Dimethyl fumarate abrogates dust mite-induced allergic asthma by altering dendritic cell function

Abstract

Introduction: Allergic asthma is the most common inflammatory disease of upper airways. Airway dendritic cells (DCs) are key antigen presenting cells that regulate T helper 2 (Th2)-dependent allergic inflammation. Recent studies have shown critical role of airway DCs in the induction of Th2-mediated allergic inflammation and are attractive therapeutic targets in asthma. However, molecular signaling mechanism that regulate DCs function to Th2 immune responses are poorly understood. Here we aim to evaluate the immunomodulatory effect of dimethyl fumarate (DMF), an FDA approved small molecule drug, in the house dust mite (HDM)-induced experimental model of allergic asthma.

Methods: DMF was administered intranasally in the challenge period of HDM-induced murine model of experimental asthma. Airway inflammation, airway hyperreactivity, Th2/Th1 cytokine were assessed. The effect of DMF on DC function was further evaluated by adoptive transfer of HDM-pulsed DMF treated DCs to wild-type naïve mice.

Results: DMF treatment significantly reduced HDM-induced airway inflammation, mucous cell metaplasia, and airway hyperactivity to inhaled methacholine. Mechanistically, DMF interferes with the migration of lung DCs to draining mediastinal lymph nodes, thereby attenuates the induction of allergic sensitization and Th2 immune response. Notably, adoptive transfer of DMF treated DCs to naïve mice with HDM challenge similarly reduces the features of allergic asthma.

Conclusion: This identifies a novel function of DMF on DC-mediated adaptive immune responses in the setting of HDM-induced airway inflammation. Taken together, our results offer a mechanistic rationale for DMF use to target DCs in local lung environment as antiasthmatic therapy.

Keywords: allergic asthma; dendritic cells; dimethyl fumarate; house dust mite.

Figure 1

Effect of local DMF treatment on asthma features. A, Mice were sensitized with two i.p. injections of 100 μg HDM on days 0 and 4 and challenged on days 8, 11, and 12 by intranasal administration of HDM (100 μg) before harvest and endpoint analysis on day 14. Thirty minutes before each HDM challenge, mice received an i.n. administration of vehicle or DMF (dosage at 0.5 mg/kg bwt) in volume of 40 µL). B, The number of total BALF inflammatory cells and inflammatory cell types (Eos, AM, PMN, and Lym from vehicle, HDM, or DMF treated mice (n = 8‐10 mice, *P < .01, vehicle/HDM‐challenged versus Vehicle/HDM/DMF, one‐way ANOVA with Sidak's multiple comparison test). C, Representative lung histology sections stained with H&E or PAS are shown. Scale bars = 100 μm for the ×100 and ×600 images. D, BALF levels of CC‐chemokine ligands from mice treated or not with DMF (n = 5‐10 mice, *P < .05, or P = NS, unpaired t‐test). Results shown are pooled data from two independent experiments. E,F, Plasma levels of HDM‐specific IgE and IgG1 (n = 9‐10 mice, *P < .01, Mann–Whitney test, vehicle/HDM‐challenged versus DMF‐treated mice). G, Airway resistance (cm H₂O per mL/s) to increasing dosage of inhaled methacholine from vehicle, HDM‐challenged or DMF treated mice (n = 8‐10 mice) (*P < .05, two‐way ANOVA). AM, alveolar macrophages; DMF, dimethyl fumarate; Eos, eosinophils; HDM, house dust mite; H&E, hematoxylin and eosin; Lym, lymphocytes; PAS, periodic acid‐Schiff; PMN, neutrophil

Figure 2

Effect of local DMF treatment on lung myeloid cell distribution. A, Changes of myeloid‐cell subsets during HDM‐induced airway inflammation and DMF treatment. Single cell suspension of enzymatically digested mouse lungs were prepared and myeloid‐cell subsets were identified and enumerated. Difference between groups were compared using one‐way ANOVA with Sidak's multiple comparison test (*P < .01 or P = NS, vehicle/HDM‐challenged versus vehicle/HDM/DMF). B, Enumeration of CD11b⁺ DCs, (C) T and B cells from lung draining mLNs or pLNs, and (D) CD25⁺/FoxP3⁺ Tregs (CD4⁺ and CD8⁺) in draining mLNs from HDM‐challenged and DMF treated mice. *P < .05 or P = NS, vehicle/HDM‐challenged versus vehicle/HDM/DMF, one‐way ANOVA with Sidak's multiple comparison test). Data are representatives of at least two independent experiments and represents means ± SEMs (n = 8‐10 mice). DMF, dimethyl fumarate; HDM, house dust mite; mLV, mediastinal lymph node; pLN, peripheral lymph node

