The Additional Effects of Clarithromycin And Pranlukast On The Cytokine Suppression By Corticosteroids Using Murine Allergic Bronchopulmonary Aspergillosis Model

Few medicines other than oral corticosteroids and anti-fungal medicines are currently known as reliable treatments for allergic bronchopulmonary aspergillosis (ABPA). The ecacies of macrolide or leukotriene receptor antagonist (LTRA) with or without corticosteroid on ABPA are unknown. Mice were sensitized to Dermatophagoides farinae (Df) allergen intranasally and infected with Aspergillus fumigatus (Af). After Af infection, corticosteroid (Dexamethasone; Dex) was administered for ve days in DfAf/Dex group. The effects of macrolide (clarithromycin; CAM) or LTRA (pranlukast; PRN) with or without Dex were also evaluated. Pathologically, the combined treatment with Dex and CAM decreased the airway inammation. The interleukin (IL)-5, IL-13 and macrophage inammatory protein (MIP)-2 concentrations in homogenized lungs were signicantly elevated in DfAf mice compared to control mice (p < 0.05, each). CAM signicantly decreased the elevations of MIP-2 of DfAf mice (p < 0.05). The addition of CAM on Dex suppressed both of the MIP-2 and IL-5 elevation (p < 0.05, each, DfAf/Dex vs DfAf/Dex/CAM group), but the addition of PRN on Dex did not. It was suggested that combination of CAM and corticosteroid enhanced the suppressing effect of both eosinophilic and neutrophilic airway inammations. This nding will give a new hope for the treatment of severe fungus-related asthma.


Background
Allergic bronchopulmonary aspergillosis (ABPA) caused by sensitization of the Aspergillus antigen, accounts for 1-4% of the asthma patients and involves in the asthma severity [1][2][3]. Exposure to fungal spores or mycelial fragments results in the formation of IgE and IgG antibodies, and T cells also play important roles in ABPA progress [4,5]. The working group of the International Society for Human and Animal Mycology (ISHAM) had suggested the disease criteria with a preceding lung disease (Bronchial asthma or cystic brosis), and have both type I allergy (type I skin reaction or speci c IgE antibody against Aspergillus antigen and/or increase of serum total IgE value) and type allergy (image ndings indicate the airway in ammation or bronchial injury [6]. Without adequate diagnosis and treatment, ABPA will progress to severe respiratory failure with bronchiectasis and brosis [7]. Systemic glucocorticoids are considered the mainstay of treatment of ABPA [8,9]. Oral corticosteroid (OCS) and antifungal therapies appear to be partially successful in certain patients with ABPA [10][11][12], which like via suppression of cytokine production [4,5,13].
Other than anti-fungal medication, we hypothesized that cysteinyl leukotriene receptor antagonist (LTRA) or macrolide would be expectable for treatment of ABPA. LTRA, e.g. pranlukast (PRN) is effective for asthma control [14], and has been recommended as additional medicine on inhaled corticosteroid (ICS) / long acting beta agonist (LABA) treatment [1]. PRN suppressed the airway eosinophilic in ammation of asthmatic patients via interleukin (IL)-5 suppression [15,16]. It is reported that macrolide improved peak expiratory ow, clinical symptoms, quality of life, and airway hyper-reactivity in asthmatics [17], and that macrolides improved the symptoms in the patients with severe neutrophilic asthma and in asthma exacerbations [18,19].
To clarify the e cacies of LTRA and macrolide on ABPA especially in cytokine level for the direct or additional effect on OCS, in this study, we investigated the single or additional effect of LTRA (pranlukast; PRN) and macrolide (clarithromycin; CAM) with or without OCS (dexamethasone; Dex) on Aspergillus fumigatus (Af) and Dermatophagoides farinae (Df) co-exposed ABPA murine model.

Animals and Experimental protocol
Six groups of mice were prepared in the present study (Fig. 1). Four-week-old female BALB/c mice (Charles River Japan, Inc, Yokohama, Japan) were immunized twice intraperitoneally on days 1 and 14 with 0.5 mg per mouse of Dermatophagoides farinae (Df) (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 that, 5 × 10 6 of Af conidia were administered i.n. on days 19, 21 and 23. Various drugs were administered from on day21 to 25 and ten groups of mice were prepared as follows. DfAf, no drug was administered; DfAf/Dex, 0.02mg of Dexamethasone (Sigma, St Louis, Mo) were injected subcutaneously (SC); DfAf/PRN, 0.5mg of pranlukast hydrate (ONO Pharmaceutical Co., Osaka, Japan) were injected subcutaneously (SC); DfAf/CAM, 4mg of clarithromycin (Taisho Toyama, Tokyo, Japan) were orally administered; DfAf/Dex/PRN, combination therapy with Dexamethasone and pranlukast hydrate; DfAf/Dex/CAM, combination therapy with Dexamethasone and clarithromycin. On day 26, all mice were sacri ced. Bronchoalveolar lavage uid (BALF) and lung tissues were obtained from each group. The procedures were reviewed and approved by Nagasaki University School of Medicine Committee on Animal Research. All experiments were repeated at least 3 times.

