Date of Award

1-1-2020

Embargo Period

4-24-2022

Document Type

Thesis

Degree Name

Master of Biomedical Science

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Chad Novince

Second Advisor

Christopher Davies

Third Advisor

Alexander Alekseyenko

Fourth Advisor

Sakamuri Reddy

Abstract

Problem: A balanced relationship between the host and oral microbiota supports periodontal health and alveolar bone homeostasis. Antibiotic perturbation of the gut microbiota critically regulates the osteoimmune response at non-oral skeletal sites. However, the impact of antibiotics on the oral microbiota and osteoimmune mechanisms regulating alveolar bone homeostasis are unknown. Considering that periodontitis driven bone loss is caused by dysbiotic shifts in the oral microbiome, antibiotic disruption of the oral microbiota may have deleterious effects on alveolar bone homeostasis. Approach: Drinking water of sex-matched C57BL/6T specific-pathogen-free (SPF) mice was supplemented with minocycline (MINO) or vehicle (VEH) control treatment from age 6 to 12 weeks. SPF mice were euthanized at age 12 weeks to assess immediate effects and at age 18 weeks to evaluate sustained minocycline effects. 16S rDNA analysis was performed to evaluate bacterial load and phylum level alterations in the oral microbiome. Micro-CT was utilized to assess linear alveolar bone loss in the maxilla and cortical/trabecular bone microarchitecture in the mandible. qRT-PCR analysis was carried out to assess pro-osteoclastic and pro-inflammatory genes in the mandible bone marrow (MBM) and gingiva. TRAP+ osteoclastic cell outcomes in alveolar bone were evaluated by in situ and in vitro approaches. Flow cytometric analysis of immune cells was performed in MBM and cervical lymph nodes (CLNs). In a separate experiment, drinking water of male C57BL/6T germ-free (GF) mice was supplemented with MINO or VEH treatment from age 6 to 12 weeks. Results: MINO treatment increased overall bacterial load and induced phylum level alterations in the oral bacteriome of 12-week-old male SPF mice. Disruption of phylum level bacterial communities were sustained in 18-week-old male SPF mice. The effects of MINO treatment on the oral microbiota were sex-dependent as no alterations were seen in female mice. MINO treatment induced linear alveolar bone loss in both male and female SPF mice at the age 12 weeks and these effects persisted at age 18 weeks. Validating that MINO-induced catabolic effects on alveolar bone is dependent on the oral microbiota, no differences were found in linear alveolar bone loss in MINO vs. VEH treated male GF mice. Cortical bone thickness was decreased in the mandible in response to MINO treatment. Osteoclast cell size and bone interface were increased in maxillary alveolar bone sections from MINO vs. VEH treated male SPF mice. Exogenous MINO stimulation in MBM cultures derived from naïve 12-week-old male SPF mice increased osteoclast size and number of nuclei. Intriguingly, these findings suggest that MINO-induced pro-osteoclastic effects could be in part independent of the microbiota. Pro-inflammatory plasmacytoid dendritic cells (DCs) were upregulated within MBM and CLNs of MINO vs. VEH treated male SPF mice. Paralleling the plasmacytoid DCs, MINO treatment profoundly increased TH1 and TH17 cell populations in the MBM and CLNs. Conclusion: The current investigation reveals that MINO disruption of oral microbiota induces a pro-inflammatory immune response, which upregulates osteoclastogenesis, and drives alveolar bone loss. This novel research shows that oral MINO therapy, a commonly prescribed antibiotic treatment, may have detrimental clinical effects on alveolar bone in the healthy periodontium.

Rights

All rights reserved. Copyright is held by the author.

Download

Share

COinS