Figure 3

Local DMF administration attenuates CD4⁺ Th2‐cytokine producing cells in lungs. A, The total number of CD4⁺ T cells, as well as (B) IL‐4⁺/CD4⁺, IL‐5⁺/CD4⁺, IL‐13⁺/CD4⁺, and IFN‐γ⁺/CD4⁺ T cells in lung, was quantified by flowcytometry (n = 9‐10 mice, *P < .01 or P = NS, Mann–Whitney test, vehicle/HDM‐challenged versus DMF‐treated mice). Results are pooled data from at least two independent experiments. DMF, dimethyl fumarate; HDM, house dust mite

Figure 4

Effect of local DMF administration on lung DC migration to the lung draining mLNs. On day 0, mice were administered with HDM extract (100 μg) labeled with Alexa Fluor 647 with or without DMF (10 μg) treatment. A, MFI and (B,C) percent of HDM‐AF647⁺ CD11b⁺ and CD11b⁻ DCs from lung and draining mLNs were enumerated 24 hours after installation of labeled HDM. SiglecF⁻/CD11c⁺/MHCII^hi/AF647⁺ DCs were gated for evaluation (n = 8‐12 mice per group, *P < .01, or P = NS, unpaired t‐test, vehicle/HDM‐challenged versus DMF‐treated mice). Data represents means ± SEMs. DC, dendritic cell; DMF, dimethyl fumarate; HDM, house dust mite

Figure 5

The adoptive transfer of HDM‐pulsed CD11c⁺ BMDCs treated with DMF have an impaired ability to induce allergen‐mediated airway inflammation. A, DMF treated BMDCs were pulsed ex vivo with vehicle or HDM (100 µg/mL) for 16 hours. CD11c⁺ DCs (0.1 × 10⁶) were adoptively transferred to naïve Balb/c recipient mice by means of intranasal administration on day 0, and intranasal HDM challenges (50 µg) were administered on day 11 through day 13, to all recipient mice before endpoint analysis on day 15. B, Enumeration of BALF inflammatory cell subtypes in recipient mice by flow cytometry. BALF lymphocytes (lym) were identified as CD3⁺/CD45R⁺/MHCII⁻ cells, and the CD3⁻/CD45R⁻ cell population were gated as CD11c⁺/MHCII⁺/F4/80⁺ alveolar macrophages (AM); F4/80⁻/SSC ^hi/CCR3⁻ neutrophil (PMN); and SSC ^hi/MHC II⁻/CCR3⁺ eosinophil (Eos) (n = 8‐16 mice per group). *P < .05, or P = NS, HDM‐pulsed DMF treated versus HDM‐pulsed, or HDM‐pulsed versus vehicle, one‐way ANOVA with Sidak's multiple comparison test. C, BALF levels of CCL24 from recipient mice that received HDM‐pulsed DMF‐treated DCs or HDM‐pulsed untreated DCs. *P < .05, HDM‐pulsed DMF treated versus HDM‐pulsed, unpaired t‐test, (n = 4‐8 mice per group). D, Representative histologic lung sections stained with H&E and PAS. Scale bars = 100 μm for the ×100 and ×600 images. E, Quantitation of PAS⁺ mucous cell metaplasia. F, Lung myeloid cells in recipient mice sensitized with HDM‐pulsed DMF‐treated DCs or HDM‐pulsed untreated DCs. Results are representative of least two independent experiments and expressed as means ± SEMs (n = 4). Difference between groups were compared using one‐way ANOVA with Sidak's multiple comparison test (*P < .01 or P = NS, HDM‐pulsed DMF treated versus HDM‐pulsed). DC, dendritic cell; DMF, dimethyl fumarate; H&E, hematoxylin and eosin; HDM, house dust mite

Figure 6

DMF treated DCs induces Th2 immune responses. The total number of (A) lung CD4⁺ T cells and (B) CD4⁺/CD25⁺/FoxP3⁺ Tregs in mediastinal LNs, as well as (C) IL‐4⁺/CD4⁺, (D) IL‐5⁺/CD4⁺, (E) IL‐13⁺/CD4⁺, and (F) IFN‐γ⁺/CD4⁺ T cells from recipient mice lungs that received HDM‐pulsed DMF‐treated or untreated DCs (n = 4‐8 mice per group). Results are representative of at least two independent experiments. One‐way ANOVA with Sidak's multiple comparison test (*P < .01 or P = NS, HDM‐pulsed DMF treated versus HDM‐pulsed). DC, dendritic cell; DMF, dimethyl fumarate; HDM, house dust mite; Th2, T helper 2