The impact of CE proteins on periodontal disease and the oral microbiome in white spot lesions patients

• Author / Creator

  Catunda, Raisa Queiroz

• Background: Periodontal disease is a pathological condition that affects the soft and hard tissues that surround teeth. Although its overall etiology is well- studied, there remains less clarity as to the etiology of the severe forms of this disease (SvP). The epithelial barrier is one of the greatest shields of our body and, at the same time, the portal of entry for many aggressors. To understand more about the etiology of SvP, we have focused on the cornified epithelium (CE), which is the outermost layer of the skin and oral mucosa. The insoluble protein, loricrin, comprises most of the protein mass and supports the barrier between environments. Loricrin downregulation has been linked to inflammatory skin disorders, which suggests that this protein may play a key role in the barrier function of the CE. Signal Transducer and Activator of Transcription 6 VT (Stat6VT) transgenic mice are a model of CE protein downregulation used to study skin disorders. These mice overexpress the transcription factor Stat6, increasing levels of T Helper Cell Type 2 cytokines, which compete for a mutual co-factor (p300/CBP) and consequently decrease expression of loricrin and filaggrin, two CE proteins. Porphyromonas gingivalis (Pg), a major periodontal disease pathogen, can also affect CE protein expression through chronic activation of Stat6VT. Our study aims to determine the impact of deficiency/mislocalization of several CE proteins on periodontal health in the context of infection by Pg and investigate the localization and expression of CE proteins in human gingival samples of patients that presented with SvP compared to healthy controls. Hypothesis: There are two hypotheses: 1. SvP patients will show downregulation/mislocalization of CE proteins accompanied by changes in epithelial differentiation and increased signs of inflammation in histological sections of uninvolved gingival tissues compared to healthy controls. 2. Stat6VT mice will develop an increased oral immune response and more significant alveolar bone loss compared to littermate controls in response to a pathogen challenge. Methods: Fifteen patients were recruited from the periodontology residency program at the University of Alberta. Patients with Stage III or IV and Grade C periodontitis (SvP group) or included as healthy controls. Discarded healthy tissue samples containing keratinized gingiva were collected during various periodontal procedures. The human samples were assessed for loricrin, filaggrin, cytokeratins 1, and 14 expressions. For the animal study, plasma was then carefully collected and used to analyze Th2 cytokines and systemic inflammatory status. Alveolar bone loss was assessed using microcomputed tomography, and tissue morphology of the soft tissues was qualitatively and quantitatively assessed by histological examination for several proteins. Results: Loricrin expression was visibly downregulated in all SvP patients when compared to controls. Filaggrin, on the other hand, showed a similar signal to controls in Stage III C patients, and a decreased signal in Stage IV C patients. CK1 presentation was more widespread but had lower expression (qualitatively) in all SvP patients. CK14 expression was also more widespread over several layers, instead of being contained to a single layer as in controls, and a decrease in expression was also observed in all SvP patients. Pathogen-infected Stat6VT mice presented increased alveolar bone loss compared to controls in 9 out of 16 examined sites. This increase in bone loss was accompanied with increased tissue inflammation, based on CD45+ cell count (35.30 ± 1.539 vs. 22.86 ± 1.067; p<0.0001). CK1 expression was dramatically decreased in Stat6VT mice (6.556 ± 0.76 vs. 19.94 ± 2.868; p<0.0001). CK14 expression was more broadly expressed in Stat6VT mice, with positive cells extending up into the granular layer, exceeding confinement to the basal layer, as observed in controls. Ki67 positive cells were decreased in Stat6VT mice (38.54 ± 2.637 vs. 53.97 ± 3.870; p:0.0013) and scattered in the distribution. Conclusions: Our study found decrease and difference in localization of important proteins of the oral epithelium, such as loricrin, filaggrin, CK1 and CK14. These changes in epithelial differentiation that may exacerbate the effects of Porphyromonas gingivalis infection in Stat6VT mice. There were similarities between the mouse model and the most severe forms of human periodontitis, suggesting similar etiologies. The hypothesis that barrier integrity may contribute to SvP needs further study. The identification of Stat6VT mice as a model of SvP may significantly impact mechanistic study.

• Subjects / Keywords

  • loricrin
  • epithelial barrier
  • periodontitis
  • white spot lesions

• Graduation date

  Fall 2023

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-k3fj-rm53

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.