Yonsei Med J.  2014 Mar;55(2):387-394.

Effects of Stress on Mouse beta-Defensin-3 Expression in the Upper Digestive Mucosa

• Kawashima
• Shimizu T
• To M
• Saruta J
• Jinbu Y
• Kusama M
• Tsukinoki

Abstract

PURPOSE
Gastrointestinal integrity and immune surveillance are affected by stress. Stress also adversely affects mucosal barrier function. beta-defensins constitute an integral component of the innate immune system as antimicrobial peptides, serving as the first line of defense against microbial pathogens at the epithelial surfaces of the upper digestive mucosa. The primary objective of this study was to determine the effects of stress on the expression profile of mouse beta-defensin-3 in the upper digestive mucosa of mice with diabetes.
MATERIALS AND METHODS
We established a mouse model of restraint stress by using NSY/Hos mice with type 2 diabetes mellitus. We used real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry to investigate the effects of stress and glucocorticoid administration on mouse beta-defensin-3 expression in the upper digestive mucosa of the gingiva, esophagus, and stomach.
RESULTS
Mouse beta-defensin-3 mRNA expression was higher in the esophagus than in the gingiva or stomach (p<0.05). In the esophagus, mouse beta-defensin-3 mRNA expression was lower in stressed mice than in non-stressed mice (p<0.05). Furthermore, immunoreactivity to mouse beta-defensin-3 protein was lower in the esophagus of stressed mice than non-stressed mice, consistent with the results of mRNA expression analysis. Systemic glucocorticoid administration also downregulated esophageal mouse beta-defensin-3 mRNA expression.
CONCLUSION
Our novel findings show that stress decreases mouse beta-defensin-3 expression in the esophagus of mice with diabetes, possibly due to increased endogenous glucocorticoid production. It appears to be highly likely that stress management may normalize mucosal antimicrobial defenses in patients with diabetes.

Keyword

Antimicrobial peptide; defensin; stress; esophagus; diabetes mellitus

MeSH Terms

Animals
beta-Defensins
Diabetes Mellitus
Diabetes Mellitus, Type 2
Esophagus
Gingiva
Humans
Immune System
Immunohistochemistry
In Situ Hybridization
Methods
Mice*
Mucous Membrane*
Peptides
Real-Time Polymerase Chain Reaction
RNA, Messenger
Stomach
Peptides
RNA, Messenger
beta-Defensins

Figure

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  Fig. 1 Blood glucose and plasma corticosterone levels in stressed and non-stressed mice. Blood glucose (A) and plasma corticosterone levels (B) in stressed and non-stressed mice (n=5), represented as mean±SD values. Statistical comparisons were made individually (between stressed and non-stressed mice). Statistical significance was observed for plasma corticosterone levels (p<0.05), but not for blood glucose levels.

• 

  Fig. 2 Expression of mBD-3 in the upper digestive mucosa. Real-time PCR results for mBD-3 in the upper digestive mucosa (n=4). The results are represented as mean±SD values. Statistical comparisons were made among the mBD-3 expression in the gingiva, esophagus, and stomach, and statistical significance was seen for mBD-3 among the 3 groups (p<0.05 for all). mBD-3, mouse β-defensin-3; PCR, polymerase chain reaction.

• 

  Fig. 3 mBD-3 mRNA expression in the upper digestive mucosa in stressed and non-stressed mice. Real-time PCR results for mBD-3 expression in gingival (n=4) (A), esophageal (n=4) (B), and stomach (n=4) (C) tissue from stressed and non-stressed mice. The results are represented as mean±SD values. Statistical comparisons were made individually (between stressed and non-stressed mice); significant differences were seen between the 2 groups for the mBD-3 mRNA expression in the esophagus but not in the gingiva or stomach (p<0.05). mBD-3, mouse β-defensin-3; PCR, polymerase chain reaction.

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  Fig. 4 mBD-3 protein expression in the esophagus of stressed and non-stressed mice by immunohistochemical stain. The localization of mBD-3 in the esophagus in stressed mice (A) and non-stressed mice (B) is shown by immunohistochemical results. Positive mBD-3 immunoreactivity was found in the keratinized layers of non-stressed mice, but not of stressed mice. Scale bar reveals 20 µm. mBD-3, mouse β-defensin-3.

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  Fig. 5 ISH analysis of mBD-3 mRNA expression in the esophagus of stressed and non-stressed mice. The mBD-3 signals in stressed (A) and non-stressed mice (B) are shown by ISH with antisense probes. Positive mBD-3 signals were found in non-stressed mice, but not in stressed mice. No mBD-3 signals were observed with sense probes (C). Scale bar reveals 20 µm. ISH, in situ hybridization; mBD-3, mouse β-defensin-3.

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  Fig. 6 mBD-3 mRNA expression in the esophagus of GC-treated and untreated mice. Real-time PCR results for mBD-3 expression in the esophagus of GC-treated and untreated mice (n=4). The results are represented as mean±SD values. Statistical comparisons were made individually (between GC-treated and untreated mice); mBD-3 expression significantly differed between GC-treated and untreated mice (p<0.05). GC, glucocorticoid; mBD-3, mouse β-defensin-3; PCR, polymerase chain reaction.

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