The aim of this study was to investigate the preventive effect of 1% dietary GOS on lung function and pulmonary inflammation in a murine model of HDM-induced allergic asthma. As is usually done for potentially new preventive and/or therapeutic agents, we compared the effectiveness with a golden standard reference treatment, the corticosteroid budesonide. To understand the underlying pathophysiology of the disease, animal models for allergic asthma are used. Here, a murine model for HDM-allergic asthma was used that mimics human features of asthmatic disease such as HDM-induced AHR, airway inflammation and pulmonary cytokine release [
11,
35,
36]. Airway responsiveness upon methacholine exposure in HDM-allergic mice was significantly increased when compared to HDM-PBS control mice. Moreover, total inflammatory cell numbers were significantly increased in the BALF of HDM-allergic mice when compared to control mice. Budesonide treatment showed no significant decrease on the development of AHR whereas dietary intervention with 1% GOS prevented AHR development in HDM-allergic mice. However, both interventions suppressed airway inflammation in HDM-allergic mice. Both 1% GOS and budesonide were effective in the suppression of airway inflammation and decreased the number of eosinophils and macrophages. Furthermore, 1% GOS prevented the increase of CCL17 and IL-33 and significantly decreased CCL5 and IL-13 concentrations in the lungs. To our knowledge, this is the first study to demonstrate that dietary intervention with 1% GOS during sensitization and challenge is as effective as treatment with budesonide on allergic HDM asthma symptoms in a murine model. IL-6 concentrations have been reported to be increased in serum, BALF and sputum of asthmatic patients [
12,
37]. This increase was also observed in the murine model of HDM-induced allergic asthma. Budesonide, but not 1% GOS, normalized HDM allergy-induced increase of IL-6 concentrations to the control level. This effect of budesonide is in agreement with clinical studies which demonstrate that glucocorticosteroids decrease IL-6 concentrations in BALF in asthmatic patients in association with decreased activation and recruitment of inflammatory cells in asthma [
38]. In our study, budesonide decreased the number of inflammatory cells as well. Besides being released by macrophages and T cells, IL-6 is highly expressed by epithelial cells obtained from allergic asthma patients [
39]. However, the exact role of IL-6 in asthma pathology is not fully understood and has to be further elucidated. In patients with asthma, CCL17 has been found in increased concentrations in serum and BALF [
40]. Previously, an important role for CCR4 and its ligand CCL17 in Th2 T cell recruitment has been demonstrated in asthma [
41]. Budesonide significantly decreased the HDM allergy-induced levels of CCL17, whereas 1% GOS showed a reduction of >20%. The latter is in agreement with studies from Leung
et al., who found higher concentrations of CCL17 in the serum of non-steroid-treated asthmatic children compared with steroid-treated asthmatic children [
42]. IL-33 is known to contribute to AHR since animal studies demonstrated that intranasally administered IL-33 results in an AHR-association with eosinophilia, goblet cell hyperplasia, and accumulation of IL-4, −5 and −13 in the lungs [
43,
44]. IL-33 is produced by epithelial cells after allergen stimulation, is a chemoattractant for Th2 cells and can activate mast cells to release CXCL8, IL-5, −6, and −13 [
45]. Indeed, IL-33 may be involved in human asthmatic disease, since it is increased in BALF of moderate asthma patients as compared to mild asthma patients or controls [
46]. Typically the concentration of IL-33 in the lung tissue obtained in our study was significantly enhanced in HDM-allergic and budesonide-treated mice compared to the control mice. In these budesonide-treated mice, the AHR was still evident. Indeed, Deckers
et al. also demonstrated that budesonide had no effect on IL-33 concentrations in asthmatic patients [
47]. However in HDM-allergic mice fed 1% GOS the HDM induced increase in IL-33 in lung tissue was prevented. Since IL-33 is one of the factors contributing to AHR [
43,
