Differential Effects of Dexamethasone and Itraconazole on Aspergillus fumigatus-Exacerbated Allergic Airway Inflammation in a Murine Model of Mite-Sensitized Asthma

Bronchial asthma is a representative airway inflammatory disorder that develops from the central to small airways, and involves various inflammatory cells and mediators in its pathogenesis [1,2]. Since human airways are directly connected to the atmosphere, numerous airborne factors are continuously reaching the airway during breathing. As a result, some of these airborne factors can potentially cause allergic airway inflammation, leading to an acute exacerbation of asthma.

Respiratory infections are the most frequent cause of asthma exacerbation in both children and adults [3,4]. Indeed, respiratory viruses have been studied exclusively for their role in the worsening of asthma symptoms [5]. Aside from viruses, the link between fungi and asthma has been long established. There is sufficient evidence to indicate that exposure to outdoor and indoor fungi are associated with the severity of asthma [6,7,8]. Aspergillus spp. are ubiquitous within the indoor and outdoor environment, and airways colonization by Aspergillus fumigatus (Af) has been demonstrated in asthmatic subjects. Allergic bronchopulmonary aspergillosis (ABPA) is a specific disease entity characterized by a highly exaggerated allergic response due to long-term colonization of the respiratory tract by Af [9]. More recently, the phrase ‘severe asthma with fungal sensitization', or SAFS, has been coined to define a particular phenotype of severe asthma, which is distinguished from ABPA [10]. In marked contrast to anti-virals, anti-fungal treatments are available and have the potential to be clinically useful in the treatment of fungus-exacerbated asthma. For instance, the benefit of oral itraconazole (ITCZ) has been shown in double-blind, placebo-controlled trials in patients with ABPA [11,12]. Additionally, oral ITCZ has been reported to be effective for treating SAFS [13,14].

The mouse is an excellent animal model for studying interactions between fungal infection and mite allergen sensitization. Previous reports used a model that was first sensitized to and then challenged with Af protein [15,16]. However, since people are continuously inhaling aeroallergens from the atmosphere they are generally sensitized with multiple allergens. Thus, atopic subjects are likely to be initially sensitized to aeroallergens, such as mites, in utero or in early life, establishing allergic airway inflammation, and are then subsequently exposed to other allergens, such as Af. Based on this understanding, we previously developed a murine model of Af-exacerbated mite allergen-sensitized asthma [17]. Utilizing this model, the present study aimed to compare the effects of ITCZ versus corticosteroids on Af-enhanced preexisting allergic airway inflammation.

Af MF-13 [17] was isolated from the sputum of a patient with pulmonary aspergilloma and was subcultured on Sabouraud dextrose agar (Becton Dickinson, Cockeysville, Md., USA) at 30°C for 7 days. The conidia were harvested with sterile saline containing 0.02% Tween 80 (Wako Pure Chemical Industries, Tokyo, Japan), then counted in a hemocytometer and diluted with sterile saline for intranasal infection [17].

As illustrated in figure 1, four groups of mice were prepared. Four-week-old female BALB/c mice (Charles River Japan Inc., Yokohama, Japan) were immunized intraperitoneally (i.p.) on both days 1 and 14 with 0.5 mg of Dermatophagoides farinae(Df; crude extract of the mite: LG-5339; Cosmo Bio, Tokyo, Japan) precipitated in aluminum hydroxide. These mice were then challenged intranasally (i.n.) with 50 μg/50 μl Df allergen (crude extract of the mite) on days 14, 16 and 18. Following Df sensitization, control groups were challenged with PBS and subsequently sham-infected i.n. with culture media. Mice in the Df-Af group were administered 5 × 10⁶Af-conidia i.n. on days 19, 21 and 23 following Df sensitization and the Df challenge. Mice in the Df-Af group were either injected subcutaneously (s.c.) with 0.02 mg of dexamethasone (Dex; Sigma, St Louis, Mo., USA) or orally administered 0.1 mg of ITCZ (Janssen Pharmaceutical K.K., Tokyo, Japan) on days 19, 21 and 23. On day 24, all mice were sacrificed. Bronchoalveolar lavage (BAL) samples and lung tissues were obtained from each group. All procedures were reviewed and approved by the Nagasaki University School of Medicine Committee on Animal Research. In every experiment, each group consisted of 2-3 mice and all experiments were repeated three times. Finally, the findings from the three independent experiments were combined and each group consisted of 8 mice.

