Effect of a chemical chaperone, tauroursodeoxycholic acid, on HDM-induced allergic airway disease

Abstract

Endoplasmic reticulum (ER) stress-induced unfolded protein response plays a critical role in inflammatory diseases, including allergic airway disease. However, the benefits of inhibiting ER stress in the treatment of allergic airway disease are not well known. Herein, we tested the therapeutic potential of a chemical chaperone, tauroursodeoxycholic acid (TUDCA), in combating allergic asthma, using a mouse model of house dust mite (HDM)-induced allergic airway disease. TUDCA was administered during the HDM-challenge phase (preventive regimen), after the HDM-challenge phase (therapeutic regimen), or therapeutically during a subsequent HDM rechallenge (rechallenge regimen). In the preventive regimen, TUDCA significantly decreased HDM-induced inflammation, markers of ER stress, airway hyperresponsiveness (AHR), and fibrosis. Similarly, in the therapeutic regimen, TUDCA administration efficiently decreased HDM-induced airway inflammation, mucus metaplasia, ER stress markers, and AHR, but not airway remodeling. Interestingly, TUDCA administered therapeutically in the HDM rechallenge regimen markedly attenuated HDM-induced airway inflammation, mucus metaplasia, ER stress markers, methacholine-induced AHR, and airway fibrotic remodeling. These results indicate that the inhibition of ER stress in the lungs through the administration of chemical chaperones could be a valuable strategy in the treatment of allergic airway diseases.

Keywords: ER stress; airway hyperresponsiveness; asthma; house dust mite; inflammation.

Copyright © 2016 the American Physiological Society.

Figures

Fig. 1.

Preventive regimen of tauroursodeoxycholic acid (TUDCA) attenuates house dust mite (HDM)-induced airway inflammation. A: schematic representing the time points of HDM or PBS instillation and TUDCA treatment. HDM (25 μg/mouse) was instilled intranasally while TUDCA (0.5, 1, and 5 mg/kg body wt) was administered via nasopharynx during the HDM challenge phase. B–F: analysis of inflammatory and immune cells in the bronchoalveolar lavage fluid (BALF). Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-untreated HDM-challenged mice. ‡P < 0.05 compared with vehicle-treated HDM-challenged mice. §P < 0.05 compared with mice treated with 0.5 mg/kg TUDCA. G–M: analysis of inflammatory cytokines and chemokines in lung lysates. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-untreated HDM-challenged mice. ‡P < 0.05 compared with vehicle-treated HDM-challenged mice.

Fig. 2.

Preventive regimen of TUDCA inhibits HDM-induced endoplasmic reticulum (ER) stress and protects from methacholine-induced airway hyperresponsiveness (AHR). A–D: mRNA expression analysis of ER stress marker genes in whole lung lysates of vehicle-treated and TUDCA-treated HDM-challenged mice. GAPDH served as housekeeping gene, and data were normalized to GAPDH. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. E–G: analysis of methacholine-induced AHR in mice. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls and #P < 0.05 compared with vehicle-treated or untreated HDM-challenged mice.

Fig. 3.

Preventive regimen of TUDCA downregulates HDM-induced increases in mucus genes and mitigates mucus metaplasia. A–C: mRNA expression analysis of the genes associated with mucus production and secretion, in whole lung lysates of vehicle-treated and TUDCA-treated HDM-challenged mice. Data were normalized to the housekeeping gene Gapdh. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls and #P < 0.05 compared with vehicle-treated HDM-challenged mice. D: representative images of periodic acid-Schiff (PAS)-stained lung tissue sections of vehicle-treated or untreated and TUDCA (1 mg/kg body wt dose)-treated HDM-challenged (×20 magnification; scale bars = 50 μm) mice and the quantification of percentage of area that positively stained for PAS staining. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-untreated HDM-challenged mice. ‡P < 0.05 compared with vehicle-treated HDM-challenged mice.

Fig. 4.

Preventive regimen of TUDCA decreases HDM-induced airway fibrosis. A: measurement of collagen by hydroxyproline assay. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls and #P < 0.05 compared with vehicle-untreated HDM-challenged mice. B: representative images of α-smooth muscle actin (α-SMA)-stained lung tissue sections of vehicle-treated or untreated and TUDCA (1 mg/kg body wt dose)-treated HDM-challenged mice (×20 magnification; scale bars = 50 μm) and the quantification of percentage of area that positively stained for α-SMA immunostaining. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls and #P < 0.05 compared with vehicle-untreated HDM-challenged mice.

Fig. 5.

Therapeutic regimen of TUDCA decreases HDM-induced airway infiltration of inflammatory cells and decreases inflammatory cytokines and chemokines. A: schematic representing the time points of HDM or PBS instillation and TUDCA treatment. HDM (25 μg/mouse) was instilled intranasally while TUDCA (0.25, 0.75, and 5 mg/kg body wt) was administered via nasopharynx after the HDM challenge phase. B–F: analysis of inflammatory and immune cells in the BALF. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with mice treated with 0.25 mg/kg TUDCA. §P < 0.05 compared with mice treated with 5 mg/kg TUDCA. G–J: ELISA for cytokines and chemokines. Data are means ± SE of 4–6 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with mice treated with 0.25 mg/kg TUDCA.

Fig. 6.

