Resolvin D1 and aspirin-triggered resolvin D1 promote resolution of allergic airways responses

Abstract

Asthma is a disease of airway inflammation that in most cases fails to resolve. The resolution of inflammation is an active process governed by specific chemical mediators, including D-series resolvins. In this study, we determined the impact of resolvin D1 (RvD1) and aspirin-triggered RvD1 (AT-RvD1) on the development of allergic airway responses and their resolution. Mice were allergen sensitized, and RvD1, AT-RvD1 (1, 10, or 100 ng), or vehicle was administered at select intervals before or after aerosol allergen challenge. RvD1 markedly decreased airway eosinophilia and mucus metaplasia, in part by decreasing IL-5 and IκBα degradation. For the resolution of established allergic airway responses, AT-RvD1 was even more efficacious than RvD1, leading to a marked decrease in the resolution interval for lung eosinophilia, decrements in select inflammatory peptide and lipid mediators, and more rapid resolution of airway hyperreactivity to methacholine. Relative to RvD1, AT-RvD1 resisted metabolic inactivation by macrophages, and AT-RvD1 significantly enhanced macrophage phagocytosis of IgG-OVA-coated beads in vitro and in vivo, a new proresolving mechanism for the clearance of allergen from the airways. In conclusion, RvD1 and AT-RvD1 can serve as important modulators of allergic airway responses by decreasing eosinophils and proinflammatory mediators and promoting macrophage clearance of allergen. Together, these findings identify D-series resolvins as potential proresolving therapeutic agents for allergic responses.

FIGURE 1

RvD1 decreases allergic lung inflammation. (A). Mice were sensitized and challenged with OVA prior to one dose of RvD1 (10 ng, i.v.) or vehicle 1 hour before BAL on protocol day 18 (inset). Total leukocytes and leukocyte subsets were enumerated (see Methods). (B) A second cohort of sensitized and challenged mice received RvD1 (1–100 ng, i.v.) or vehicle 30 minutes prior to OVA aerosol challenge on 4 successive days (protocol days 14–17) (inset) and BALF leukocytes were enumerated on protocol day 18. The dashed black line indicates the cells from OVA naïve, non-allergic control mice. Results represent the mean ± s.e.m of two or more independent experiments with three or more mice per group per experiment. * P < 0.05 vs. vehicle.

FIGURE 2

RvD1 prevents the development of allergic airway responses. Allergen sensitized mice received RvD1 (10 ng, i.v.) or vehicle 30 minutes prior to OVA aerosol challenge on 4 successive days (protocol day 14–17). On protocol day 18, (A) lung tissue inflammation was assessed by hematoxylin and eosin staining (H&E, original magnification, 200×) and the presence of mucus metaplasia was determined by periodic acid–Schiff reagent (original magnification, 400×) staining of lung sections. Arrows indicate mucus (magenta) containing goblet cells; Br, bronchus. Quantitative measures of (B) inflammation and (C) mucus metaplasia were determined in lung sections (see Methods). (D) BALF levels of peptide and lipid mediators were measured by immunoassay and (E) lung IκBα was determined by Western blotting (see Methods). (F) Mean lung resistance (% of baseline) after aerosolized methacholine was determined by Flexivent (see Methods). (G) Representative images for identification of ALX/FPR2 receptors by imunohistochemistry in lung sections from control mice and protocol days 18 and 21 (magnification: 400×). (H) Relative abundance of lung ALX/FPR2 staining in the lung. Results represent the mean ± s.e.m of two or more independent experiments with three or more mice per group per experiment. * P< 0.05 vs. vehicle. ^# P< 0.05 vs. control.

