Polybacterial Periodontal Infection Alters oxidative and Inflammatory Biomarkers in Primary Human Aortic Endothelial Cell (pHAECs)

Abstract

Background: Periodontitis (PD) is a highly prevalent inflammatory disease associated with complex microbial infection in the subgingival cavity. We have previously shown that polybacterial periodontal infection led to aortic atherosclerosis and lipid profile modulation; however, the underlying mechanism(s) has not been yet demonstrated.

Methods: Primary human aortic endothelial cells (pHAECs) were infected periodically for 48 hours either with P. gingivalis (monobacterial infection) or polybacterial periodontal pathogens, P. gingivalis (Pg), T. forsythia (Tf), T. denticola (Td), and F. nucleatum (Fn), using HoxBan coculture technique. Cell viability was assessed using MTT assay. Nitric oxide synthesis was measured in the form of total nitrite released into the media after incubation period. Inflammatory and oxidative gene expression were evaluated in the cell lysate using quantitative real-time polymerase chain reaction (qRT-PCR) at each time point (12-48h). Statistical analysis was performed using two-way ANOVA.

Results: pHAEC cell viability was significantly (p<0.05) reduced in both mono and poly-bacterial infection when compared with non-infected cells in a time dependent manner. Nitrite levels in the media were significantly impaired in both infection groups. mRNA expression for cytokines (TLR-4, IL-6, and TNF-α,) and inducible nitric oxide synthase (iNOS) were significantly (p<0.05) elevated in both experimental groups. In contrast, endothelial (e) NOS, tetrahydrobiopterin (BH₄) synthesis, GCH -1, Nrf2, and Nqo 1 were significantly (p<0.05) reduced in both experimental groups. Finally, polybacterial infection group showed greater changes in cell viability, nitrite levels, cytokines, eNOS/BH₄/Nrf2 expression in a time dependent manner.

Conclusions: Our study highlights the adverse effects of in vitro PD infection in NO mediated vascular endothelial cell function.

Keywords: nitric oxide; nitric oxide synthase; polybacterial periodontal pathogens; primary human aortic endothelial cells.

Figure 1:. Mono and polybacterial cytotoxicity against pHAEC’s.

The cell growth inhibitory rate was measured using MTT assay. The values are expressed as percentage of the control (uninfected with mono or polybacterial) at each time point, respectively (0 to 48 h). The results are from three independent experiments are represented in the form of means ± SD (n = 3, three independent experiments and, in each experiment, we had duplicates values for all the results). ^a p< 0.05 compared with control (uninfected) cells. ^b p< 0.05 compared with monobacterial infected cells. Pg—P. gingivalis; Poly – polybacterial.

Figure 2:. Mono and polybacterial induced nitric oxide changes in pHAEC’s.

The nitric oxide concentration in the supernatant at each time point (0 to 48 h), respectively was determined by the Griess reaction. The results are from three independent experiments are represented in the form of means ± SD (n = 3, three independent experiments and, in each experiment, we had duplicates values for all the results). ^a p< 0.05 compared with control (uninfected) cells. ^b p< 0.05 compared with monobacterial infected cells. Pg—P. gingivalis; Poly – polybacterial.

Figure 3:. Time-dependent effect of mono and polybacterial infection on mRNA expressions.

(A) iNOS, (B) eNOS and (C), GCH-1, mRNA expressions in pHAECs at each time point (0 to 48 h), respectively. Data were normalized with housekeeping gene (β-actin). All data are represented as 2^−ΔΔCt relative to vehicle. The results are from three independent experiments are represented in the form of means ± SD, three independent experiments and, in each experiment, we had duplicates values for all the results). ^a p< 0.05 compared with control (uninfected) cells. ^b p< 0.05 compared with monobacterial infected cells. Pg—P. gingivalis; Poly – polybacterial; iNOS - inducible nitric oxide synthase; eNOS - endothelial nitric oxide synthase; GCH-1 - GTP cyclohydrolase-1.

Figure 4:. Mono and polybacterial induces inflammatory cytokines in pHAEC’s.

(A) TLR-4, (B) IL-6, (C) TNF α and (D) MCP-1 at each time point, respectively (0 to 48 h). Data were normalized with housekeeping gene (β-actin). All data are represented as 2^−ΔΔCt relative to vehicle. The results are from three independent experiments are represented in the form of means ± SD, three independent experiments and, in each experiment, we had duplicates values for all the results). ^a p< 0.05 compared with control (uninfected) cells. ^b p< 0.05 compared with monobacterial infected cells. Pg—P. gingivalis; Poly – polybacterial; TLR-4 – Toll like receptor 4; IL-6 – interleukin 6; TNF α – tumor necrosis factor alpha; MCP-1 - Monocyte Chemoattractant Protein-1.

Figure 5:. Mono and polybacterial suppresses Nrf2, and NQO1, expression in pHAEC’s.

(A) Nrf2, and (B) NQO1 at each time point (0 to 48 h), respectively. Data were normalized with housekeeping gene (β-actin). All data are represented as 2^−ΔΔCt relative to vehicle. The results are from three independent experiments are represented in the form of means ± SD, three independent experiments and, in each experiment, we had duplicates values for all the results). ^a p < 0.05 compared with control (uninfected) cells. ^b p< 0.05 compared with monobacterial infected cells. Pg—P. gingivalis; Poly – polybacterial; Nrf2 - Nuclear factor erythroid-2 related factor 2; NQO1 - NADPH quinone dehydrogenase.