Twenty-four hours after final Af conidia or sham infection, mice were sacrificed. BAL was conducted in the immediate postmortem period with 1 ml of PBS, and the obtained BAL sample was centrifuged. Differential cell counts were performed using the cytocentrifuged BAL sample stained with May-Grünwald-Gìemsa. Concentrations of Th2 cytokine (IL-5 and IL-13), MIP-2 (murine cytokine corresponding to human IL-8), and IFN-γ (Th1 cytokine), in the supernatants of the BAL sample were measured by enzyme-linked immunosorbent assay, according to the manufacturer's directions (Endogen, Wobum, Mass., USA). The detection limits for IL-5, IL-13, MIP-2 and IFN-γ were 5, 1.5, 1.5 and 10 pg/ml, respectively. Formaldehyde fixative was gently infused through the lavage catheter set in the trachea. Resected lungs were fixed for an additional 24 h and embedded in paraffin. Sections (4 μm) were stained with hematoxylin eosin (HE), and with periodic acid-Schiff (PAS). Gomori methenamine-silver staining was also performed in order to identify Af in the lung tissues and to confirm the presence of fungal spores in Df-Af mice (data not shown).

In a different set of experiments, another four groups of BALB/c mice were prepared (fig. 2). Control and Df mice were prepared as described above. Dex and Df-Dex mice, which were also prepared as control and Df mice, respectively, were injected s.c. with a daily dose of 0.02 mg of Dex on days 15-19. On day 20, lung tissues were harvested from all groups of mice, then chopped with sterile scissors, digested for 2 h in a 37°C water bath containing 1.5 mg/ml collagenase A (type 1A; Boehringer Mannheim, Mannheim, Germany), and finally filtered with a metal mesh. After three washes with a RPMI-1640 medium (Gibco-BRL Life Technology Inc., Grand Island, N.Y., USA) containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (hereafter referred to as cRPMI), cells were resuspended in cRPMI. Mononuclear cells were isolated using a density gradient method with Ficoll (Amersham Pharmacia Biotech). FBS was put into a dish and incubated at 37°C for 15 min. After the FBS was discarded, suspended cells were placed in the dish and incubated overnight at 37°C. Thereafter, cells in the dish were collected using PBS containing EDTA. Aliquots (1 ml) of cell suspension (10⁶ cells/ml) were mixed with 1 ml of Af suspension (10⁶ cells/ml) opsonized with 100 μl of normal serum, and subsequently incubated for 60 min at 33°C. Ten minutes before the completion of incubation, methylene blue (0.01%) was added. Three areas were randomly selected per well in a blinded fashion. In each area, the number of phagocytosed conidia in two hundred conidia was counted. Results were expressed as an index representing the percentage of phagocytosed Af conidia (phagocytosed conidia/600 conidia).

Results are expressed as mean ± standard error of the mean (SEM). Differences between groups were examined for statistical significance using repeated measures analysis of variance (ANOVA) followed by a Bonferroni correction. A p value <0.05 was considered statistically significant.

Figure 3 shows some examples of pulmonary pathology observed in the study. The results of quantitative analysis of airway inflammation by BAL are shown in table 1. By comparison to control mice, Df-Af mice exhibited significantly increased levels of all cellular components, especially eosinophils and neutrophils. Among Df-Af mice, Dex significantly decreased the level of macrophages and eosinophils, but not neutrophils. However, ITCZ was found to significantly decrease the levels of macrophages and eosinophils, as well as neutrophils in Df-Af mice. As illustrated in figure 3, neither Dex nor ITCZ significantly reduced goblet cell hyperplasia in Df-Af mice, as determined by PAS staining.

Analysis of cytokine concentrations in BAL revealed that pulmonary concentrations of Th2 cytokines (IL-5 and IL-13), MIP-2 (murine cytokine corresponding to human IL-8) and IFN-γ were significantly elevated in Df-Af mice by comparison to control mice (table 2). In Df-Af mice, Dex significantly decreased IL-5, while ITCZ significantly decreased MIP-2. However, neither Dex nor ITCZ significantly affected pulmonary IL-13 and IFN-γ concentration.

To determine the effects of in vivo Dex treatment on anti-fungal innate immunity in allergic asthma, phagocytosis of live Af conidia by alveolar macrophages (AM) was compared between control, Df, Dex and Df-Dex mice. By comparison to AM isolated from control mice, AM isolated from Df mice revealed a significant decrease in phagocytosis (fig. 4). This observation suggested that preexisting allergic airway inflammation attenuates the phagocytotic activity of AM. Although Dex also significantly reduced phagocytotic activity in naïve mice, it was found to reverse phagocytotic activity against Af conidia in the AM of Df mice. In our preliminary experiments, phagocytosis of live Af conidia by AM was comparable between control and ITCZ mice, indicating that ITCZ does not have a significant effect on AM (data not shown).