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

Bronchoalveolar lavage and lung pathology
On day 26, mice were sacri ced and BAL was conducted utilizing 1 ml of PBS in the immediate postmortem period. The obtained BALF was centrifuged. Differential cell counts were performed using cytocentrifuged BALF stained with May-Grünwald-Gìemsa. Formaldehyde xative was gently infused through the lavage catheter set in the trachea. Resected lungs were xed for an additional 24 hours and embedded in para n. Sections (4 µm) were stained with hematoxylin and eosin (HE).

Analysis of cytokines concentrations in homogenized lung
Lung homogenates were prepared by homogenizing a freshly excised lung. Concentrations of IL-5, IL-13 and MIP-2 in homogenized lung samples were measured using enzyme-linked immunosorbent assay using the methods described by the manufacturer. Detection limits for IL-5, IL-13 and MIP-2 were 5 pg/ml, 1.5 pg/ml and 5 pg/ml respectively.

Statistical analysis
Results are expressed as mean (standard error of mean). Differences between groups were examined for statistical signi cance using repeated-measures ANOVA with a Bonferroni multiple comparison test. p values of < 0.05 were considered signi cant.

Results
The pulmonary pathology ndings and the cell differentiation in BALF of ABPA Mice Figure 2 shows some examples of the pulmonary pathology ndings. DfAf mice had severe airway in ammation ( Fig. 2A), and this in ammation was suppressed by combination of Dex and CAM (Fig. 2F).

Discussion
In this study, we found mainly two results. Firstly, our DfAf co-exposed murine ABPA model showed airway in ammation with increased eosinophils, neutrophils, and lymphocytes. Of these cells, neutrophil rate was In ABPA management, it is still problem that there are only two creditable medications, OCS and anti-fungal medicines [1][2][3][4][5]. In this study, we expected the effect of LTRA and macrolide on airway eosinophilic or neutrophilic in ammation with or without corticosteroid. Several evidences suggested that LTRA reduces airway in ammation and remodeling [20][21][22]. It is known that cysteinyl LTs are not only critically involved in the pathogenesis of asthma but are also produced in the airway during respiratory infection and are involved in protection against respiratory pathogens. [23]. In this study, however, PRN and Dex/PRN did not decrease the elevated cytokines in this ABPA model. In a previous study, PRN did not signi cantly reduce cytokine production regardless of infection (aspergillus fumigatus or respiratory syncytial; RS virus) and increased IL-10 and IL-12 in Df sensitized mice after RS virus infection but not after aspergillus infection [24]. The role of PRN might not to be so effective on ABPA therapy.
In previous studies, ABPA showed both of eosinophilic and neutrophilic in ammation in the airways [25,26], and which increased after withdrawal of the treatment in patients with severe asthma [29]. Macrolides have been known to have anti-in ammatory effect on bronchiectasis [30] or panbronchitis [31]. Macrolides have also been reported to reduce airway hyper-responsiveness and improve pulmonary function [32,33]. Taking these ideas together, we hypothesized that CAM might be effective to ABPA which has both of neutrophilic and eosinophilic The experimental protocol All mice were immunized twice intraperitoneally on days 1 and 14 with   The cytokine concentrations in homogenized lung of the groups. The concentrations of IL-5 and IL-13 (Th2 cytokines) and that of MIP-2 (murine cytokine corresponding to human IL-8) were signi cantly elevated in DfAf mice compared to control mice (p < 0.05, each, †: p<0.05). Dex or PRN did not suppress the measured cytokine levels. CAM suppressed the elevation of MIP-2 solely (p < 0.05, DfAf/CAM group vs DfAf group, ‡: p<0.05).
Interestingly, the combination of Dex and CAM suppressed both of the MIP-2 elevation and the IL-5 elevation (p < 0.05, each DfAf/Dex/CAM group vs DfAf/Dex group, §: p<0.05). None of Dex, PRN, or CAM affected the IL-13 concentration.