Influence of periodontal pathogens on induction of epithelial-mesenchymal transition (EMT) in oral keratinocytes

Shoker, Satvir Singh (2022). Influence of periodontal pathogens on induction of epithelial-mesenchymal transition (EMT) in oral keratinocytes. University of Birmingham. Ph.D.

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Abstract

Epithelial-mesenchymal transition (EMT) is a process where epithelial cells gain migratory and invasive properties and is associated with dissolution of epithelial integrity. Inflammatory mediators released as a host response to periodontal pathogens are known to induce EMT. This project investigated the ability of these periodontal pathogens to induce EMT in vitro and to identify whether the natural EMT-inhibitor curcumin could interfere with this process. A 2D- oral epithelium model using H400 oral keratinocytes was exposed to Escherichia coli lipopolysaccharide (LPS) (positive control) and heat-killed (HK) or oxygen-killed (OK) periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum and Treponema denticola for 8-days. EMT-related changes were assessed by quantitative real time-polymerase chain reaction (qRT-PCR), enzyme linked immunosorbent assay (ELISA), immunofluorescence, Ibidi culture-insert (2-well) wound healing assay and trans-epithelial electrical resistance (TEER). Scanning electron microscopic analysis showed that heat-killing resulted in loss of T. denticola morphology but was maintained after oxygen-killing. Further analysis by mass spectrometry-based proteomics of T. denticola isolated membrane, revealed that heat-killing was more detrimental to T. denticola proteins than oxygen-killing. Stimulation of oral keratinocytes by E. coli LPS and all HK and OK pathogens for 8-days, increased secretion of pro-inflammatory cytokines interleukin (IL)-1β, IL-8 and transforming growth factor-β1 (TGF-β1) and gene expression of EMT-related transcription factors snail-1, snail-2 and twist-1, resulting in down-regulated expression of epithelial markers, E-cadherin, desmoplakin-1 and cytokeratin 5. Concomitantly, expression of mesenchymal markers, N- cadherin, vimentin and matrix metalloproteinase-9 (MMP-9), were up-regulated. Molecular changes were associated with compromised epithelial barrier integrity and increased cell migration. Simultaneous treatment with 5μM curcumin suppressed gene expression of OK pathogen induced pro-inflammatory cytokines and snail-1. In addition, curcumin inhibited OK pathogen induced down-regulation of epithelial markers and inhibited up-regulated expression of mesenchymal markers. Curcumin inhibited OK P. gingivalis and T. denticola induced cell migration. These data indicated that naturally derived EMT-inhibitors, such as curcumin, may inhibit periodontal pathogen induced EMT, therefore maintaining the epithelial barrier and potentially preventing the progression of periodontitis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):