Allergy Asthma Immunol Res.  2018 Jan;10(1):25-33. 10.4168/aair.2018.10.1.25.

Alteration in Claudin-4 Contributes to Airway Inflammation and Responsiveness in Asthma

• Lee PH
• Kim BG
• Lee SH
• Lee JH
• Park SW
• Kim DJ
• Park CS
• Leikauf GD
• Jang AS

• KMID: 2428847
• DOI: http://doi.org/10.4168/aair.2018.10.1.25

Abstract

PURPOSE
Claudin-4 has been reported to function as a paracellular sodium barrier and is one of the 3 major claudins expressed in lung alveolar epithelial cells. However, the possible role of claudin-4 in bronchial asthma has not yet been fully studied. In this study, we aimed to elucidate the role of claudin-4 in the pathogenesis of bronchial asthma.
METHODS
We determined claudin-4 levels in blood from asthmatic patients. Moreover, using mice sensitized and challenged with OVA, as well as sensitized and challenged with saline, we investigated whether claudin-4 is involved in the pathogenesis of bronchial asthma. Der p1 induced the inflammatory cytokines in NHBE cells.
RESULTS
We found that claudin-4 in blood from asthmatic patients was increased compared with that from healthy control subjects. Plasma claudin-4 levels were significantly higher in exacerbated patients than in control patients with bronchial asthma. The plasma claudin-4 level was correlated with eosinophils, total IgE, FEV1% pred, and FEV1/FVC. Moreover, lung tissues from the OVA-OVA mice showed significant increases in transcripts and proteins of claudin-4 as well as in TJ breaks and the densities of claudin-4 staining. When claudin-4 was knocked down by transfecting its siRNA, inflammatory cytokine expressions, which were induced by Der p1 treatment, were significantly increased.
CONCLUSIONS
These findings thus raise the possibility that regulation of lung epithelial barrier proteins may constitute a therapeutic approach for asthma.

Keyword

Asthma; epithelial barrier; claudin-4

MeSH Terms

Animals
Asthma*
Claudin-4*
Claudins
Cytokines
Eosinophils
Epithelial Cells
Humans
Immunoglobulin E
Inflammation*
Lung
Mice
Ovum
Plasma
RNA, Small Interfering
Sodium
Claudin-4
Claudins
Cytokines
Immunoglobulin E
RNA, Small Interfering
Sodium

Figure

• 

  Fig. 1 Comparison of plasma claudin-4 level between stable and exacerbated states of asthmatic subjects. *P<0.05; **P<0.005 compare to control; #P<0.05 Stable vs exacerbated asthma.

• 

  Fig. 2 Relationship of plasma Claudin-4 and clinical variables. Correlation of plasma Claudin-4 level with (A) Forced Expired Volume 1 second (FEV1) (percent predicted), (B) Forced Expired Volume 1 second/Forced Vital Capacity (FEV1/FVC), (C) eosinophils, and (D) total IgE.

• 

  Fig. 3 Lung claudin-4 transcripts and protein levels in ovalbumin-sensitized and -challenged (OVA) mice. (A) Penh was measured following inhalation of increasing doses of methacholine, (B) Numbers of bronchoalveolar lavage fluid (BALF) cells, (C) Analysis of BALF cytokines, (D) Lung claudin-4 transcripts, (E) Protein levels. Densitometry was determined with 3 immunoblots and normalized to β-actin. Values (normalized to β-actin) means±SEM. *P<0.05 OVA vs sham.

• 

  Fig. 4 (A) Immunohistochemistry and immunofluorescence staining of the lung sections of OVA/OVA and sham mice. Disrupted integrity of claudin-4 in the lung of the ovalbumin-sensitized and -challenged (OVA) mice. Tight junctions form a continuous ring that circumscribes individual cells is more evident but appear disrupted in the NHBE cells of OVA-sensitized/challenged mice. Disrupted integrity of claudin-4 in the lung. (B) Quantitation of claudin-4 was determined with 3 immunostains. *P<0.05 OVA/OVA vs sham.

• 

  Fig. 5 Effects of claudin-4 knockdown on Der p1-induced inflammatory responses. (A) Representative image of siRNA directed claudin-4 in NHBE cells decreased (left panel) transcript determined by PCR (right panel) proteins determined by Western blot. (B) Claudin-4 transcripts increased in NHBE cells following 10 µg/mL Der p1 treatment for 8 hour. This response was inhibited by 10 µM dexamethasone (DEX) treatment. Small interference RNA directed at claudin-4 decreased claudin-4 mRNA. (C) Trans-epithelial electrical resistance (TEER) increased in NHBE cells following 10 µg/mL Der p1 treatment. This response was delayed in NHBE cells treated with 10 µM DEX treatment. siRNA directed at claudin-4 diminished the Der p1-induced increase in TEER. *P<0.05.

• 

  Fig. 6 Total RNAs extracted to measure mRNA expression levels of inflammatory cytokines (IL-4, 5, 13) by real-time PCR. (A) Interleukin 4 (IL-4), (B) IL-5 and (C) IL-13 transcript increased in NHBE cells following 10 µg/mL Der p1 treatment for 8 or 24 hours. This response was inhibited by 10 µM DEX treatment. The siRNA directed at claudin-4 increased the response to Derp1 stimulation. *P<0.05.

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