAM+DXM group. (B) IL-5 in BALF, marker of T_H2 inflammation, was not significantly decreased in DXM or CAM+DXM group. (C) IL-17, marker of T_H17 inflammation, was significantly decreased in DXM and CAM+DXM group. (D) IL-13, marker of T_H2 inflammation, was evaluated in lung tissue. There was no significant decrease in DXM or CAM+DXM group. (E, F) Proinflammatory cytokine was decreased in combination of CAM+DXM and it was not decreased in monotherapy of CAM or DXM. O+L vs. all (^*p<0.05, ^**p<0.01, ^***p<0.001), O+L+C vs. O+L+D+C (^†p<0.05, ^†††p<0.001). BALF: bronchoalveolar lavage fluid; O: ovalbumin (OVA); L: lipopolysaccharide (LPS); C: clarithromycin (CAM); D: dexamethasone (DXM); IL: interleukin; IFN-γ: interferon γ; TNF-α: tumor necrosis factor α.

Figure 5. Macrolide has potent effects of recovering HDAC2 activity with corticosteroid. (A) HDAC2 activity was decreased after OVA+LPS challenge. It was not recovered after CAM treatment, but it was recovered after DXM or CAM+DXM treatment. (B) DXM or DXM+CAM group showed higher density than OVA+LPS or CAM group in western blot analysis. (C) Relative density of HDAC2/β-actin was well correlated these tendency and CAM+DXM group was further increased in density of HDAC2/β-actin than DXM group. O+L vs. all (^*p<0.05, ^**p<0.01), O+L+C vs. O+L+D+C (^†p<0.05, ^†††p<0.001), O+L+D vs. O+L+D+C (^‡‡p<0.01). HDAC2: histone deacetylase-2; O: ovalbumin (OVA); L: lipopolysaccharide (LPS); C: clarithromycin (CAM); D: dexamethasone (DXM).