In agreement with our previous report [17], we found that Af infection enhanced both eosinophilic and neutrophilic airway inflammation, along with mucous hyper-production in mite allergen-sensitized mice. Additionally, the current study reported that Dex and ITCZ differently regulated inflammation and the cytokine profile in the airway. Finally, while phagocytotic activity against Af was reduced in the asthmatic airway, in vivo Dex treatment prior to Af infection was able to reverse this effect.

A key characteristic of fungal-associated asthma is the increased severity of the asthma. A number of studies reported that fungal-sensitized asthma has been associated with a risk of death, and admission to the hospital and intensive care unit [6,7,8]. Why fungal allergens, in particular, are implicated in such severe cases of asthma remains unclear. One key difference between fungal allergens versus other aeroallergens is that house dust mite or grass pollen is only a source of an allergenic protein, while fungi also have the capability to actively germinate and colonize the airway. Thus, fungi may have a much greater impact on patients in terms of triggering a host's defense against pathogens and producing toxins and enzymes that may play an accessory role in triggering allergy [10].

Recently, the critical role of neutrophils in the allergic airway inflammation has been reconsidered [18] and a distinct pathological feature of fungal-associated asthma is neutrophilic airway inflammation. For instance, in the present study, pulmonary pathology demonstrated significant increases in neutrophils as well as eosinophils in the airway of Af-Df mice. While ABPA is characterized by eosinophilia, it has also been correlated with increased sputum neutrophils and increased levels of IL-8 [19]. The detection of Af in sputum is associated with sputum neutrophils, as well as Af IgE sensitization and reduced lung function [20]. The role of neutrophilic airway infiltration and MMP-9 activity in the development of airway allergy was confirmed in a murine model of fungal-sensitized asthma [21]. Neutrophilic airway inflammation caused by Af may at least partially explain the increased severity of fungus associated asthma. Indeed, current anti-inflammatory therapies for asthma, including inhaled corticosteroids, are effective in managing eosinophilic airway inflammation, but have little or no impact on neutrophilic airway inflammation [22,23]. Accordingly, additive treatment, which has an impact on neutrophilic inflammation, is required for fungus-associated asthma. Due to this fact, antifungal drugs have been attracting increasing attention, and the benefits of ITCZ in ABPA have been reported in a number of studies [11,12]. Additionally, ITCZ was reported to improve asthma-related quality of life in patients with fungal sensitization in severe asthma but free of ABPA [13]. Another retrospective cohort study also demonstrated that antifungal therapy with oral ITCZ or voriconazole significantly reduced serum total and Af-specific IgE as well as blood eosinophils in patients with severe asthma with fungal sensitization and ABPA [14]. In the present study, Dex was only effective at reducing eosinophilic but not neutrophilic inflammation, while ITCZ significantly reduced neutrophilic infiltration into the airway as well as eosinophilic infiltration.

To determine the molecular actions of each drug, we compared the cytokine profile in BALF between Dex- and ITCZ-treated Df-Af-mice. While Dex significantly decreased IL-5, ITCZ significantly decreased MIP-2. Thus, each drug was found to differentially regulate the Af- exacerbated allergic airway inflammation. The fact that ITCZ significantly reduced eosinophils without decreasing IL-5 suggests that it might inhibit other cytokines, such as IL-17, which is critical in both allergy and infection [24]. Both drugs did not significantly influence IL-13 or IFN-γ. Since IL-13 is associated with mucous hypersecretion, it appears that both Dex and ITCZ failed to inhibit goblet cell metaplasia.

A clinical concern with utilizing corticosteroids for fungus-exacerbated asthma is the inhibition of innate immunity. Indeed, in the present study, phagocytosis against Af by AM was significantly reduced in Df mice and Dex-treated naïve mice. This finding agrees with a previous report which showed that AM function is compromised in poorly controlled asthmatics and is characterized by decreased phagocytosis [25]. AM are the main phagocytotic cells in the lungs with innate immunity against Af and their phagocytotic activity can be attenuated by corticosteroids [26]. Unexpectedly, AM isolated from Dex-treated Df mice showed phagocytotic activity similar to those found in control mice. Thus, at least in terms of maintaining phagocytotic activity of AM, the inhibitory effects of corticosteroids on allergic inflammation may dominate over their suppressive effects on innate immunity.

In conclusion, the present study observed that anti-fungal therapy could have a benefit in fungus-exacerbated allergic asthma, which is characterized by neutrophilic inflammation that is resistant to corticosteroids. Since both Dex and ITCZ failed to inhibit mucous hypersecretion, a macrolide, which can modulate neutrophil accumulation and activation in the airways of patients with refractory asthma [27], could be another therapeutic candidate. The combination of Dex and ITCZ for the treatment of fungus-exacerbated asthma should also be evaluated in a future study.

This study was supported by Grants-in-Aid for Scientific Research (No. 17607009 and 21590968) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

None of the authors have conflicts of interest to declare in relation to this study.