FIGURE 3

RvD1 and AT-RvD1 promote the resolution of airway inflammation. (A) RvD1 (10 or 100 ng), AT-RvD1 (100 ng) or vehicle was administered (i.v.) to OVA sensitized and challenged mice on protocol days 18–20 (inset) after cessation of OVA exposure, and (B) BALF total cells, eosinophils, macrophages and lymphocytes were evaluated on protocol day 21. The dashed black line indicates the cells from OVA naïve, non-allergic control mice. (C) The resolution interval (R_i) for RvD1 (100ng) and (D) AT-RvD1 (100 ng) for BALF eosinophils was determined. (E) RvD1 (100 ng), AT-RvD1 (100 ng) or vehicle was administered directly to the airway (intranasal) in OVA sensitized and challenged mice on protocol days 18–20 after cessation of OVA exposure. BALF total cells, eosinophils, macrophages and lymphocytes were evaluated on protocol day 21. Results represent the mean ± S.E.M. for two or more independent experiments with three or more mice per group per experiment. * P < 0.05 vs. vehicle, ^#P < 0.05 vs. RvD1.

FIGURE 4

Impact of RvD1 and AT-RvD1 on the resolution of airway inflammation, mucus and hyper-reactivity. (A) Lung tissue sections were obtained at protocol day 21 from mice given RvD1 (100 ng), AT-RvD1 (100 ng) or vehicle and stained with hematoxylin and eosin (H&E, original magnification, 200×) or periodic acid–Schiff reagent (PAS, original magnification, × 400) (see Methods). Arrows indicate examples of mucus (magenta) containing goblet cells; Br, bronchus. (B) Quantitative analyses of lung inflammation and bronchial PAS-positive cells in lung sections were performed (see Methods). (C) Airway hyper-responsiveness to aerosolized methacholine was assessed on day 21 by measuring the mean lung resistance (% of baseline) (see Methods). Results represent the mean ± s.e.m for two or more independent experiments with three or more mice per group per experiment. *P < 0.05 vs. vehicle.

FIGURE 5

RvD1 and AT-RvD1 selectively regulate inflammatory mediators during resolution of allergic inflammation. (A–I) BALF levels of peptide and lipid mediators were measured in OVA naïve, non-allergic control mice (white) and on protocol day 21 in materials obtained from mice given RvD1 (100 ng, grey), AT-RvD1 (100 ng, black) or vehicle (hatched) (see Methods). (M) Lung tissue was obtained on protocol day 21 during resolution and was subjected to Western blotting analyses for IκBα (see Methods). Results represent the mean ± s.e.m for two or more independent experiments with three or more mice per group per experiment. *P< 0.05 vs. vehicle. nd, not detected.

FIGURE 6

AT-RvD1 resists metabolic inactivation by macrophages and stimulates macrophage clearance of allergen. Murine alveolar macrophages cells (AMJ2-C8) were incubated (30 minutes, 37°C) in the presence of RvD1 (in some cases with indomethacin) or AT-RvD1. Lipids were extracted and (A) RvD1 metabolites were identified and quantitated by LC-MS/MS analyses (see Methods). (B) Values are expressed as the relative percent 17-oxo-RvD1 metabolite relative to the total starting compound. (C) Macrophage phagocytosis of allergen was determined using rabbit anti-OVA IgG-coated beads (2 µm) that are detectable by light microscopy. In non-permeabilized cells, a fluorophore tagged antibody (FITC-conjugated goat anti-rabbit antibody) was used to distinguish adherent (fluorescent) from internalized (non-fluorescent) beads. A phagocytosis index was calculated after 15 min in the presence of RvD1, AT-RvD1 (0.1, 10 and/or 100 nM), vehicle (V) or media alone (M) using (D) AMJ2-C8 cells in vitro or (E,F) alveolar macrophages (mAlvMacs) ex vivo that were isolated from BALF of (E) control or (F) OVA-sensitized and –challenged mice (Protocol day 21) (see Methods). The dashed black line indicates the baseline phagocytosis index. (G) The phagocystosis index was determined in vivo in OVA-sensitized or control mice 15 min after i.p. injection of OVA IgG-coated beads. In some animals, RvD1 (100 ng), AT-RvD1 (100 ng) or vehicle (0.1% ethanol) were given i.p. 5 min before introduction of the beads. Results represent the mean ± S.E.M. for two or more independent experiments. *P< 0.05 vs. vehicle, ^#P < 0.05 vs. RvD1.