Allergy Asthma Immunol Res.  2019 Jul;11(4):560-571. 10.4168/aair.2019.11.4.560.

Protease-Activated Receptors 2-Antagonist Suppresses Asthma by Inhibiting Reactive Oxygen Species-Thymic Stromal Lymphopoietin Inflammation and Epithelial Tight Junction Degradation

• Kim HJ
• Lee SH
• Jeong S
• Hong SJ

• KMID: 2448758
• DOI: http://doi.org/10.4168/aair.2019.11.4.560

Abstract

PURPOSE
Protease-activated receptor 2 (PAR2) reportedly triggers the immune response in allergic asthma. We aimed to investigate the mechanism on allergic inflammation mediated by PAR2.
METHODS
Human lung epithelial cells (A549 cells) were used for in vitro, and the German cockroach extract (GCE)-induced mouse model was developed for in vivo studies.
RESULTS
In A549 cells, the levels of reactive oxygen species (ROS) and thymic stromal lymphopoietin (TSLP) were significantly increased by GCE treatment, but were suppressed by PAR2-antagonist (PAR2-ant) or N-acetylcysteine (NAC) treatment. Claudin-1 was degraded by GCE, and was restored by PAR2-ant or NAC in the cells. In the mouse model, the clinical appearance including bronchial hyperresponsiveness, bronchoalveolar lavage fluid analysis and total immunoglobulin E were significantly suppressed by PAR2-ant or NAC. Moreover, TSLP levels in the lung were suppressed by the same treatments in the lung. Claudin-1 was also degraded by GCE, and was restored by PAR2-ant or NAC.
CONCLUSIONS
ROS generation and epidermal tight junction degradation are triggered by protease, followed by the induction of TSLP in allergic asthma. Our findings could suggest that PAR2-ant or anti-oxidants could be considered for allergic diseases as preventive alternatives.

Keyword

Asthma; protease-activated receptor 2; thymic stromal lymphopoietin; tight junction; reactive oxygen species

MeSH Terms

Acetylcysteine
Animals
Asthma*
Blattellidae
Bronchoalveolar Lavage Fluid
Claudin-1
Epithelial Cells
Humans
Immunoglobulin E
Immunoglobulins
In Vitro Techniques
Inflammation*
Lung
Mice
Oxygen*
Reactive Oxygen Species
Receptor, PAR-2
Receptors, Proteinase-Activated*
Tight Junctions*
Acetylcysteine
Claudin-1
Immunoglobulin E
Immunoglobulins
Oxygen
Reactive Oxygen Species
Receptor, PAR-2
Receptors, Proteinase-Activated

Figure

• 

  Fig. 1 GCE induces inflammation and degrades tight junctions in human bronchial epithelial cells (A549 cells). The 2′, 7′-DCF intensity is significantly increased following GCE exposure and suppressed following PAR2-ant and NAC treatment in A549 cells (1 hour) (A) and 2 hours (B). The expression of TSLP is significantly increased following GCE and PAR2-ago treatment and suppressed following PAR2-ant and NAC treatment in A549 cells after 2 hours (C). The expression of claudin-1 is significantly decreased by GCE and PAR2-ago treatment, but is significantly restored by PAR2-ant and NAC treatment in A549 cells (D). (A, B) Fluorescence intensity measurement at wavelengths of 450 nm and 520 nm; (C, D) real-time polymerase chain reaction (Taqman method). GCE, German cockroach extract; NAC, N-acetylcysteine; DCF, dichlorofluorescein; PAR2-ant, protease-activated receptor 2-antagonist; PAR2-ago, protease-activated receptor 2 agonist; TSLP, thymic stromal lymphopoietin. *P < 0.01 and †P < 0.001. The experiments were repeated 6 times.

• 

  Fig. 2 GCE induces allergic inflammation in the mouse model of asthma. (A) BHR is significantly increased following GCE treatment and suppressed following PAR2-ant and NAC treatment. ‡P < 0.001 compared to saline. (B) The total number of cells in bronchoalveolar lavage fluid is also significantly increased following GCE treatment and suppressed following PAR2-ant and NAC treatment. (C) The numbers of macrophages, eosinophils and neutrophils are significantly increased following GCE treatment and suppressed following PAR2-ant and NAC treatment. (D) The total immunoglobulin E level is also significantly increased following GCE treatment and suppressed following PAR2-ant and NAC treatment. (E) Lung inflammation (hematoxylin and eosin staining 200 × magnification) and (F) peribronchial inflammation is significantly increased following GCE treatment and suppressed following PAR2 and NAC treatment. BHR, bronchial hypersensitivity; GCE, German cockroach extract; CE, cockroach extract; NAC, N-acetylcysteine; PAR2-ant, protease-activated receptor 2-antagonist. *P < 0.05, †P < 0.01, and ‡P < 0.001. The experiments were repeated 5 times.

• 

  Fig. 3 GCE induces T helper 2-allergic inflammation in a mouse model of asthma. The levels of IL-4 and IL-13 are significantly increased following GCE treatment and suppressed following PAR2-ant and NAC treatment in the lung (A and C). Moreover, the level of IFN-ν is significantly decreased following GCE treatment and increased following PAR2-ant and NAC treatment (B). Data were measured using real-time polymerase chain reaction. GCE, German cockroach extract; IL, interleukin; IFN, interferon; PAR2-ant, protease-activated receptor 2-antagonist; NAC, N-acetylcysteine. *P < 0.05; †P < 0.01. The experiments were repeated 5 times.

• 

  Fig. 4 GCE significantly suppresses GSTP1 expression in a mouse model. GSTP1 expression is significantly increased by PAR2-ant and NAC treatment in the lung, as indicated by immunohistopathology (immunohistochemistry, 200 × magnification) (A) and intensity quantification (B). GCE, German cockroach extract; GSTP1, glutathione S-transferase 1; NAC, N-acetylcysteine; PAR2-ant, protease-activated receptor 2-antagonist. *P < 0.01; †P < 0.001. The experiments were repeated 5 times.

• 

  Fig. 5 GCE significantly increases TSLP expression in a mouse model. TSLP expression is significantly suppressed by PAR2-ant and NAC treatment in the lung, as indicated by immunohistochemistry (200 × magnification) (A) and intensity quantification (B). GCE, German cockroach extract; TSLP, thymic stromal lymphopoietin; NAC, N-acetylcysteine; PAR2-ant, protease-activated receptor 2-antagonist. *P < 0.001. The experiments were repeated 5 times.

• 

  Fig. 6 GCE significantly disturbs claudin-1 expression in a mouse model. Claudin-1 expression is significantly restored by PAR2-ant and NAC treatment in the lung, as indicated by immunohistochemistry (200 × magnification) (A) and intensity quantification (B). GCE, German cockroach extract; PAR2-ant, protease-activated receptor 2-antagonist; NAC, N-acetylcysteine. *P < 0.01; †P < 0.001. The experiments were repeated 5 times.

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