[Effects of Fusobacterium nucleus derived outer membrane vesicles on claudin-4 expression in oral epithelial cells].

PURPOSE: To investigate the effect of outer membrane vesicles (OMVs) secreted by Fusobacterium nucleatum (F.n) on Claudin-4 of human oral keratinocytes (HOK) and oral epithelial barrier function. METHODS: Fusobacterium nucleatum was cultured under anaerobic conditions. The OMVs were extracted by dialysis and characterized by nanosight and transmission electron microscopy (TEM). HOK were stimulated with OMVs at different mass concentrations(0-100 µg/mL) for 12 h, and stimulated with 100 µg/mL OMVs for 6 h and 12 h respectively. The expression of Claudin-4 at gene and protein level was analyzed by RT-qPCR and Western blotting. Inverted fluorescence microscope was used to observe co-localization of HOK and OMVs and localization and distribution of Claudin-4 protein. Human oral epithelial barrier was constructed by Transwell apical chamber. Transepithelial electrical resistance(TER) of barrier was measured with a transmembrane resistance measuring instrument(EVOM2), and the permeability of the barrier was evaluated by transmittance of fluorescein isothiocyanate-dextran(FD-4). Statistical analysis was performed with GraphPad Prism 8.0 software package. RESULTS: Compared with the control group, the expression of Claudin-4 at protein and gene level in the HOK of OMVs stimulated group was significantly reduced (P＜0.05), and immunofluorescence showed that the continuity of Claudin-4 fluorescence among cells was destroyed. OMVs stimulation decreased TER value of oral epithelial barrier(P＜0.05) and increased the transmittance of FD-4(P＜0.05). CONCLUSIONS: OMVs derived from Fusobacterium nucleatum may damage oral mucosal epithelial barrier function through inhibiting the expression of Claudin-4.

Intralesional and peripheral plasma of oral lichenoid reactions exhibit different cytokine profiles: A preliminary study.

BACKGROUND/PURPOSE: Oral lichenoid reactions (OLRs) are commonly characterized by the infiltration and activation of inflammatory cells at the interface of the oral mucosa. This study aimed to compare the cytokine profiles between intralesional and peripheral plasma from patients with OLRs and elucidate the cytokine profile in the OLR microenvironment. MATERIALS AND METHODS: A total of 26 paired intralesional and peripheral plasma samples were collected from patients with OLRs. A panel of 15 cytokines was measured using a Luminex assay. The reticular, erythema, and ulcerative score was used to evaluate the degree of OLR severity. RESULTS: IL-10 was detected in a fewer number of intralesional samples (19/26) compared to peripheral samples (26/26, p = 0.01). The intralesional plasma exhibited significantly elevated levels of granzyme B (median 108.94 vs. 16.00), TGF-ß1 (mean 30448.92 vs. 10199.04), TGF-ß2 (mean 1659.73 vs. 1308.49), and TGF-ß3 (mean 914.33 vs. 573.13) compared to the peripheral plasma (p = 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively). The levels of intralesional IL-2 (median 2.84 vs. 3.45, p = 0.019) and TNF-α (median 7.66 vs. 10.34, p = 0.048) were significantly lower in the intralesional plasma compared to the peripheral plasma. CONCLUSION: The intralesional concentrations of granzyme B and TGF-ß were elevated, whereas IL-2 and TNF-α were decreased in the OLR microenvironment compared to the peripheral plasma. These findings may contribute to establishing a panel of biomarkers that can be used to monitor the disease activity of OLRs in a large cohort study in future.

Potential association between Fusobacterium nucleatum enrichment on oral mucosal surface and oral lichen planus.

OBJECTIVE: We determined the bacterial community structure of the buccal mucosa in patients with oral lichen planus (OLP) and evaluated the potential association of Fusobacterium nucleatum with OLP. SUBJECTS AND METHODS: We collected buccal mucosal swab samples of patients with OLP (n = 20) and healthy controls (n = 10) and performed 16S rRNA gene sequencing and real-time PCR to determine potentially different bacteria. Damaged and adjacent non-damaged mucosal swab samples of 25 OLP patients were used to detect the amount of F. nucleatum by real-time PCR. RESULTS: Compared with healthy controls, enrichment of Fusobacterium and Granulicatella was more abundant in patients with OLP (p = .0146 and 0.0034). The abundance of Fusobacterium and F. nucleatum was significantly enriched on buccal mucosa of patients with OLP compared with healthy controls (p = .0043 and 0.0235). Compared with adjacent non-damaged buccal mucosa of OLP patients, the amount of F. nucleatum in the damaged mucosa was significantly increased (p = .001). We examined third-level KEGG pathways for bacteria on mucosal surface and found that genes controlling sporulation and ether lipid metabolism were enriched in patients with OLP. CONCLUSIONS: A high amount of F. nucleatum may be associated with OLP. Further studies are required to investigate the precise association of F. nucleatum with OLP.

