The oral commensal Streptococcus mitis activates the aryl hydrocarbon receptor in human oral epithelial cells

Abstract

Streptococcus mitis (S. mitis) is a pioneer commensal bacterial species colonizing many of the surfaces of the oral cavity in healthy individuals. Yet, not much information is available regarding its interaction with the host. We used examination of its transcriptional regulation in oral keratinocytes to elucidate some of its potential roles in the oral cavity. Transcription factor analysis of oral keratinocytes predicted S. mitis-mediated activation of aryl hydrocarbon receptor (AhR). Activation and functionality of AhR was confirmed through nuclear translocation determined by immunofluorescence microscopy and real-time polymerase chain reaction with reverse transcription analysis of CYP1A1, the hallmark gene for AhR activation. Addition of Streptococcus mutans or Streptococcus gordonii did not induce CYP1A1 transcription in the keratinocyte cultures. Introduction of an AhR-specific inhibitor revealed that S. mitis-mediated transcription of CXCL2 and CXCL8 was regulated by AhR. Elevated levels of prostaglandin E2 (enzyme-linked immunosorbent assay) in supernatants from S. mitis-treated oral epithelial cells were also attenuated by inhibition of AhR activity. The observed AhR-regulated activities point to a contribution of S. mitis in the regulation of inflammatory responses and thereby to wound healing in the oral cavity. The concept that the oral commensal microbiota can induce AhR activation is important, also in view of the role that AhR has in modulation of T-cell differentiation and as an anti-inflammatory factor in macrophages.

Figure 1

S. mitis activates AhR in oral epithelial cells. (a) Nuclear translocation of AhR was examined by immunofluorescence microscopy of human primary oral keratinocytes, cultivated and exposed to live S. mitis for 20 min (right) or left untreated (left). Cells were stained for AhR (upper row; green) and DAPI (middle row; red). Merged pictures in the lower row. (b) Fold changes in the transcription of CYP1A1, the hallmark gene of AhR activation, determined by real-time RT-PCR in human primary oral keratinocytes exposed to live S. mitis (left), S. gordonii (middle) and S. mutans (right) for 90 min, with or without the AhR-specific inhibitor CH-223191 (n=3 or 4). Brackets marked with asterisks indicate statistically significance (ANOVA for repeated measures followed by Holm–Sidak adjustment for multiple comparisons; P<0.05). ANOVA, analysis of variance.

Figure 2

S. mitis shows AhR-dependent induction of pro-inflammatory mediators CXCL2 and CXCL8. Fold changes in the transcription of chemoattractants CXCL1 (a), CXCL2 (b) and CXCL8 (c) were determined by real-time RT-PCR in human primary oral keratinocytes exposed to live S. mitis for 90 min, with or without the AhR-specific inhibitor CH-223191 (n=4). Brackets marked with asterisks indicate statistically significance (ANOVA for repeated measures followed by Holm–Sidak adjustment for multiple comparisons; P<0.05). ANOVA, analysis of variance.

Figure 3

Oral epithelial cells exposed to S. mitis show increased secretion of PGE2. Human primary oral keratinocytes were exposed for 6 h to live S. mitis and/or the AhR-specific inhibitor CH-223191 or left untreated. Supernatants were collected and their PGE2 content was measured by ELISA (n=4). Brackets marked with asterisks indicate statistically significance (ANOVA for repeated measures followed by Holm–Sidak adjustment for multiple comparisons; P<0.05). ANOVA, analysis of variance.