Artepillin C Reduces Allergic Airway Inflammation by Induction of Monocytic Myeloid-Derived Suppressor Cells

Abstract

Propolis is a natural product produced by bees that is primarily used in complementary and alternative medicine and has anti-inflammatory, antibacterial, antiviral, and antitumoral biological properties. Some studies have reported the beneficial effects of propolis in models of allergic asthma. In a previous study, our group showed that green propolis treatment reduced airway inflammation and mucus secretion in an ovalbumin (OVA)-induced asthma model and resulted in increased regulatory T cells (Treg) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) frequencies in the lungs, two leukocyte populations that have immunosuppressive functions. In this study, we evaluated the anti-inflammatory effects of artepillin C (ArtC), the major compound of green propolis, in the context of allergic airway inflammation. Our results show that ArtC induces in vitro differentiation of Treg cells and monocytic MDSC (M-MDSC). Furthermore, in an OVA-induced asthma model, ArtC treatment reduced pulmonary inflammation, eosinophil influx to the airways, mucus and IL-5 secretion along with increased frequency of M-MDSC, but not Treg cells, in the lungs. Using an adoptive transfer model, we confirmed that the effect of ArtC in the reduction in airway inflammation was dependent on M-MDSC. Altogether, our data show that ArtC exhibits an anti-inflammatory effect and might be an adjuvant therapy for allergic asthma.

Keywords: M-MDSC; allergic asthma; artepillin C; propolis; therapies.

Figure 1

ArtC isolation. (a) ¹H NMR spectrum of ArtC (CD3OD, 300 MHz); (b) chemical structure of ArtC.

Figure 2

ArtC reduces allergic airway inflammation. (a) Experimental design; (b) body weight variation of mice exposed to the allergen before and after treatment with ArtC; (c) frequency of eosinophils (EOS), lymphocytes (LYM), mononuclear cells (MON) and polymorphonuclear cells (PMN) in the BALF; (d) IL-5 in the BALF (n.d.: not detected); (e) representative images of lung inflammation (magnification 200×, scale bar 100 µM) and (f) mucus production (magnification 200×, scale bar 100 µM); (g) lung inflammation score (0—without inflammation; 1—mild; 2—moderate to severe inflammation); (h) mucus score (0—without mucus secretion; 1—mild). Data are representative of two independent experiments (n = 4–5/group/experiment), except for (d) (one representative experiment), and expressed by mean ± SD. * p < 0.05 and **** p < 0.0001.

Figure 3

ArtC induces Treg cells differentiation in vitro. (a) Gate strategy for Treg cell (CD4⁺Foxp3⁺) characterization in vitro by flow cytometry; (b) frequency of live cells (FVS780⁻) in culture of CD4⁺ T cells stimulated with ArtC versus control (CTL); (c) representative dot plot of Treg cells generated in vitro in the presence or absence of ArtC; (d) percentage of Treg cells generated in vitro. Data are representative of three independent experiments (n = 3–4/group/experiment) and expressed by mean ± SD; (e) gating strategy for Treg cell characterization in vivo; (f) representative dot plot of Treg cell in the lungs of mice exposed to the allergen and treated or not (vehicle) with ArtC; (g) frequency of Treg cells in the lungs. Data are representative of two independent experiments (n = 4–5/group/experiment) and expressed by mean ± SD. * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Figure 4

ArtC induces M-MDSC differentiation in vitro. (a) Gate strategy for M- (Ly6G⁻Ly6C⁺) and PMN-MDSC (Ly6G⁺Ly6C^int) characterization in vitro; (b) frequency of live cells (FVS780−) in culture of bone marrow cells stimulated with rIL-6, GM-CSF and ArtC versus control (CTL); (c) representative dot plot of M- and PMN-MDSC generated in vitro in the presence of ArtC (10 µM); (d) percentage of M-MDSC, and (e) PMN-MDSC. Data are representative of four independent experiments (n = 3–4/group/experiment) and expressed by mean ± SD. * p < 0.05 and **** p < 0.0001.

Figure 5

ArtC induces M-MDSC in the lungs of mice exposed to the allergen. (a) Gate strategy for Ly6G⁻Ly6C⁺ (M-MDSC) and Ly6G⁺Ly6C^int (PMN-MDSC) characterization in vivo; (b) representative dot plot of Ly6G⁻Ly6C⁺ and Ly6G⁺Ly6C^int in the lungs of mice exposed to the allergen treated with or not (Vehicle) with ArtC; (c) frequency, and (d) absolute number of Ly6G⁻Ly6C⁺ in the lungs; (e) frequency, and (f) absolute number of Ly6G⁺Ly6C^int in the lungs; (g) correlation between BALF eosinophils and lung Ly6G⁻Ly6C⁺. Data are representative of two independent experiments (n = 4–5/group/experiment) and expressed by mean ± SD. * p < 0.05 and *** p < 0.001.

Figure 5

ArtC induces M-MDSC in the lungs of mice exposed to the allergen. (a) Gate strategy for Ly6G⁻Ly6C⁺ (M-MDSC) and Ly6G⁺Ly6C^int (PMN-MDSC) characterization in vivo; (b) representative dot plot of Ly6G⁻Ly6C⁺ and Ly6G⁺Ly6C^int in the lungs of mice exposed to the allergen treated with or not (Vehicle) with ArtC; (c) frequency, and (d) absolute number of Ly6G⁻Ly6C⁺ in the lungs; (e) frequency, and (f) absolute number of Ly6G⁺Ly6C^int in the lungs; (g) correlation between BALF eosinophils and lung Ly6G⁻Ly6C⁺. Data are representative of two independent experiments (n = 4–5/group/experiment) and expressed by mean ± SD. * p < 0.05 and *** p < 0.001.

Figure 6

ArtC-induced M-MDSC reduces the allergic airway inflammation in mice. (a) Experimental design; (b) sorted M-MDSC, before cell transfer; (c) frequency and (d) number of eosinophils (EOS), lymphocytes (LYM), mononuclear cells (MON) and polymorphonuclear cells (PMN) in the BALF post cell transfer; (e) IL-4 and (f) IL-5 levels in the lungs; (g) representative dot plot of M- and PMN-MDSC in the lungs of post cell transfer; (h) percentage and (i) number of M-MDSC in the lungs; (j) representative images of lung inflammation (magnification 200×, scale bar 100 µM) and (k) mucus production (magnification 200×, scale bar 100 µM); (l) Lung inflammation score (0—without inflammation; 1—mild; 2—moderate to severe inflammation); (m) Mucus score (0—without mucus secretion; 1—mild). Data are representative of two independent experiments (n = 5–6/group/experiment), except for (e–h) (one representative experiment), and expressed by mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 6

ArtC-induced M-MDSC reduces the allergic airway inflammation in mice. (a) Experimental design; (b) sorted M-MDSC, before cell transfer; (c) frequency and (d) number of eosinophils (EOS), lymphocytes (LYM), mononuclear cells (MON) and polymorphonuclear cells (PMN) in the BALF post cell transfer; (e) IL-4 and (f) IL-5 levels in the lungs; (g) representative dot plot of M- and PMN-MDSC in the lungs of post cell transfer; (h) percentage and (i) number of M-MDSC in the lungs; (j) representative images of lung inflammation (magnification 200×, scale bar 100 µM) and (k) mucus production (magnification 200×, scale bar 100 µM); (l) Lung inflammation score (0—without inflammation; 1—mild; 2—moderate to severe inflammation); (m) Mucus score (0—without mucus secretion; 1—mild). Data are representative of two independent experiments (n = 5–6/group/experiment), except for (e–h) (one representative experiment), and expressed by mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.