Fig 2: Proposed model for the mechanisms whereby the gram- and gram+ bacterial pathogens induce the expression of the antimicrobial protein, S100A15, in the oral mucosa. The colonization of gram- and gram+ bacterial pathogens result in the activation of both TLR4 and TLR2, respectively. As a consequence, TRAF6 plays a central role in mediating TLR4 and TLR2-induced signaling to NF-kB/ASK1-JNK-AP-1/ASK1-p38-ATF-2 pathways to enhance the transcriptional activation of S100A5, which subsequently functions as an antimicrobial protein to protect oral mucosa from bacterial pathogens.

Fig 3: Immunohistochemical analysis of S100A15 protein in oral mucosa of patients with inflammatory gingiva. (A), Gingival tissues of healthy individuals (n = 10) were stained with anti-S100A15 antibodies. (B) Gingival tissues of patients with inflammatory gingiva (n = 10) were stained with anti-S100A15 antibody. Original magnification × 100. Scale bar indicates 100 µm. (C) Statistical Analysis of S100A15 expression in gingival tissues of patients with inflammatory gingiva (n = 10) as a relative ratio to the expression of S100A15 in gingival tissues of healthy individuals. Bars represent mean ± SD from inflammatory (n = 10) and healthy (n = 10) gingival tissues, * p < 0.05. (D,E) Detection of oral bacterial pathogens (gram- P. gingivalis and gram+ S. sanguinis) in the saliva of patients with inflammatory gingiva by conventional PCR. Detection of both P. gingivalis and S. sanguinis in the saliva of patients with inflammatory gingiva by PCR. The PCR was performed using specific primers to amplify a 480 bp fragment of the 16S rRNA gene as a marker for P. gingivalis (D) and specific primers to amplify a 473 bp fragment of the UDP-N-acetylglucosamine-like protein gene as a marker for S. sanguinis (E). PCR products were separated on 2% agarose and stained with ethidium bromide. (F) Analysis of band intensity as the relative ratio of the amplified DNA fragment of 16S rRNA gene, the marker of P. gingivalis, and the amplified of the UDP-N-acetylglucosamine-the marker of S. sanguinis to the corresponding positive control. Bars represent the mean ± SD from three blots, * p < 0.05.

Fig 4: Bacterial pathogens-induced expression of S100A15 in oral mucosa-derived cells is mediated by the MAP kinase and NF-?B pathways. (A) GF and KB cells were pre-treated with Thioredoxin 1 h prior to stimulation with either heat-inactivated gram- P. gingivalis or heat-inactivated gram+ S. sanguinis for 48 h. Total RNA was extracted from treated and control cells and subsequently subjected to qRT-PCR analysis to assess the relative transcription levels of S100A15. (B) GF and KB cells were pre-treated with inhibitors of JNK (SP600125), p38 (SB-203580), or NF-?B (Bay11-7082) 1 h prior to stimulation with either heat-inactivated P. gingivalis or heat-inactivated S. sanguinis for 48 h. Total RNA was extracted from treated and control cells and subsequently subjected to qRT-PCR analysis to assess the relative transcription levels of S100A15. (C) GF and KB cells were pre-treated with SP600125, SB-203580, or Bay11-7082 1 h prior to stimulation with heat-inactivated P. gingivalis or heat-inactivated S. sanguinis for 48 h. Total RNA was extracted from treated and control cells and subsequently subjected to qRT-PCR analysis to assess relative transcription levels of S100A15. Densitometric quantification of S100A15 mRNA over actin mRNA transcript. Each bar represents the mean ± SD of three independent experiments performed in duplicate, * p < 0.05.

Fig 5: Inhibition of induced expression of S100A15 by neutralization of toll-like receptors. (A) GF and KB cells were pre-treated with anti-TLR2, anti-TLR4, or control IgG 1 h prior to the stimulation with either heat-inactivated P. gingivalis or heat-inactivated S. sanguinis for 48 h. Total RNA was extracted from treated and control cells and subsequently subjected to qRT-PCR analysis to assess the relative transcription levels of S100A15. (B) GF and KB cells were pre-treated with anti-TLR2, anti-TLR4, or control IgG 1 h prior to stimulation with either LPS (100 ng/mL) or LTA (50 ng/mL) for 48 h. Total RNAs were extracted from treated and control cells and subsequently subjected to qRT-PCR analysis to assess the relative transcription levels of S100A15. Densitometric quantification of S100A15 mRNA over actin mRNA transcript. The data were normalized to the level of treated and untreated control cells in each sample. Each bar represents the mean ± SD of three independent experiments performed in duplicate, * p < 0.05.