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

doi:10.19439/j.sjos.2023.02.003

Abstract

Abstract: 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.

Key words: Oral lichen planus, Epithelial barrier function, Fusobacterium nucleatum, Outer membrane vesicles, Claudin-4

CLC Number:

R781.5

YANG Cheng-long, YANG Meng, WANG Yu-feng, SONG Chen-cheng, DU Guan-huan, TANG Guo-yao. Effects of Fusobacterium nucleus derived outer membrane vesicles on claudin-4 expression in oral epithelial cells[J]. Shanghai Journal of Stomatology, 2023, 32(2): 126-131.

References

[1] Alrashdan MS, Cirillo N, Mccullough M.Oral lichen planus: a literature review and update[J]. Arch Dermatol Res, 2016, 308(8): 539-551.
[2] Baek K, Choi Y.The microbiology of oral lichen planus: is microbial infection the cause of oral lichen planus?[J]. Mol Oral Microbiol, 2018, 33(1): 22-28.
[3] Du G, Wang Y, Chen J, et al.Potential association between Fusobacterium nucleatum enrichment on oral mucosal surface and oral lichen planus[J]. Oral Dis, 2020, 26(1): 122-130.
[4] Han YW.Fusobacterium nucleatum: a commensal-turned pathogen[J]. Curr Opin Microbiol, 2015, 23: 141-147.
[5] Toyofuku M, Nomura N, Eberl L.Types and origins of bacterial membrane vesicles[J]. Nat Rev Microbiol, 2019, 17(1): 13-24.
[6] Brunner Pm, Leung Dym, Guttman-Yassky E.Immunologic, microbial, and epithelial interactions in atopic dermatitis[J]. Ann Allergy Asthma Immunol, 2018, 120(1): 34-41.
[7] Takahashi N, Sulijaya B, Yamaha-Hara M, et al.Gingival epithelial barrier: regulation by beneficial and harmful microbes[J]. Tissue Barriers, 2019, 7(3): e1651158.
[8] Günzel D, Yu AS.Claudins and the modulation of tight junction permeability[J]. Physiol Rev, 2013, 93(2): 525-569.
[9] Hämäläinen L, Soiny Y, Pasonen-Seppänen S, et al.Alterations in the expression of EMT-related proteins claudin-1, claudin-4 and claudin-7, E-cadherin, TWIST1 and ZEB1 in oral lichen planus[J]. J Oral Pathol Med, 2019, 48(8): 735-744.
[10] Yang M, Liu X, Luo Q, et al.An efficient method to isolate lemon derived extracellular vesicles for gastric cancer therapy[J]. J Nanobiotechnology, 2020, 18(1): 100-110.
[11] Groeger S, Jarzina F, Windhorst A, et al.Influence of retinoic acid on human gingival epithelial barriers[J]. J Periodontal Res, 2016, 51(6): 748-757.
[12] Michikawa H, Fujita-Yoshigaki J, Sugiya H.Enhancement of barrier function by overexpression of claudin-4 in tight junctions of submandibular gland cells[J]. Cell Tissue Res, 2008, 334(2): 255-264.
[13] Liu W, Li M.The role of claudin-4 in the development of gastric cancer[J]. Scand J Gastroenterol, 2020, 55(9): 1072-1078.
[14] Yang G, Seok JK, Kang H, et al.Skin barrier abnormalities and immune dysfunction in atopic dermatitis[J]. Int J Mol Sci, 2020, 21(8): 2867-2881.
[15] Liu H, Hong X, Sun T, et al.Fusobacterium nucleatum exacerbates colitis by damaging epithelial barriers and inducing aberrant inflammation[J]. J Dig Dis, 2020, 21(7): 385-398.
[16] Groeger S, Doman E, Chakraborty T, et al.Effects of Porphyromonas gingivalis infection on human gingival epithelial barrier function in vitro [J]. Eur J Oral Sci, 2010, 118(6): 582-589.
[17] Maerz JK, Steimle A, Lange A, et al.Outer membrane vesicles blebbing contributes to B. vulgatus mpk-mediated immune response silencing[J]. Gut Microbes, 2018, 9(1): 1-12.
[18] Chmiela M, Walczak N, Rudnicka K.Helicobacter pylori outer membrane vesicles involvement in the infection development and Helicobacter pylori-related diseases[J]. J Biomed Sci, 2018, 25(1): 78-89.
[19] He Y, Shiotsu N, Uchida-Fukuhara Y, et al.Outer membrane vesicles derived from Porphyromonas gingivalis induced cell death with disruption of tight junctions in human lung epithelial cells[J]. Arch Oral Biol, 2020, 118: 104841.
[20] Alvarez CS, Badia J, Bosch M, et al.Outer membrane vesicles and soluble factors released by probiotic Escherichia coli Nissle 1917 and Commensal ECOR63 enhance barrier function by regulating expression of tight junction proteins in intestinal epithelial cells[J]. Front Microbiol, 2016, 7: 1981-1995.
[21] Martin-Gallausiaux C, Malabirade A, Habier J, et al.Fusobacterium nucleatum extracellular vesicles modulate gut epithelial cell innate immunity via FomA and TLR2[J]. Front Immunol, 2020, 11: 583644.