The Impact of Loricrin Deficiency on Porphyromonas gingivalis Induced Periodontitis

• Author / Creator

  Ho, Karen K

• Background: According to the 2017 periodontal classification, severe forms of periodontitis (SvP) are characterized as stage 4, grade C, and hold the greatest extent of destruction to the tissues and bones surrounding teeth. Underlying mechanisms in the etiology of SvP remain to be discovered. Some studies suggest that the causative factor(s) is related to host defense, while others suggest it is related to the pathogenic bacteria. We have focused on the cornified epithelium (CE), which is the outermost layer of the skin and oral mucosa. Within this epithelial layer, an insoluble protein, called loricrin, comprises 70-80% of the total protein mass, and helps maintain the barrier between the external and internal environments. Down regulation of loricrin has been shown to be involved in inflammatory skin disorders, which suggests that this protein plays a key role in the barrier function of the CE. An animal model used to study these skin disorders, Signal Transducer and Activator of Transcription 6 VT (Stat6VT) transgenic mice, overexpress the transcription factor Stat6, causing higher levels of T Helper Cell Type 2 (Th2) cytokines and consequently, decreased loricrin expression. Hypothesis: Stat6VT mice, due to an impaired epithelial barrier as a result of loricrin deficiency, will develop an exaggerated immune response and much greater bone loss compared with littermate control mice, both in response to normal bacteria in the oral cavity, and even more so in response to a periodontal disease pathogen challenge. Specific Aims: Aim 1. To determine if there is a reduction in the expression of loricrin in the oral epithelium of Stat6VT mice compared to controls. Aim 2. To investigate Stat6VT mice for alveolar bone loss, histological evidence of inflammation, and changes in tissue structure in the oral epithelium. Aim 3. To infect Stat6VT and control mice with Porphyromonas gingivalis (Pg) and compare alveolar bone loss, histological evidence of inflammation, and changes in tissue structure in the oral epithelium. Methods and Expected Outcomes: A longitudinal study was performed on unchallenged Stat6VT mice and controls to determine whether, similar to skin loricrin, there is a down-regulation of oral loricrin that corresponds with disease development. Based on the literature, mice were euthanized at weeks 6-8-, 10-13-, and 18-week time points and gingiva, palate, heads and blood were collected for experimentation. Cardiac puncture was performed to draw blood for analysis of Th2 cytokines and systemic inflammatory status (cytokine array). An enzyme-linked immunosorbent assay (ELISA) was used to determine loricrin expression in gingival tissue. Alveolar bone loss was assessed using microcomputed tomography (microCT) and tissue morphology of the palate was qualitatively assessed by histological examination. Additionally, we performed immunohistochemical assessments of leukocyte infiltration (antibody to cluster of differentiation 45 or CD45), antigen KI-67 (Ki67 antibody), keratin (cytokeratin 1 and 14 antibodies) and loricrin expression (loricrin antibody). Based on information from Aim 1, a time frame was chosen to orally infect mice with Pg by oral lavage every other day for 2 weeks. At endpoint, blood, heads and tissue were collected for assessment using methods similar to those in Aim 1. Results: The longitudinal study was made up of 9-11 male and 9-11 female Stat6VT and control mice for each time point. Phenotypical observations confirmed that the time points (6-8-, 10-13-, and 18 weeks) were appropriate for our study. Lesion onset occurred at 10-13-weeks; therefore, this time point was chosen for Pg infection. In both the uninfected and Pg infected groups, microCT analysis demonstrated a reduction in alveolar bone levels in the Stat6VT mice when compared to controls. Additionally, histological examination demonstrated increased signs of inflammation in the Stat6VT mice compared to controls. The ELISA did not demonstrate any significant differences in the 10-13- or 18-week time point. Cytokine array profiling identified 5 cytokines in the uninfected group: Cluster of Differentiation 30 Ligand (CD30L), Eotaxin 2, Monocyte Chemotactic Protein 5 (MCP5), Monokine Induced Gamma Interferon (MIG) and B Lymphocyte Chemoattractant (BLC). 3 cytokines were identified in the Pg-infected group: Osteopontin (OPN), Insulin-like Growth Factor Binding Protein 2 (IGFBP2), and Intercellular Adhesion Molecule 1 (ICAM1). Significance: Our studies suggested increased inflammation/bone loss in some cases that could be a result of an impaired epithelial barrier. SvP studies have been greatly hindered by the lack of a suitable animal model, and Stat6VT mice showed aspects of the disease, such as accelerated bone loss in response to a periodontal pathogen. Further study is necessary, but our results suggest that they could provide an important new tool in the field.

• Subjects / Keywords

  • Loricrin
  • Porphyromonas gingivalis
  • periodontitis

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-98k7-6294

• 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.