Therapeutic effects of co-inhaled roflumilast or formoterol and fluticasone on asthma-induced ultrastructural changes in murine airways

Purpose: To investigate the therapeutic effects of "inhaled" roflumilast and formoterol separately or combined with fluticasone on the ultrastructural airway changes in ovalbumin-induced asthmatic mice. Methods: The asthmatic mice were divided randomly into seven groups (n = 8): positive control, vehicle, and five treated groups. The following treatments were given by inhalation (15 min once/day) for seven days: roflumilast (500 μg/kg), formoterol (50 μg/kg), fluticasone (1000 μg/kg), roflumilast + fluticasone (500 + 1000 μg/kg), and formoterol + fluticasone (50 + 1000 μg/kg). Ultrathin lung sections (50 70 nm thick) were examined by transmission electron microscopy. Results: The asthmatic mice showed marked degenerative changes in bronchiolar epithelial cells. The alveolar septal walls were thickened with cellular changes and capillary congestion. The basement membranes showed marked thickening and the airway lumens contained abundant mucinous secretions. These ovalbumin-induced ultrastructural airway changes were markedly-reversed in the roflumilast + fluticasone group, moderately-reversed in the roflumilast, fluticasone, and formoterol + fluticasone groups, but were not affected in the formoterol group. Conclusion: Co-inhalation of roflumilast + fluticasone significantly improved the ultrastructural airway changes than co-inhalation of formoterol + fluticasone in ovalbumin-asthmatic mice due to its antiinflammatory and antifibrotic effects.


INTRODUCTION
In bronchial asthma the injured airway epithelium enhances airway inflammation and remodeling through secretion of proinflammatory mediators [1].Airway remodeling occurs partly as a result of chronic inflammation.It is manifested by epithelial changes, goblet cell hyperplasia, sub-epithelial fibrosis, thickened smooth muscle layer, and increased vascularity.It leads to airway narrowing, hyperresponsiveness, edema, progressive declining of lung functions, and more need for medications [2].The airway smooth muscle also secretes inflammatory mediators and its mass increases due to hypertrophy, hyperplasia, and deposition of extracellular matrix proteins [3].In asthma, airway inflammation generally responds to treatments but unfortunately airway remodeling is resistant [4].Inhaled corticosteroids (ICS) are the mainstay in asthma therapy; however, systemic side effects may occur at high doses [5].
Formoterol is a long-acting β 2 agonist (LABA) with rapid onset of bronchodilation.LABAs do not have clinically important anti-inflammatory effects, thus in treatment of asthma they are always combined with ICS [6].Roflumilast is a selective phosphodiesterase (PDE) inhibitor which is approved as an add-on treatment for chronic obstructive pulmonary disease (COPD).PDE-4 inhibitors are ineffective bronchodilators but they are powerful anti-inflammatory agents [7].In a recently-published work [8], it was concluded that co-inhalation of roflumilast and fluticasone significantly improved the inflammatory and histopathological changes than that of formoterol plus fluticasone in ovalbumin-asthmatic mice.
Studying the ultrastructural changes at cellular and subcellular levels using transmission electron microscopy (TEM) reveals more details and hence helps more understanding of the effects of the different treatments especially the anti-remodeling effect.Consequently, the current study was designed in OVA-sensitized and challenged mice to investigate the therapeutic effects of inhaled roflumilast and formoterol separately or combined with fluticasone on the ultrastructural airway changes.

EXPERIMENTAL Animals
The study was carried out according to the International Guidelines for the Care and Use of Laboratory Animals [9].The protocol was approved by the institutional Research Ethics Committee (ref no. 198-15).Drugs and chemicals were purchased from Sigma-Aldrich Corp. (St.Louis, MO, USA) unless mentioned otherwise.Female BALB/c mice (8 -10 weeks old and about 30 g weight) were housed in cages at 24 °C in a 12 h light-dark cycle and were acclimatized for a week before experimentation.Animal feed and water were available ad libitum.

Ovalbumin sensitization and challenge
The mice were sensitized by intraperitoneal injection of 20 µg of ovalbumin (OVA) in 0.1 mL of Alum (aluminium hydroxide powder) on days 0 and 14.The phosphate-buffered saline (PBS) was used as a vehicle for OVA.On days 27, 28, 47, 61, and 73 -75, mice were subjected to intranasal OVA (500 µg) or PBS in the control group [10,11].