The high expression level of programmed death-1 ligand 2 in oral lichen planus and the possible costimulatory effect on human T cells.

BACKGROUND: Oral lichen planus (OLP) is a T-cell-mediated chronic autoimmune disease whose precise etiology is unknown. The recently identified costimulatory programmed death-1 (PD-1) molecule and its ligands, PD-L1 and PD-L2, have been identified as CD28-B7 family molecules and constitute a regulatory pathway of potential therapeutic use in immune-mediated diseases. METHODS: We examined the expression of two ligands of PD-1 at both the protein and gene level in the focal mucosa and peripheral blood of OLP patients using immunohistochemistry and real-time PCR. Next, we used the PD-L2.Ig fusion protein and observed its effects on T cells, which were co-cultured with IFN-γ-treated keratinocytes (KCs) in the presence of PHA. RESULTS: We found that the expression of PD-L2 at both the gene and protein level was statistically different in peripheral blood and local lesion tissue of patients with OLP compared to the normal controls. The proliferation ability of T cells and the expression level of IFN-γ in the supernatant of the above co-culture model were significantly augmented (P < 0.05). PD-L2.Ig fusion protein significantly aggravated the apoptosis of T cells, inhibited the proliferation of T cells and decreased the release levels of IL-2 and IFN-γ in the model (P < 0.05). CONCLUSION: These data show that the increased expression of PD-L2, as a costimulatory molecule, may have an important modulatory function on the local immune responses of OLP in vivo.

[Establishment of a correlated cell model of oral lichen planus in vitro].

PURPOSE: To establish a correlated cocultured model of keratinocytes and T lymphocytes in vitro to simulate the immune response circumstance of oral lichen planus(OLP) lesions. METHODS: Keratinocytes prepared with enzyme digestion from normal oral mucosa and T lymphocytes prepared with negative isolation procedure by magnetic beads were co-cultured. Proliferation of T lymphocytes in cocultured cells was detected using MTT assay and the expression of IFN-γ was detected by ELISA. All the results were analyzed with independent samples t test using SAS6.12 software package. RESULTS: The proliferation level of T lymphocytes in the cocultured group of T lymphocytes and keratinocytes which expressed PD-L1 and PD-L2 was higher than that in the group of T lymphocytes (P < 0.05). The expression of IFN-γ in the cocultured group was significantly higher than that in the group of T lymphocytes (P < 0.01). CONCLUSIONS: The cocultured model of T lymphocytes and keratinocytes which express PD-L1 and PD-L2 can simulate the immune response circumstance of OLP lesions to a certain extent. Supported by National Natural Science Foundation of China (30872888),Research Fund of Science and Technology Commission of Shanghai Municipality(08DZ2271100) and Shanghai Leading Academic Discipline Project (S30206).

[Study on the expression and potential effect of PD-L1mRNA and PD-L2mRNA in oral lichen planus].

PURPOSE: To study the expression of PD-L1 and PD-L2mRNA in oral lichen planus(OLP) patients' peripheral blood and focal mucosa, and the different expression of target molecules in gene level in different clinical and pathological OLP groups. METHODS: The expression of PD-L1 and PD-L2 mRNA in OLP patients, which included 35 reticular and 25 atrophic-erosive OLP patients, 38 cases without dysplasia and 22 cases with dysplasia, was examined by real-time PCR. Peripheral blood and mucosa from 10 volunteers were used as control. All the results were analysed with Wilcoxon test by SAS.6.12 software package. RESULTS: The expression of PD-L2mRAN, but not PD-L1, was significantly higher in oral mucosa of OLP patients (P<0.01), while decreased in OLP patients' peripheral blood (P=0.0415). The gene expression of PD-L2 differed between different clinical types, and had highly significant correlation between the OLP patients' focal mucosa and peripheral blood(r=0.6976, P<0.0001). CONCLUSIONS: PD-L2 may have some potential effect on the pathogenesis of OLP at systemic and local level.