Antimicrobial barrier of an in vitro oral epithelial model

Abstract

Objective: Oral epithelia function as a microbial barrier and are actively involved in recognizing and responding to bacteria. Our goal was to examine a tissue engineered model of buccal epithelium for its response to oral bacteria and proinflammatory cytokines and compare the tissue responses with those of a submerged monolayer cell culture.

Design: The tissue model was characterized for keratin and beta-defensin expression. Altered expression of beta-defensins was evaluated by RT-PCR after exposure of the apical surface to oral bacteria and after exposure to TNF-alpha in the medium. These were compared to the response in traditional submerged oral epithelial cell culture.

Results: The buccal model showed expression of differentiation specific keratin 13, hBD1 and hBD3 in the upper half of the tissue; hBD2 was not detected. hBD1 mRNA was constitutively expressed, while hBD2 mRNA increased 2-fold after exposure of the apical surface to three oral bacteria tested and hBD3 mRNA increased in response to the non-pathogenic bacteria tested. In contrast, hBD2 mRNA increased 3-600-fold in response to bacteria in submerged cell culture. HBD2 mRNA increased over 100-fold in response to TNF-alpha in the tissue model and 50-fold in submerged cell culture. Thus, the tissue model is capable of upregulating hBD2, however, the minimal response to bacteria suggests that the tissue has an effective antimicrobial barrier due to its morphology, differentiation, and defensin expression.

Conclusions: The oral mucosal model is differentiated, expresses hBD1 and hBD3, and has an intact surface with a functional antimicrobial barrier.

Figure 1. Histology and immunohistology of oral tissue model

A. Buccal tissue, H&E stained section. B. Oral tissue model, H&E stained section. C. – G. Oral tissue model immunohistochemical staining with antibodies to Keratin 14 (C); Keratin 13 (D);. hBD1 (E); hBD2 (F); hBD3 (G). Note that the differentiation specific keratin 13 is expressed in the upper portion of the tissue. hBD1 and hBD3 are also expressed in the upper layers, while hBD2 is not detected. Original magnification 10X.

Figure 2. Expression of β-defensins and IL-8 in the oral tissue model in response to commensal and pathogen oral bacteria

The tissue model was exposed for 24 h to P. gingivalis, S. gordonii, or F. nucleatum (as indicated by +) or to growth medium (−) placed on the tissue surface as described in Methods section. Total RNA was extracted and analyzed for various markers of innate immunity. A. RT-PCR analysis for hBD1–3, IL8 and RPO in two independent experiments. Note that hBD1 is constitutively expressed, while hBD2 is upregulated with all bacteria and hBD3 is upregulated with the commensals (S. gordonii and F. nucleatum). B. Quantitative PCR for change in hBD2 mRNA expression in the oral tissue model in response to bacteria at 24 h exposure. Expression of hBD2 is shown relative to GAPDH and compared to the growth medium for each bacterial species (results are the average of 3–5 independent experiments).

Figure 3

Expression of the receptors TLR2, TLR4, PAR1, and PAR2 mRNA in the oral tissue model in response to commensal and pathogen oral bacteria as in Figure 2. T=0, control tissue at the time of initial challenge; T=+24, control tissue with no challenge.

Figure 4

hBD2 immunostaining in TNF-α stimulated (A) and control (B) buccal tissue model. Note hBD2 staining in distinct cells within the lower half of the epithelial tissue model in response to TNF-α placed in the medium under the tissue. Exposure to TNF-α was for 48 hr. Original magnification 10X