Transmission electron microscopy
Mice were sacrificed by cervical dislocation.Immediately after animal dissection, lung specimens (1 -2 mm 3 ) were fixed in 2.5 % cold glutaraldehyde.Specimens were washed in cacodylate buffer (pH 7.2) three times (20 min each), post-fixed in 1 % osmium tetraoxide for 2 h, dehydrated in rising grades of ethanol, and embedded in Epon-araldite mixture.Semithin sections (1.0 µm thick) were cut from the embedded blocks, stained with toluidine blue, and then examined by light microscopy.The areas suitable for examination by electron microscopy were determined and ultrathin sections (50 -70 nm thick) were obtained by the LKB ultramicrotome.The sections were stained with uranyl acetate and lead citrate.They were examined by transmission electron microscope (TEM) (100 CXII;JEOL, Ltd., Tokyo, Japan), and photomicrographed by a digital camera (XR -41) [16].

Statistical analysis
Data are expressed as mean ± SEM and were analysed with SPSS 18. Comparisons were made using Student's t-test for two groups and one-way analysis of variance (ANOVA) with Tukey's test for more than two groups.P < 0.05 was considered statistically significant.

RESULTS
In the toluidine blue stained sections (Figure 1), TEM images of bronchioles and alveolar tissue (Figure 2 and Figure 3), PC group showed marked ultrastructural changes as compared to NC group.These changes were markedlyreversed in the roflumilast + fluticasone group and moderately-reversed in the roflumilast, fluticasone, and formoterol + fluticasone groups while not-reversed in the formoterol group.
The increases in the airway basement membrane thickness and alveolar septal wall thickness were marked in the PC and formoterol groups, moderate in the roflumilast, fluticasone, and formoterol + fluticasone groups, and mild in the roflumilast + fluticasone group (Table 1).

DISCUSSION
In the current study, the asthmatic mice showed marked degenerative changes of bronchiolar epithelium cells.The alveolar septal walls were thickened with cellular changes and capillary congestion.The basement membranes showed marked thickening and the airway lumens contained abundant mucinous secretions.Previously, similar ultrastructural airway changes were detected in chronic OVA-asthmatic mice [17].The numbers of Clara cells and ciliated epithelial cells decreased while the numbers of goblet cells increased with numerous secretory granules containing mucus.The basement membrane was thickened due to deposition of collagen.The myofibroblast sheath was also thickened due to high content of collagen and myofibroblasts in addition to smooth muscle hypertrophy and hyperplasia.The bronchial epithelium was shrunken with pyknotic nuclei.Moreover, there were platelet activation and inflammatory cell infiltration in the perivascular and peribronchiolar areas.In the airway smooth muscle, the mitohcondria increased in number with ultrastructural changes. .In another study in OVA -sensitized and -challenged mice, changes of the epithelial phenotype were detected especially in the proximal airways.The toluidine blue-stained sections showed a significant increase in goblet cells which completely compensated the significant decrease in Clara cells and the non-significant decrease of ciliated cells.Consequently, the epithelial thickness significantly increased due to replacement of the smaller Clara cells by the larger mucous cells (mucous cell metaplasia).In the distal airways, there were no significant differences regarding the total number of epithelial cells per unit area of the basement membrane, the thickness of epithelium, and the different cell types [16].In another study, the EM examination of biopsies obtained by fibreoptic endoscopy showed that airway myositis was characterized by a direct interaction between airway smooth muscle cells, mast cells, and lymphocytes.The smooth muscle remodeling was manifested by muscle cell hypertrophy and abnormal extracellular matrix deposition [18].
The current results showed that the ovalbumininduced ultrastructural airway changes were markedly-reversed in the roflumilast + fluticasone group, moderately-reversed in the roflumilast, fluticasone, and formoterol + fluticasone groups, while not-reversed in the formoterol group.Previously it was found that ICS have a little effect on airway remodeling in vivo as shown by failure to decrease collagen deposition in lungs of asthmatic patients which may be due to inability to inhibit TGF-β1 expression [19].Moreover, in OVA-sensitized rats, concomitant inhalation of fluticasone during OVA exposure partly inhibits airway remodeling and hyperresponsiveness while its inhalation after OVA exposure had no effect on remodeling [20].Fortunately, roflumilast was found to decrease airway inflammation, deposition of subepithelial collagen, and epithelial thickening in a murine model of asthma [21].In addition, oral roflumilast effectively reduced bleomycin-induced lung α-I collagen transcripts and fibrosis in both preventive and therapeutic protocols in rodents while oral glucocorticoids were ineffective in the therapeutic protocol [22].
Failure of long acting β2 agonists (formoterol) to improve remodeling in the current study agrees with a previous study which showed that in OVA -sensitized and -challenged BALB/c mice, LABA treatment (salmeterol) exaggerated airway inflammation, remodeling, and hyperresponsiveness due to mucus metaplasia, while fluticasone separately or combined with salmeterol reduced airway inflammation and remodeling [10].Moreover, aerosolized combined salmeterol and fluticasone in rats reversed goblet cell hyperplasia, but increased fibronectin and collagen in the airway wall [23].In addition, in asthmatic patients adding salmeterol to ICS reduced airway muscular tone and improved expiratory flows, but exerted a little effect on remodeling shown by decreasing number of vessels in the airway lamina propria [24].

