In vivo beta-defensin gene expression in rat gingival epithelium in response to Actinobacillus actinomycetemcomitans infection

Abstract

Background and objective: Human beta-defensins have been identified in the oral cavity and are predicted to play a role in the defense against pathogenic bacteria. Homologous rat beta-defensins (RBDs) have been identified, but their expression in the oral cavity has not been examined. Therefore, the aim of this study was to investigate the expression of innate immune mediators in the rat gingival epithelium.

Material and methods: Rats were pretreated with antibiotics to depress the normal oral flora, followed by the introduction of Actinobacillus actinomycetemcomitans in their food to allow colonization and the development of periodontal disease. At various time points, animals were killed and the gingival epithelium was extracted. Semiquantitative reverse transcription-polymerase chain reaction was performed to measure RBD and Toll-like receptor (TLR) mRNA levels.

Results: Three beta-defensins (RBD-1, -2 and -5) and two TLRs (TLR-3 and -4) are expressed in normal rat gingival epithelium. After the introduction of A. actinomycetemcomitans, RBD-1 and RBD-2 mRNA levels increased for the first week followed by a return to basal levels. No change in TLR mRNA levels was observed.

Conclusion: The rat model provides a good system for experimental analysis of the innate immune response to periopathogenic bacteria in the oral cavity, as well as the potential role of beta-defensins in the host response to colonization.

Fig. 1

β-defensin expression in rat gingival epithelium. mRNA was isolated from rat gingiva and amplified by reverse transcription–polymerase chain reaction. The amplification products were subjected to agarose-gel electrophoresis then examined for the presence of rat β-defensin (RBD)-1, RBD-2 and Defb5. Kidney and lung were included as positive controls. Glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) was included as a housekeeping gene.

Fig. 2

Map of the promoter regions in rat β-defensins (RBDs). The 5′ flanking regions (to–1000 bp) for RBD-1, -2 and -5 were analyzed for the presence of putative transcription factor binding sequences. Flanking regions for human β-defensin (HBD)-1, -2 and -3 are shown for comparison. CEBP, CCAAT/enhancer binding protein; IRF, Interferon regulatory factor; ISRE, Interferon-stimulated response element; NF-κB, nuclear factor-κB; STAT, signal transducers and activators of transcription.

Fig. 3

Toll-like receptor (TLR) expression in rat gingival epithelium. mRNA from rat gingiva was analyzed for the expression of TLR-2, -3, -4, -5, -6, -7 and -9. The only positive signals obtained by gel electrophoresis were for TLR-3 and -4 (shown). Bone marrow (BM) is included as the positive control for TLRs (positive signals were observed for all TLRs; data not shown), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as a housekeeping gene.

Fig. 4

Expression of rat β-defensins in the gingival epithelium in response to bacterial challenge. Rats were treated with antibiotic for 4 d prior to feeding with either Actinobacillus actinomycetemcomitans (Aa)-incorporated food or normal food, as described in the Material and methods. Rats (n = 3) were killed on the day indicated, and mRNA was isolated from the gingival epithelium and amplified by the semiquantitative reverse transcription-polymerase chain reaction. Levels of mRNA for rat β-defensin 1 (RBD-1) (A) and RBD-2 (B) are shown relative to the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Error bars + standard error of the mean are shown. Asterisks represent statistical significance, determined by the Student's t-test (*p < 0.04; **p < 0.03; ***p < 0.01).