The presence of bacteria within tissue provides insights into the pathogenesis of oral lichen planus

Oral lichen planus (OLP) is a chronic T cell-mediated mucocutaneous disease of unknown etiopathogenesis. Although various antigens have been considered, what actually triggers the inflammatory response of T cells is unknown. In the present study, we propose that intracellular bacteria present within tissues trigger T cell infiltration and provide target antigens. Sections of OLP (n = 36) and normal (n = 10) oral mucosal tissues were subjected to in situ hybridization using a universal probe targeting the bacterial 16S rRNA gene and immunohistochemistry with anti-CD3, anti-CD4, anti-CD8, and anti-macrophage-specific antibodies. Bacteria were abundant throughout the epithelium and the lamina propria of OLP tissues, which exhibited positive correlations with the levels of infiltrated CD3+, CD4+, and CD8+ cells. Furthermore, bacteria were detected within the infiltrated T cells. Pyrosequencing analysis of the mucosal microbiota from OLP patients (n = 13) and control subjects (n = 11) revealed a decrease in Streptococcus and increases in gingivitis/periodontitis-associated bacteria in OLP lesions. Using the selected bacterial species, we demonstrated that certain oral bacteria damage the epithelial physical barrier, are internalized into epithelial cells or T cells, and induce production of T cell chemokines CXCL10 and CCL5. Our findings provide insights into the pathogenesis of OLP.

at MOI 500 for 24 hours. Next, 20 μl of CCK-8 solution was applied to each well, and the cells were further incubated for 1 hour at 37 °C. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated as a relative percentage relative to the vehicle control.

Bacterial internalization into human cells
HOK-16B cells were plated at a density of 3 x 10 4 cells cm -2 onto 24-mm diameter glass cover slips. The cells were infected at 70% confluence with the CFSE-labeled bacteria at an MOI of 1,000 for 24 hours. Purified human CD4 + , CD8 + , or CD14 + cells (2.5 x 10 5 cells) in RPMI medium with 10% fetal bovine serum (FBS) were infected with the CFSE-labeled bacteria at an MOI of 1,000 for 1 hour in the absence of antibiotics.
For confocal microscopic examination, the infected cells were fixed, permeabilized, and then stained with rhodamine-phallodin (Molecular Probes) and Hochest 33342 (Molecular Probes). The leukocytes were attached onto collagen-coated slides after staining. Mounted slides were imaged using a Zeiss LSM700 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with serial z-sections.
For flow cytometric analysis, the infected cells were washed with PBS and resuspended in trypan blue (400 mg ml -1 prepared in 0.85% saline solution) to quench the fluorescence of the bacteria bound on the surface. The cells were analyzed using a FACSCalibur (BD Biosciences).
The cells were gated first on the appropriate population based on the forward vs. side scatters.
Then, live cells were gated based on the FL-3 fluorescence of trypan blue. Non-infected live cells and the cells fixed with 3.7% formaldehyde and infected with the same MOI of CFSElabeled bacteria served as negative controls that were subtracted from the values by live cells. Figure 1. The levels of bacteria, CD4 + cells, CD8 + cells, and macrophages in the areas with different degrees of inflammatory infiltration. Serial sections of OLP tissues were subjected to in situ hybridization with a eubacterial 16S rRNA probe and immunohistochemical staining of CD4, CD8, and macrophage. By comparison of each slide with H&E stained section, the same locations with heavy (left panels) or low (right panels) inflammatory infiltration were chosen and photographed.    1 Hyperorthokeratosis in the epithelium 2 Hyperparakeratosis in the epithelium 3 Atrophy (reduction in thickness more than 1/3 of normal area) in the epithelium 4 Acanthosis (broadening of rete ridges more than two time normal width for area) in the epithelium 5 Simple hyperplasia (thickness more than 1 1/2 time normal thickness for area, excluding stratum corneum) in the epithelium 6 Atrophy alternating with hyperplasia in the epithelium 7 Finger-like rete ridges 8 Sawtooth rete ridges 9 Thick stratum granulosum more than five cell layers thick in the epithelium 10 Easily visible small groups or heavy infiltration by leucocytes in the epithelium 11 Liquefaction degeneration of basal layer of the epithelium 12 Intraepithelial vesicles: subepithelial vesicles excluded epithelial cellular changes 13 Colloid bodiescivatte bodies in the epithelium 14 Multinucleated epithelial cells: cells containing three or more nuclei