The Combination Therapy of Dietary Galacto-Oligosaccharides With Budesonide Reduces Pulmonary Th2 Driving Mediators and Mast Cell Degranulation in a Murine Model of House Dust Mite Induced Asthma

Abstract

Background: Dietary non-digestible galacto-oligosaccharides (GOS) suppress allergic responses in mice sensitized and challenged with house dust mite (HDM). Budesonide is the standard therapy for allergic asthma in humans but is not always completely effective. Aim: To compare the efficacy of budesonide or different doses of GOS alone or with a combination therapy of budesonide and GOS on HDM-allergic responses in mice. Methods:BALB/c mice were sensitized and challenged with HDM, while fed a control diet or a diet supplemented with 1 or 2.5 w/w% GOS, and either or not oropharyngeally instilled with budesonide. Systemic and local inflammatory markers, such as mucosal mast cell protease-1 (mMCP-1) in serum, pulmonary CCL17, CCL22, and IL-33 concentrations and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) were determined. Results: Budesonide or GOS alone suppressed the number of eosinophils in the BALF of HDM allergic mice whereas budesonide either or not combined with GOS lowered both eosinophil and lymphocyte numbers in the BALF of HDM-allergic mice. Both 1 w/w% and 2.5 w/w% GOS and/or budesonide suppressed serum mMCP-1 concentrations. However, budesonide nor GOS alone was capable of reducing Th2 driving chemokines CCL17, CCL22 and IL-33 protein levels in supernatants of lung homogenates of HDM allergic mice, whereas the combination therapy did. Moreover, IL-13 concentrations were only significantly suppressed in mice treated with budesonide while fed GOS. A similar tendency was observed for the frequency of GATA3⁺CD4⁺ Th2 and CD4⁺RORγt⁺ Th17 cells in the lungs of the allergic mice. Conclusion: Dietary intervention using GOS may be a novel way to further improve the efficacy of anti-inflammatory drug therapy in allergic asthma by lowering Th2 driving mediators and mast cell degranulation.

Keywords: allergy; asthma; budesonide; galacto-oligosaccharides; house dust mite.

Figure 1

Induction of house dust mite allergy in mice. BALB/c mice were intranasally (i.n.) sensitized with PBS or house dust mite (HDM) on day 0 and challenged on days 7 to 11 intranasally with PBS or HDM. Mice were fed control diet (AIN93G, contr) or 1 w/w% or 2.5 w/w% GOS from day −14 to 14 and either or not oropharyngeally instilled with budesonide on days 7, 9, 11, and 13. All mice were sacrificed on day 14.

Figure 2

Combination of dietary GOS and budesonide effectively reduces eosinophilic inflammation in the lungs of HDM-allergic mice. Inflammatory cell influx in the BALF of house dust mite allergic mice. Total BAL cells (A), absolute number of lymphocytes (B), eosinophils (C). Results are shown as mean ± SEM. Statistical significance of differences was tested using post hoc Bonferroni's multiple comparisons test after One-Way ANOVA. ^*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the HDM-contr group, + P < 0.05 n = 8–9 mice/group. Representative photomicrographs of the lungs stained with H&E. PBS- control diet (D), HDM-control diet (E), HDM- control diet and budesonide treatment (F), HDM-1 w/w% GOS diet (G), HDM-1 w/w% GOS diet and budesonide treatment (H), inflammation score of histological photomicrographs; the percentage of tissue surface area that was infiltrated with inflammatory cells was scored blinded as follows: score 0 no inflammation (0%), score 1 mild inflammation (>0– < 30%), score 2 moderate inflammation (>30– < 60%), score 3 severe inflammation (>60–100%) (I). Results are shown as mean ± SEM. Statistical significance of differences was tested using Kruskall Wallis test. Magnification 200x, BALF n = 8–9 mice per group, histology n = 5 mice/group. PBS, PBS- sensitized and PBS- challenged mice (white bars); HDM, HDM -sensitized and challenged mice (gray bars); Contr, control diet; GOS, 1 w/w% GOS or 2.5 w/w% GOS diet; Bud, budesonide.

