Mitoquinone mesylate attenuates pathological features of lean and obese allergic asthma in mice

Abstract

Obesity is associated with severe, difficult-to-control asthma, and increased airway oxidative stress. Mitochondrial reactive oxygen species (mROS) are an important source of oxidative stress in asthma, leading us to hypothesize that targeting mROS in obese allergic asthma might be an effective treatment. Using a mouse model of house dust mite (HDM)-induced allergic airway disease in mice fed a low- (LFD) or high-fat diet (HFD), and the mitochondrial antioxidant MitoQuinone (MitoQ), we investigated the effects of obesity and ROS on HDM-induced airway inflammation, remodeling, and airway hyperresponsiveness (AHR). Obese allergic mice showed increased lung tissue eotaxin, airway tissue eosinophilia, and AHR compared with lean allergic mice. MitoQ reduced airway inflammation, remodeling, and hyperreactivity in both lean and obese allergic mice, and tissue eosinophilia in obese-allergic mice. Similar effects were observed with decyl triphosphonium (dTPP⁺), the hydrophobic cationic moiety of MitoQ lacking ubiquinone. HDM-induced oxidative sulfenylation of proteins was increased particularly in HFD mice. Although only MitoQ reduced sulfenylation of proteins involved in protein folding in the endoplasmic reticulum (ER), ER stress was attenuated by both MitoQ and dTPP⁺ suggesting the anti-allergic effects of MitoQ are mediated in part by effects of its hydrophobic dTPP⁺ moiety reducing ER stress. In summary, oxidative signaling is an important mediator of allergic airway disease. MitoQ, likely through reducing protein oxidation and affecting the UPR pathway, might be effective for the treatment of asthma and specific features of obese asthma.

Keywords: ER stress; MitoQ; obese allergic asthma.

Graphical abstract

Figure 1.

MitoQ attenuates acute responses to HDM: A: schematic for the acute HDM/MitoQ response. B–F: BAL total inflammatory cells, BAL macrophages, BAL neutrophils, BAL eosinophils, and BAL lymphocytes. G–K: analysis of lung tissue protein levels of innate cytokines IL-1β, IL-33, Eotaxin, CCL20, and mRNA levels of Th2 cytokine Il-4. P values <0.05 were regarded as discovery or statistically significant. Error bars ± SE. *Significant differences between PBS and HDM groups and number sign (#) indicates significant differences between HDM and HDM + MQ groups. HDM, house dust mite.

Figure 2.

Tissue eosinophilia is elevated in HDM-challenged HFD mice and is attenuated with MitoQ treatment. A: representative images of EPX immunohistochemistry. Scale bars: 100 µm. B: quantification for the EPX staining. C: ELISA for EPX in the lung tissue. D: lung tissue IL-5 measured by ELISA. P values < 0.05 were regarded as discovery or statistically significant. Error bars ± SE. *Significant differences between PBS and HDM groups. #Significant differences between HDM and HDM + MQ groups. EPX, eosinophil peroxidase; HDM, house dust mite; HFD, high-fat diet.

Figure 3.

Mucus production is increased by HDM and decreased by MitoQ. A: representative images of PAS staining. Scale bars: 100 µM. B: quantification of PAS staining. C and D: qRT-PCR of total lung RNA for genes involved in mucus production: Gob5 and Agr2. P values < 0.05 were regarded as discovery or statistically significant. Error bars ± SE. *Significant differences between PBS and HDM groups. #Significant differences between HDM and HDM + MQ groups. HDM, house dust mite; PAS, Periodic acid Schiff.

Figure 4.

HDM-induced fibrosis is reduced by MitoQ treatment. A: Masson’s Trichrome staining (MT) for collagen. Scale bars: 100 µM. B: MT staining quantification. C and D: BAL TGF-β and BAL periostin levels. P values < 0.05 were regarded as discovery or statistically significant. Error bars ± SE. *Significant differences between PBS and HDM groups. #Significant differences between HDM and HDM + MQ groups. HDM, house dust mite.

Figure 5.

Analysis of methacholine-induced AHR in mice. A: central airway resistance (R_n) in response to methacholine. B: tissue resistance or damping G response to methacholine. C: tissue elastance H response to methacholine. P values < 0.05 were regarded as discovery or statistically significant. Error bars ± SE. *Significant differences between PBS and HDM groups. #Significant differences between HDM and HDM + MQ groups. AHR, airway hyperresponsiveness; HDM, house dust mite.

Figure 6.

A and C: DCP Bio-1 pull-down samples from various conditions (PBS, HDM, HDM + MitoQ from lean and obese groups) were separated on SDS-PAGE. After in-gel digestion and mass spectrometry, protein abundances (reflecting sulfenylation) were analyzed by spectral counting and represented in volcano plots and heat maps. Log₂ ratios (vs. PBS) of the total spectral counts for those proteins with increased sulfenylation in HDM (P < 0.05) are represented in heat maps, with protein accessions listed according to the corresponding average log2 fold-changes (from high to low). B and D: proteins with increased sulfenylation on HDM treatment and decreased with HDM + MitoQ treatment are highlighted in red in the volcano plot, whereas those with sulfenylation upregulated by HDM, but not subsequently diminished by MitoQ, are highlighted in green. Cutoffs of HDM/PBS at twofold (log₂ −2 = −1 and log₂ 2 = 1) and P value at 0.05 (–log₁₀ 0.05 = 1.301) are indicated by dotted line(s) on the x- and y-axis, respectively. E: validation of ER-stress-related proteins: cysteine sulfenylation of GRP94 and PDIA3. HDM sample without DCP-Bio1 labeling was used as a negative control. F: comparison of cysteine sulfenylation of GRP94 and PDIA3 between MitoQ and dTPP⁺. G: detection of ER stress. Activation of ER stress through the IRE, ATF6, and PERK pathways was observed by detection of phospho-IRE1, ATF6⁵⁰, and phospho-EIF2α. β-Tubulin was used as the loading control. ER, endoplasmic reticulum; HDM, house dust mite.