Cholesterol crystals enhance TLR2- and TLR4-mediated pro-inflammatory cytokine responses of monocytes to the proatherogenic oral bacterium Porphyromonas gingivalis

Abstract

Cholesterol deposits and pro-inflammatory cytokines play an essential role in the pathogenesis of atherosclerosis, a predominant cause of cardiovascular disease (CVD). Epidemiological evidence has linked periodontal disease (PD) with atherosclerotic CVD. Accordingly, viable periodontal pathogens, including Porphyromonas gingivalis, have been found in atherosclerotic plaques in humans and mice. We aimed to determine whether cholesterol crystals (CHCs) and oral bacteria synergize in the stimulation of human monocytes. Incubation of human monocytes with CHCs induced secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8. Moreover, CHCs markedly enhanced secretion of IL-1β by monocytes stimulated with the toll-like receptor (TLR) 4 agonist Escherichia coli lipopolysaccharide (LPS), and the TLR2 agonist Staphylococcus aureus lipoteichoic acid. Notably, CHCs also enhanced IL-1β secretion induced by P. gingivalis LPS and IL-1β secretion induced by whole P. gingivalis bacteria. This enhancement was abrogated by the NLRP3 inflammasome inhibitors Z-YVAD-FMK and glibenclamide. CHCs had no effect on cytokine production induced by P. gingivalis gingipains. Taken together, our findings support that CHCs, via stimulation of NLRP3 inflammasomes, act in synergy with the periodontal pathogen P. gingivalis to promote monocyte secretion of pro-atherogenic cytokines.

Fig 1. Influence of cholesterol crystals (CHCs) on E. coli (Ec)-lipopolysaccharide (LPS)-induced cytokine responses.

(A–E) Isolated monocytes were stimulated with CHCs (2mg/mL) for 20 hours. Interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and IL-8 in supernatants were measured after 20 hours of incubation, and are shown as mean±SD for experiments using 9 healthy donors. (F–J) Isolated monocytes were stimulated with Ec-LPS at two concentrations; 0.01 (low) and 1.0 (high) μg/mL in the absence (black circles) or presence (white circles) of CHCs (2mg/mL). Concentrations of IL-1β, TNF-α, IL-6, IL-10, and IL-8 in supernatants after 20 hours of incubation are shown as mean±SD for experiments using 4 healthy donors.

Fig 2. Influence of cholesterol crystals (CHCs) on toll-like receptor (TLR) 2-induced cytokine responses.

(A–E) Isolated monocytes were stimulated with the TLR2 agonist lipoteichoic acid from Staphylococcus aureus (Sa-LTA) at two concentrations; 0.1 (low) and 1.0 μg/mL (high) in the absence (black circles) or presence (white circles) of CHCs (2 mg/mL). Concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and IL-8 in supernatants after 20 hours of incubation are shown as mean±SD for experiments using 5 healthy donors.

Fig 3. Influence of cholesterol crystals (CHCs) on cytokine responses induced by P. gingivalis lipopolysaccharide (Pg-LPS).

(A–E) Isolated monocytes were stimulated with CHCs in different concentrations in the absence (black circles) or presence (white circles) of Pg-LPS (10 μg/mL). Concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10 and IL-8 in supernatants after 20 hours are shown as means±SD for experiments using 3 healthy donors. (F-I) Isolated monocytes were cultured with no stimulation, Arg-gingipain, CHCs, and Arg-gingipain and CHCs combined. The content of IL-1β, TNF-α, Il-6, and IL-10 in supernatants after 20 hours is shown as means±SD for experiments using 6 healthy donors.

Fig 4. Role of the NLRP3 inflammasome in induction of cytokine responses to cholesterol crystals (CHCs) and P. gingivalis lipopolysaccharide (Pg-LPS).

(A–E) Freshly isolated monocytes were cultured in presence of Pg-LPS (10 μg/mL) and CHCs, and the caspase-1 inhibitor Z-YVAD-FMK was added in concentrations of 5, 25 and 50 μg/mL. Concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and IL-8 in supernatants after 20 hours of incubation were measured, and data were normalized to P. gingivalis+CHCs. Data are shown as means±SD for experiments using 3 healthy donors. (F–J) Freshly isolated monocytes were cultured in presence of P. gingivalis lipopolysaccharide (Pg-LPS) (10 μg/mL) and CHCs (2 mg/mL), and the ATP-sensitive potassium channel inhibitor glibenclamide was added at concentrations of 1, 10 and 100 μM. Concentrations of IL-1β, TNF-α, IL-6, IL-10, and IL-8 in supernatants after 20 hours of incubation were measured, and data were normalized to P. gingivalis+CHCs. Data are shown as means±SD for experiments using 5 healthy donors.

Fig 5. Effect of interleukin (IL)-1β inhibition on P. gingivalis lipopolysaccharide (Pg-LPS)-induced cytokine production.

(A–E) Isolated monocytes were cultured without stimuli, or in the presence of Pg-LPS (10 μg/mL) or Pg-LPS in combination with IL-1 receptor antagonist (IL-1RA). Concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and IL-8 in supernatants after 20 hours are shown as means±SD for experiments using 7 healthy donors. (F) Isolated monocytes were cultured with Pg-LPS (10 μg/mL) and CHCs (2 mg/mL), and concentrations of IL-1β and TNF-α in supernatants were measured after 1, 3 and 7 hours of incubation. Data are shown as average and range for experiments using 2 healthy donors, normalized to the values obtained after 7 hours of incubation.

Fig 6. Toll-like receptor (TLR) 2-, TLR4- and caspase-dependent cytokine secretion of monocytes stimulated with P. gingivalis and cholesterol crystals (CHCs).

(A–E) Isolated monocytes incubated with P. gingivalis (Pg), as whole bacteria, and CHCs in the presence of various combinations of an antibody blocking TLR2 (anti-TLR2), the TLR4 antagonist lipopolysaccharide (LPS) from Rhodobacter sphaeroides (Rs-LPS), and the pan-caspase inhibitor Z-VAD-FMK. Supernatant concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and IL-8 after 20 hours of incubation are shown as means±SD for experiments using 4 healthy donors. Background values obtained in the absence of stimuli were subtracted, and data normalized to values obtained after incubation with P. gingivalis alone.