44], this could relate to the 1% GOS-induced abrogation of the AHR response of these mice. CCL5 is produced at high concentrations within the airway epithelium of human asthmatics and in turn will target eosinophils to the airways [
48]. In this study, both dietary 1% GOS as well as budesonide treatment showed a significant decrease in CCL5 concentrations in lung tissue of HDM-allergic mice. Dampening of pulmonary CCL5 concentrations could be the mechanism by which GOS and budesonide treatment induces an abrogation of eosinophil infiltration in the lungs of HDM-allergic mice. In humans, the release of the Th2 cytokine IL-5 leads to activation of the eosinophil/basophil lineage. Increased eosinophil cell influx and AHR are strongly associated in asthmatic patients and can be provoked after inhalation of IL-5. However, there are many studies in humans demonstrating that treatment with anti-IL-5- specific antibodies reduced the number of eosinophils in sputum and blood of mild and severe asthmatic patients, although the AHR was not affected [
49]. Thus, inflammatory pathways underlying IL-5 alone are not sufficient for the development of AHR in allergic asthma. In our study, IL-5 tended to be increased in the BALF of HDM-allergic mice whereas this did not occur after dietary intervention with 1% GOS or treatment with budesonide (Additional file
1: Figure S1). Indeed, IL-5 in BALF was positively correlated with eosinophil numbers (Additional file
1: Figure S1). The concentration of another Th2 cytokine, IL-13, in lung tissue was significantly increased in HDM-allergic mice compared to the control mice. These data are in agreement with previous studies that show a central role for IL-13 in generating the murine allergic AHR following sensitization and challenge of HDM [
50]. Many of the processes involved in allergic asthma can be directed to IL-13. Besides being secreted by Th2 cells, IL-13 is also secreted by mast cells and innate lymphoid cells. In the current study, IL-13 concentrations in lung tissue were positively correlated with BALF lymphocyte numbers, suggesting this subset to be an important source of IL-13. IL-13 also triggers macrophage and eosinophil activation which, in turn, can contribute to AHR [
51]. Furthermore, IL-13 is increased in BALF and bronchial biopsy specimens of asthmatic patients and known to be inhibited by glucocorticoids [
51,
52]. Both dietary 1% GOS as well as budesonide showed a significant decrease in IL-13 concentrations in lung homogenates of HDM-allergic mice, hereby dampening a major contributor to asthmatic inflammation. In previous studies
, Bifidobacterium breve or
Lactobacillus rhamnosus, either or not combined with specific oligosaccharides, suppressed airway inflammation in a murine model for OVA induced chronic asthma [
27,
28]. Our studies show similar effects of only GOS in an acute model for HDM induced asthma. As shown in earlier studies with dietary oligosaccharides, it is known that they have a positive effect on the composition of microbiota [
21-
23]. A potential mechanism of GOS could be that by changing the microbiota, immunomodulation via intestinal epithelial signaling occurs leading to systemic effects resulting in a decreased HDM immune response, as has been suggested by several studies [
17,
20]. In conclusion, in our study budesonide suppressed inflammatory cell numbers and cytokine concentrations of IL-6, CCL17, CCL5 and IL-13 in HDM allergic mice. However, budesonide did not modulate the HDM-allergy induced AHR and increased the pulmonary tissue concentrations of IL-33. Interestingly, dietary intervention with 1% GOS prevented the development of AHR and suppressed airway eosinophilia in HDM allergic mice. Moreover, 1% GOS prevented the increase in IL-33 and abrogated the HDM-induced CCL17, CCL5 and IL-13 release in the lungs of HDM-allergic mice. Dietary intervention with 1% GOS may be beneficial in the prevention of HDM-induced allergic asthma, and may offer a potential novel strategy with less side effects than current therapeutic treatments. However, more research is needed to demonstrate this beneficial effect. In addition, the mechanism of the immune modulating functions of 1% GOS needs to be further elucidated, as well as the most effective dose.