Vitamin D-mediated induction of innate immunity in gingival epithelial cells

Abstract

Human gingival epithelial cells (GEC) produce peptides, such as β-defensins and the cathelicidin LL-37, that are both antimicrobial and that modulate the innate immune response. In myeloid and airway epithelial cells, the active form of vitamin D(3) [1,25(OH)(2)D(3)] increases the expression and antibacterial activity of LL-37. To examine the activity of vitamin D on the innate immune defense of the gingival epithelium, cultured epithelial cells were treated with either 10(-8) M 1,25(OH)(2)D(3) or ethanol for up to 24 h. A time-dependent induction of LL-37 mRNA up to 13-fold at 24 h in both standard monolayer and three-dimensional cultures was observed. Induction of the vitamin D receptor and the 1-α-hydroxylase genes was also observed. The hydroxylase was functional, as LL-37 induction was observed in response to stimulation by 25(OH)D(3). Through microarray analysis of other innate immune genes, CD14 expression increased 4-fold, and triggering receptor expressed on myeloid cells-1 (TREM-1) was upregulated 16-fold after 24 h of treatment with 1,25(OH)(2)D(3). TREM-1 is a pivotal amplifier of the innate immune response in macrophages, leading to increased production by inflammatory response genes. Activation of TREM-1 on the GEC led to an increase in interleukin-8 (IL-8) mRNA levels. Incubation of three-dimensional cultures with 1,25(OH)(2)D(3) led to an increase in antibacterial activity against the periodontal pathogen Aggregatibacter actinomycetemcomitans when the bacteria were added to the apical surface. This study is the first to demonstrate the effect of vitamin D on antibacterial defense of oral epithelial cells, suggesting that vitamin D(3) could be utilized to enhance the innate immune defense in the oral cavity.

Fig. 1.

Induction of LL-37 in gingival epithelial cells in response to 1,25(OH)₂D₃. OKF6/TERT (A and B) or primary gingival epithelial cells (C) were cultured in the presence of 10⁻⁸ M 1,25(OH)₂D₃ or ethanol (0.1%) for increasing times (A and C) or for 24 h in the presence of increasing concentrations of 1,25(OH)₂D₃ (B). Total mRNA was isolated, and LL-37 mRNA levels were quantified by QRT-PCR. Results are mRNA levels of 1,25(OH)₂D₃-stimulated cultures compared to those of ethanol-treated cultures at each time point, normalized to the level for β₂M (n = 3; bars indicate mean ± standard deviation). (D) LL-37 protein levels were visualized by Western blot analysis of whole-cell lysates of OKF6/TERT cells cultured in an air-liquid interface (ALI) and stimulated with 1,25(OH)₂D₃ for 18 h. Tubulin was visualized as a loading control. The increase in LL-37 mRNA levels in panel A is significant at 24 h as quantified by t test (P < 0.001). The dose response in panel B is significant as measured by analysis of variance (ANOVA) (P < 0.0001).

Fig. 2.

Expression of the vitamin D pathway in GEC. (A) Expression of vitamin D-related genes, i.e., those encoding VDR (lane 1) and Cyp27B1 (1-α-hydroxylase) (lane 2), in unstimulated cultures was observed by gel electrophoresis of RT-PCR products. (B) Cultures of OKF6/TERT cells were incubated for 2 h with 10⁻⁸ M 1,25(OH)₂D₃ or ethanol. Total mRNA was isolated, and relative levels of VDR and Cyp27B1 were quantified by QRT-PCR as described in the text. Results are means ± standard deviations for triplicate cultures, compared to ethanol-treated cultures, normalized to the result with β₂M. The increase in VDR mRNA levels is significant (*, P = 0.0001). The increase in Cyp27B1 mRNA levels is significant (**, P = 0.03). (C) Functionality of the hydroxylase is shown by incubation of OKF6/TERT cells with either ethanol or 10⁻⁸ M 25(OH)D₃ for 24 h and quantification of LL-37 mRNA levels as described above. The increases in LL-37 mRNA levels with 25(OH)D₃ are significant at 24 h (*, P = 0.0003) and 48 h (**, P = 0.00005) as measured by Student's t test.

Fig. 3.

Induction of TREM-1 in GEC. OKF6/TERT cells were grown in 3D cultures for increasing times in the presence of either ethanol or 10⁻⁸ M 1,25(OH)₂D₃. (A and B) Total mRNA was isolated, and TREM-1 mRNA levels were observed by gel electrophoresis of RT-PCR products of 24-h-stimulated cultures (A) and quantified by QRT-PCR of triplicate cultures at each time point as described in the text (B). (C) Western blot analysis of whole-cell lysates using anti-TREM-1 antibody. (D) Immunofluorescence of nonpermeabilized OKF6/TERT cultures grown on coverslips and treated with anti-TREM-1 antibody, visualized with an Alexa Fluor-coupled secondary antibody. Magnification, ×400. Time-dependent induction of TREM-1 was significant as measured by ANOVA (P < 0.0001).

Fig. 4.

Antibacterial host defense response of gingival epithelial cells. Three-dimensional cultures of OKF6/TERT cells were grown as described in the text. (A) Antibacterial activity was measured on the surface of OKF6/TERT cells cultured in ALI, stimulated for 24 h with either ethanol or 10⁻⁸ M 1,25(OH)₂D₃ (D3). Bacteria were incubated on the surface of the 3D cultures for 3 h and plated to quantify viable colonies. Results are shown as the mean reduction in viable colonies compared to that of the control result (± standard deviation) for triplicate cultures. The reduction in colonies is significant by a paired t test (n = 4 independent experiments of triplicate cultures; P = 0.04). (B) Cultures were treated with either ethanol or 10⁻⁸ M 1,25(OH)₂D₃ for 24 h, followed by the addition of 500 CFU live bacteria to the surface for 1 h. Anti-TREM-1 antibody or an isotype control was added to the surface for a further 3 h, and total mRNA was isolated as described above. Levels of IL-8 mRNA were quantified by QRT-PCR and are shown relative to that of the isotype/ethanol control, normalized to β₂-microglobulin. Induction of IL-8 mRNA is significant in anti-TREM-1/ethanol-treated cultures, compared with that of the control (*, P = 0.001), and in anti-TREM-1/vitamin D-treated cultures (**, P = 0.02) by t test.