Figure 1 :
Figure 1: Micrographs of lung stained by toluidine blue (x200) in untreated and treated ovalbumin-asthmatic mice.(A) Normal control group showing that the bronchiolar wall formed by a thin fibrous layer and lined by Clara cells and few ciliated cells.The lumen (L) is free from secretions.The peribronchiolar alveoli show empty alveolar lumen (x).The alveolar septa are thin and formed by flattened elongated cells (pneumocyte type I) (1), few large cells (pneumocyte type II) (2), and septal capillaries (c).(B) Positive control: (B1, B2) group: [B1] showing marked degenerative changes of the bronchiolar epithelium.The lumen (L) contains abundant amounts of secretions (Se), [B2] showing that the alveolar septa are thickened and the population of pneumocyte type II (2) is increased with marked hypertrophy.The alveolar lumens (L) are mostly narrow.(C) Roflumilast group, (E) Fluticasone group, and (G) Formoterol + fluticasone group showing that the bronchioles have swollen Clara cells with deeply stained cytoplasmic granules.The ciliated cells are numerous and deeply stained.The bronchiolar wall is formed by a thin fibrous layer.The peribronchiolar alveoli show distention and their septal walls show a membranous structure (arrow), slight thickening, congestion of septal capillaries, and few pneumocytes (D) Formoterol group showing that the bronchiolar lining epithelium is formed from an increased population of Clara cells which appear faintly stained with deeply stained granules.The ciliated cells are compressed and embedded between Clara cells.The lumen (L) contains secretory material.The peribronchiolar alveoli show thickened septal wall and congestion of septal capillaries.The alveolar lumen contains faintly stained material and cellular debris (x).(F) Roflumilast + fluticasone group showing that the bronchiolar wall is formed by a thin fibrous wall and is lined by Clara cells containing deeply stained granules.The ciliated cells appear deeply stained, and embedded between Clara cells.The lumen (L) is empty.The alveolar septa are thin

Figure 2 :
Figure 2: Transmission electron microscopy (TEM) micrographs of lung bronchioles stained by uranyl acetate and lead citrate (x2900) in untreated and treated ovalbumin-asthmatic mice.(A) Normal control group: showing that the epithelial lining of the bronchiole is formed from few ciliated cells (Ci) and numerous Clara cells (Cc) which have large vesicular nuclei (N) and a large amount of cytoplasmic electron dense (Ed) secretory granules beside the cell organelles.Both types of cells are situated on the basement membrane (B).The bronchiolar wall is formed from a thin fibrous layer containing a capillary (C).(B) Positive control group: showing swollen and sometimes ruptured Clara cells (Cc) and the electron dense (Ed) secretory granules are markedly reduced.The ciliated epithelial cells (Ci) appear compressed and more electron dense, and some of them contain numerous vacuoles (v).The lumen (L) contains light electron dense mucinous secretion.(C) Roflumilast group, (E) Fluticasone group, and (G) Formoterol + fluticasone group showing that Clara cells (Cc) contain moderate amount of electron dense (Ed) secretory granules, dilated rough endoplasmic reticulum (RER), and numerous large vacuoles (v).The ciliated cells (Ci) show numerous variable size electron dense (Ed) cytoplasmic bodies.The bronchiolar lumen contains a small amount of secretion (x).(D) Formoterol group showing swollen and vacuolated Clara cells (Cc) with marked reduction of the cytoplasmic electron dense (Ed) secretory granules and occasional pyknotic nuclei (N).The ciliated cells (Ci) are compressed with increased cytoplasmic electron density.(F) Roflumilast + fluticasone group showing that the bronchiolar epithelium is formed from both Cc and Ci cells situated on the basement membrane.The Clara cells (Cc) contain large vesicular nuclei (N) and a large amount of cytoplasmic electron dense (Ed) secretory granules.The ciliated cells (Ci) are shorter with more electron dense cytoplasm and vesicular nuclei.The bronchiolar wall is formed from thin fibrous collagen layer (Co).Scale bar, 2 μm.

Table 1 :
Effects of treatments on the airway basement membrane thickness (nm) and alveolar septal wall thickness (µm) in ovalbumin-induced asthma in mice