Therapeutic regimen of TUDCA attenuates HDM-induced ER stress and AHR. A: Western blot analysis for ER stress markers in whole lung lysates of vehicle-treated or untreated and TUDCA-treated HDM-challenged mice. β-Actin was used as a loading control. B–E: protein bands were subjected to densitometry, and fold change is represented after normalizing to β-actin. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with HDM-challenged mice. ‡P < 0.05 compared with mice treated with 0.25 mg/kg TUDCA. F–H: analysis of methacholine-induced AHR in mice. Data are means ± SE of 8–10 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice.

Fig. 7.

Therapeutic regimen of TUDCA downregulates the expression of HDM-induced mucin genes and mucus production. A–C: mRNA analysis of the genes associated with mucus production and secretion in whole lung lysates of vehicle-treated and TUDCA-treated HDM-challenged mice. Data are means ± SE of 5–6 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with mice treated with 0.25 mg/kg TUDCA. D: representative images of PAS-stained lung tissue sections of vehicle-treated or untreated and TUDCA-treated HDM-challenged mice (×20 magnification; scale bars = 50 μm) and the quantification of percentage of area that positively stained for PAS staining. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with mice treated with 0.25 mg/kg TUDCA. §P < 0.05 compared with mice treated with 5 mg/kg TUDCA.

Fig. 8.

Therapeutic regimen of TUDCA does not decrease HDM-induced airway fibrosis. A: representative images of α-SMA-stained lung tissue sections of vehicle-treated and TUDCA-treated HDM-challenged mice (×20 magnification; scale bars = 50 μm) and the quantification of percentage of area that positively stained for α-SMA immunostaining. Statistical differences were not significant between groups. B: measurement of collagen by hydroxyproline assay. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. Statistical differences were not significant between vehicle-treated and TUDCA-treated HDM-challenged mice.

Fig. 9.

Therapeutic regimen of TUDCA decreases HDM-induced airway infiltration of inflammatory cells and decreases inflammatory cytokines and chemokines, regardless of HDM rechallenge. A: schematic representing the time points of HDM or PBS instillation and TUDCA treatment. HDM (25 μg/mouse) was instilled intranasally while TUDCA (0.75 mg/kg body wt) was administered via nasopharynx after the HDM challenge phase. B–F: analysis of inflammatory and immune cells in the BALF. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with HDM-challenged mice treated with 0.75 mg/kg TUDCA. G–M: ELISA for cytokines and chemokines. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with HDM-challenged mice treated with 0.75 mg/kg TUDCA.

Fig. 10.

Therapeutic regimen of TUDCA attenuates HDM-induced ER stress and AHR, regardless of HDM rechallenge. A–D: mRNA expression analysis of ER stress marker genes in lung lysates of vehicle-treated and TUDCA-treated HDM-challenged mice. Data were normalized to the housekeeping gene GAPDH. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. E–G: analysis of methacholine-induced AHR in mice. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls and #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with HDM-challenged mice treated with 0.75 mg/kg TUDCA.

Fig. 11.

Therapeutic regimen of TUDCA downregulates the expression of HDM-induced mucin genes and mucus production, regardless of HDM rechallenge. A–C: mRNA analysis of the genes associated with mucus production and secretion in whole lung lysates of vehicle-treated and TUDCA-treated HDM-challenged mice. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with HDM-challenged mice treated with 0.75 mg/kg TUDCA. D: representative images of PAS-stained lung tissue sections of vehicle-treated and TUDCA-treated HDM-challenged mice (×20 magnification; scale bars = 50 μm) and the quantification of percentage of area that positively stained for PAS staining. *P < 0.05 compared with their respective PBS controls. #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with HDM-challenged mice treated with 0.75 mg/kg TUDCA.

Fig. 12.

Therapeutic regimen of TUDCA decreases HDM-induced airway fibrotic remodeling, regardless of HDM rechallenge. A: representative images of α-SMA-stained lung tissue sections of vehicle-treated and TUDCA (0.75 mg/kg body wt dose)-treated HDM-challenged mice (×20 magnification; scale bars = 50 μm) and the quantification of percentage of area that positively stained for α-SMA immunostaining. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls and #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with HDM-challenged mice treated with 0.75 mg/kg TUDCA. B: measurement of collagen by hydroxyproline assay. Data are means ± SE of 6–8 mice/group. *P < 0.05 compared with their respective PBS controls and #P < 0.05 compared with vehicle-treated HDM-challenged mice. ‡P < 0.05 compared with HDM-challenged mice treated with 0.75 mg/kg TUDCA.

Fig. 13.

Comparison of total cell counts and AHR between HDM-challenged mice. A: percentage of difference in total cell counts in HDM-challenged mouse BALF from preventive (PR) and therapeutic (TR) regimen and therapeutic regimen with subsequent HDM rechallenge (TRRC) experiments. *P < 0.05 compared with PR and #P < 0.05 compared with TR. B: percent difference in methacholine-induced central airway resistance (Rn) of HDM-challenged mice from preventive and therapeutic regimen and therapeutic regimen with subsequent HDM rechallenge experiments. #P < 0.05 compared with TR. C: percent difference in hydroxyproline content of HDM-challenged mice from preventive and therapeutic regimen and therapeutic regimen with subsequent HDM rechallenge experiments. *P < 0.05 compared with PR and #P < 0.05 compared with TR.