Figure 3

Combination of dietary GOS and budesonide suppresses mMCP-1 serum concentration and Th2 driving mediators in lung homogenates of HDM allergic mice. mMCP-1 (A) (pg/mL in serum) and IL-33 (B), CCL17 (C), CCL22 (D) (pg/mg protein in supernatant of lung homogenates) concentrations were measured. Statistical significance of differences was tested using post hoc Bonferroni's multiple comparisons test after One-Way ANOVA. ^*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the HDM-contr group, +P < 0.05, ++P < 0.01, + + ++P < 0.0001, and ##P < 0.01, ###P < 0.001 compared to the PBS-contr group, n = 6 mice/group. Results are shown as mean ± SEM. PBS: PBS–sensitized and–challenged mice (white bars), HDM, HDM-sensitized and–challenged mice (gray bars). Contr, control diet; GOS, 1 w/w% GOS or 2.5 w/w% GOS diet; Bud, budesonide. Results are shown as mean ± SEM.

Figure 4

Combination of dietary GOS and budesonide reduces IL-13 concentrations in the lungs of HDM-allergic mice. IL-13 concentrations were measured in supernatants of lung homogenates (pg/mg protein) (A) and in supernatants of lung cell suspensions upon ex vivo HDM restimulation (pg/mL) (B). Correlation of IL-13 concentration in lung homogenates and the number of lymphocytes between the HDM-groups (C). Results are shown as mean ± SEM. Statistical significance of differences was tested using post hoc Bonferroni's multiple comparisons test after One-Way ANOVA. ^*P < 0.05, ***P < 0.001, ****P < 0.0001 compared to the HDM-contr group n = 6 mice/group. Correlation was assessed using the Spearman correlation test. PBS, PBS–sensitized and–challenged mice (white bars); HDM, HDM-sensitized and–challenged mice (gray bars). Contr, control diet; GOS, 1 w/w% GOS or 2.5 w/w% GOS diet; Bud, budesonide.

Figure 5

The frequency of activated Th cells and Th2 and Th17 cells decreases after dietary intervention with 2.5 w/w% GOS combined with budesonide. Representative dot plots and histograms of gating strategy of lung T helper cell subsets (E). Lymphocytes were gated based on FSC-SSC pattern, and T helper cells were gated based on expression of CD4. Within the CD4⁺ population the frequency of GATA3 (Th2 cells), RORγt (Th17 cells) and Tbet (Th1 cells) was analyzed. In the histogram the blue line represents FMO control, red line isotype control and green line MFI of the specific antibody. Percentage of activated CD4⁺ cells (A), GATA3⁺ of CD4⁺ cells (B), RORγt⁺ of CD4⁺ cells (C), and Tbet⁺ of CD4⁺ cells (D) was calculated. Results are shown as mean ± SEM. Statistical significance of differences was tested using post hoc Bonferroni's multiple comparisons test after One-Way ANOVA. ^*P < 0.05, ^****P < 0.0001 compared to the HDM-contr group, +P < 0.05, ++P < 0.01, n = 6 mice/group.

Figure 6

Overview of the effects of the dietary intervention with GOS combined with glucocorticosteroid budesonide treatment. After the initial exposure to HDM mMCP-1 is released by mast cells, and IL-33 is known to be secreted by the airway epithelium, which can also activate mast cells as well as DC and ILC2. Combination of the GOS diet with budesonide treatment reduced mMCP-1 and IL-33 concentrations. CCL17 and CCL22 are secreted by activated DC, which can differentiate naïve T cells into Th2 cells and are chemo-attractants for Th2 cells. The combination therapy significant suppressed the production of both chemokines. Th2 cells as well as ILC2 and mast cells are able to produce IL-13. Concentrations of IL-13 were reduced after the treatment of both GOS and budesonide. The number of lymphocytes and eosinophils was decreased by treatment with budesonide, whereas GOS alone suppressed the number of eosinophils. The combination of dietary GOS with budesonide treatment effectively suppressed both leukocyte subtypes. Only dietary intervention with GOS in combination with budesonide tended to suppress the Th2 and Th17 frequency in lung cell suspensions. Small arrows, tendency to reduce; big arrows, significant reduced.