Effect of Aging on Periodontal Inflammation, Microbial Colonization, and Disease Susceptibility

Abstract

Periodontitis is a chronic inflammatory disease induced by a biofilm that forms on the tooth surface. Increased periodontal disease is associated with aging. We investigated the effect of aging on challenge by oral pathogens, examining the host response, colonization, and osteoclast numbers in aged versus young mice. We also compared the results with mice with lineage-specific deletion of the transcription factor FOXO1, which reduces dendritic cell (DC) function. Periodontitis was induced by oral inoculation of Porphyromonas gingivalis and Fusobacterium nucleatum in young (4 to 5 mo) and aged (14 to 15 mo) mice. Aged mice as well as mice with reduced DC function had decreased numbers of DCs in lymph nodes, indicative of a diminished host response. In vitro studies suggest that reduced DC numbers in lymph nodes of aged mice may involve the effect of advanced glycation end products on DC migration. Surprisingly, aged mice but not mice with genetically altered DC function had greater production of antibody to P. gingivalis, greater IL-12 expression, and more plasma cells in lymph nodes following oral inoculation as compared with young mice. The greater adaptive immune response in aged versus young mice was linked to enhanced levels of P. gingivalis and reduced bacterial diversity. Thus, reduced bacterial diversity in aged mice may contribute to increased P. gingivalis colonization following inoculation and increased periodontal disease susceptibility, reflected by higher TNF levels and osteoclast numbers in the periodontium of aged versus young mice.

Keywords: DNA-seq; bacteria; dendritic cell; lymphocyte; osteoclast; periodontitis.

Figure 1.

Aging and dendritic cell (DC) FOXO1 deletion reduce the number of DCs in lymph nodes and reduce DC migration. DCs in the lymph nodes were identified by immunohistochemistry with a specific antibody to CD205. DC migration was examined in DC2.4 cells or bone marrow–derived DCs after treatment with bovine serum albumin (BSA) or advanced glycation end products (AGEs) and stimulated with CCL19 or CCL21 added to the bottom chamber in a Transwell assay. (A) Images of CD205-positive immunostaining in cervical lymph nodes. (B) Quantification of CD205-positive immunostaining in lymph nodes. (C) Migrated DC2.4 DCs stimulated with CCL19. (D) Migrated bone marrow–derived DCs stimulated with CCL21. Values are expressed as the mean ± SE, n = 4 to 6. ^*Significant difference (P < 0.05) between young mice and corresponding aged mice or between DCs migrated from treatment with CCL19 or CCL21 and vehicle. ⁺Significant difference (P < 0.05) between wild-type mice and age-matched DC FOXO1 deletion mice. ^#Significant difference (P < 0.05) between DCs migrated from treatment with AGE and control BSA. This was done by 2 or 3 independent experiments.

Figure 2.

Aging enhances activation of the adaptive immune response. Serum antibody (IgG1) to Porphyromonas gingivalis was measured by ELISA. Paraffin sections of neck lymph nodes from young or aged dendritic cell (DC) FOXO1-deleted mice or control littermates mice were stained with CD138-specific antibody for plasma cells or IL-12 by immunofluorescence. Matched control antibody was negative for immunofluorescent staining. (A) Serum anti–P. gingivalis IgG1 antibody measured by ELISA. (B) Quantification of CD138-positive immunostaining in lymph nodes. (C) Quantification of IL-12-positive immunostaining in lymph nodes. Values are expressed as the mean ± SE, n = 4 to 6. *Significant difference (P < 0.05) between young mice and corresponding aged mice. ⁺Significant difference (P < 0.05) between wild-type mice and age-matched DC FOXO1-deleted mice. n = 5 or 6 mice per group.

Figure 3.

Aging increases colonization of Porphyromonas gingivalis and reduces bacterial diversity. Bacteria were separated from the surface of tooth crown, and the number of P. gingivalis and total bacteria were determined with quantitative real-time polymerase chain reaction (PCR). DNA collected from bacteria on tooth surfaces was sequenced with the MiSeq Sequencing System. (A) P. gingivalis on the tooth surface was determined by quantitative real-time PCR of the ISPg1 gene. CFU, colony-forming units. (B) Total oral bacteria loading on the tooth surface was determined by quantitative real-time PCR of the 16S rRNA gene. (C) Alpha diversity and rarefaction plots were computed with Faith’s phylogenetic diversity (PD_whole_tree). (D) Alpha diversity and rarefaction plots were computed with Shannon index biodiversity. (E) Bar charts represent the relative abundance of the main microbial phyla related to the periodontal diseases in mice subjected to different treatment. Phylogenetic orders are represented by different colors: Green indicates commensal bacteria, including Ruminococcaceae and Lactobacillus; yellow and red indicate pathogenic bacteria, including Bacillus, Staphylococcus, Prevotella, Pseudomonas, Acinetobacter, Fusobacterium, and Porphyromonas. Values are expressed as the mean ± SE, n = 4 to 6. ^*Significant difference (P < 0.05) between young mice and corresponding aged mice. ⁺Significant difference (P < 0.05) between wild-type mice and age-matched DC FOXO1 deletion mice.

Figure 4.

Aging reduces the amount of alveolar bone. Maxillary teeth were scanned by micro–computed tomography. (A) Micro-CT of maxilla. (B) Remaining bone area/total area between the first and second molars. Values are expressed as the mean ± SE, n = 5 or 6 mice per group. ^*Significant difference (P < 0.05) between young mice and corresponding aged mice. ⁺Significant difference (P < 0.05) between wild-type mice and age-matched dendritic cell FOXO1 deletion mice.

Figure 5.

Aging increases susceptibility to periodontal disease. Serial paraffin sections that included maxillary first and second molars and alveolar bone were prepared. Osteoclast number was analyzed by tartrate-resistant acid phosphatase (TRAP) staining, and TNF expressed in the gingival epithelium was determined by immunofluorescence. (A) Histomorphometric analysis of osteoclast number analyzed by TRAP staining (blue arrow indicates the osteoclast). (B) Number of osteoclasts per bone length was measured in alveolar bone adjacent to the second molar. (C) Images of TNF-positive staining in gingival epithelium between the first and second molars. The upper part of the white line indicates epithelium, and the lower part indicates connective tissue. (D) The percentage TNF-positive cells was measured in gingival epithelium by immunofluorescence. Values are expressed as the mean ± SE, n = 5 or 6 per group. ^*Significant difference (P < 0.05) between young mice and corresponding aged mice. ⁺Significant difference (P < 0.05) between wild-type mice and age-matched dendritic cell FOXO1 deletion mice.