Immune response of macrophages from young and aged mice to the oral pathogenic bacterium Porphyromonas gingivalis

Abstract

Periodontal disease is a chronic inflammatory gum disease that in severe cases leads to tooth loss. Porphyromonas gingivalis (Pg) is a bacterium closely associated with generalized forms of periodontal disease. Clinical onset of generalized periodontal disease commonly presents in individuals over the age of 40. Little is known regarding the effect of aging on inflammation associated with periodontal disease. In the present study we examined the immune response of bone marrow derived macrophages (BMM) from young (2-months) and aged (1-year and 2-years) mice to Pg strain 381. Pg induced robust expression of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10, chemokines; neutrophil chemoattractant protein (KC), macrophage colony stimulating factor (MCP)-1, macrophage inflammatory protein (MIP)-1α and regulated upon activation normal T cell expressed and secreted (RANTES), as well as nitric oxide (NO, measured as nitrite), and prostaglandin E2 (PGE2) from BMM of young mice. BMM from the 2-year age group produced significantly less TNF-α, IL-6 and NO in response to Pg as compared with BMM from 2-months and 1-year of age. We did not observe any difference in the levels of IL-1β, IL-10 and PGE2 produced by BMM in response to Pg. BMM from 2-months and 1-year of age produced similar levels of all chemokines measured with the exception of MCP-1, which was reduced in BMM from 1-year of age. BMM from the 2-year group produced significantly less MCP-1 and MIP-1α compared with 2-months and 1-year age groups. No difference in RANTES production was observed between age groups. Employing a Pg attenuated mutant, deficient in major fimbriae (Pg DPG3), we observed reduced ability of the mutant to stimulate inflammatory mediator expression from BMMs as compared to Pg 381, irrespective of age. Taken together these results support senescence as an important facet of the reduced immunological response observed by BMM of aged host to the periodontal pathogen Pg.

Figure 1

Age dependent cytokine production from mouse BMM in response to Pg. Cytokine analysis in cell culture supernatant fluids of mouse BMM from 2-months (n = 6), 1-year (n = 3) and 2-years (n = 3) of age. Cells were cultured with Pg 381 (filled bars), Pg DPG3 (gray bars) at multiplicity of infection (MOI) of 100, or medium only control (open bars) for 24 h. Cytokine levels were determined by multiplex analysis and the data presented as pg/mL mean ± standard error of the mean. A. TNF-α; B. IL-1β; C. IL-6; and D. IL-10. Asterisks indicate statistically significant (P < 0.05) as determined by Two way ANOVA with Bonferroni post-test. NS = not significant.

Figure 2

Chemokine response of young and aged mouse BMM to Pg. Chemokine levels in cell culture supernatant fluids of mouse BMM from 2-months (n = 6), 1-year (n = 3) and 2-years (n = 3) of age. BMM were challenged with Pg 381 (filled bars), Pg DPG3 (gray bars) at multiplicity of infection (MOI) of 100, or medium only control (open bars) for 24 h. Chemokine levels were determined by multiplex analysis and the data presented as pg/mL mean ± standard error of the mean. A. KC; B. MCP-1; C. MIP-1α; and D. RANTES. Asterisks indicate statistically significant (P < 0.05) as determined by Two way ANOVA with Bonferroni post-test analysis. NS = not significant.

Figure 3

Nitric oxide levels from young and aged mouse BMM in response to Pg. NO present in cell culture supernatant fluids from mouse BMM of 2-months (n = 6), 1-year (n = 3) and 2-years (n = 3) of age. BMM were cultured with Pg 381 (filled bars), Pg DPG3 (gray bars) at multiplicity of infection (MOI) of 100, or medium only control (open bars) for 24 h. Secreted NO levels were inferred by measuring μM nitrite levels using Greiss reaction as previously described [36]. Asterisks indicate statistically significant (P < 0.05) as determined by Two way ANOVA with Bonferroni post-test analysis. NS = not significant.

Figure 4

Prostaglandin E2 levels from young and aged mouse BMM in response to Pg. PGE2 present in cell culture supernatant fluids of mouse BMM from 2-months (n = 6), 1-year (n = 3) and 2-years (n = 3) of age. Cells were challenged with Pg 381 (filled bars), Pg DPG3 (gray bars) at multiplicity of infection (MOI) of 100, or medium only control (open bars) for 24 h. PGE2 levels were measured by ELISA and the data presented as pg/mL mean ± standard error of the mean. Statistically was determined by Two way ANOVA with Bonferroni post-test analysis. NS = not significant.