Correspondence analysis.

<p>Correspondence analysis visualized 2-dimensionally with axes expressed as the 2 most crucial inertia values accounting for a cumulative inertia of 37.6%. Individual 1: blue square, individual 2: red square, individual 3: green triangle, individual 4: purple cross, individual 5: light blue cross.</p

Cluster analysis.

<p>Cluster analysis based on Spearman Rank Correlation. Participant 1: green, Participant 2: blue, Participant 3: red, Participant 4: yellow, Participant 5: purple. Sample denotation: P1-P5 (Participant 1-Participant 5), D1-D2 (Day 1-Day 2), S1-S6 (Sample 1-Sample 6).</p

Principal component analysis.

<p>Principal component analysis visualized 2-dimensionally with axes expressed as the second and third most crucial components accounting for 36.6% of the variation of the dataset. Individual 1: blue square, individual 2: red square, individual 3: green triangle, individual 4: purple cross, individual 5: light blue cross.</p

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

<div><p>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 <i>Porphyromonas gingivalis</i>, 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 <i>Escherichia coli</i> lipopolysaccharide (LPS), and the TLR2 agonist <i>Staphylococcus aureus</i> lipoteichoic acid. Notably, CHCs also enhanced IL-1β secretion induced by <i>P</i>. <i>gingivalis</i> LPS and IL-1β secretion induced by whole <i>P</i>. <i>gingivalis</i> bacteria. This enhancement was abrogated by the NLRP3 inflammasome inhibitors Z-YVAD-FMK and glibenclamide. CHCs had no effect on cytokine production induced by <i>P</i>. <i>gingivalis</i> gingipains. Taken together, our findings support that CHCs, via stimulation of NLRP3 inflammasomes, act in synergy with the periodontal pathogen <i>P</i>. <i>gingivalis</i> to promote monocyte secretion of pro-atherogenic cytokines.</p></div

Frequency of viable bacteria in donor blood.

<p>Freshly drawn blood from 60 healthy blood donors was fractioned into plasma and RBCs and plated on trypticase soy blood agar plates under aerobic or anaerobic conditions, and on blue lactose plates under aerobic conditions. RBC- or plasma-fractions were defined as positive if at least 1 colony was observed on at least one of the six plates. Shown are the frequencies of donors for whom bacteria were found in the RBC-fraction only, in the plasma-fraction only, in both fractions, or in none of the fractions.</p

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

<p>(A–E) Isolated monocytes incubated with <i>P</i>. <i>gingivalis</i> (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 <i>Rhodobacter sphaeroides</i> (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 <i>P</i>. <i>gingivalis</i> alone.</p

Influence of cholesterol crystals (CHCs) on <i>E</i>. <i>coli</i> (Ec)-lipopolysaccharide (LPS)-induced cytokine responses.

<p>(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.</p

Role of the NLRP3 inflammasome in induction of cytokine responses to cholesterol crystals (CHCs) and <i>P</i>. <i>gingivalis</i> lipopolysaccharide (Pg-LPS).

<p>(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 <i>P</i>. <i>gingivalis</i>+CHCs. Data are shown as means±SD for experiments using 3 healthy donors. (F–J) Freshly isolated monocytes were cultured in presence of <i>P</i>. <i>gingivalis</i> 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 <i>P</i>. <i>gingivalis</i>+CHCs. Data are shown as means±SD for experiments using 5 healthy donors.</p

Influence of cholesterol crystals (CHCs) on cytokine responses induced by <i>P</i>. <i>gingivalis</i> lipopolysaccharide (Pg-LPS).

<p>(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.</p

Microbial profile comparisons of saliva, pooled and site-specific subgingival samples in periodontitis patients

<div><p>Objectives</p><p>The purpose of this study was to compare microbial profiles of saliva, pooled and site-specific subgingival samples in patients with periodontitis. We tested the hypotheses that saliva can be an alternative to pooled subgingival samples, when screening for presence of periopathogens.</p><p>Design</p><p>Site specific subgingival plaque samples (n = 54), pooled subgingival plaque samples (n = 18) and stimulated saliva samples (n = 18) were collected from 18 patients with generalized chronic periodontitis. Subgingival and salivary microbiotas were characterized by means of HOMI<i>NGS</i> (Human Oral Microbe Identification using Next Generation Sequencing) and microbial community profiles were compared using Spearman rank correlation coefficient.</p><p>Results</p><p>Pronounced intraindividual differences were recorded in site-specific microbial profiles, and site-specific information was in general not reflected by pooled subgingival samples. Presence of <i>Porphyromonas gingivalis</i>, <i>Treponema denticola</i>, <i>Prevotella intermedia</i>, <i>Filifactor alocis</i>, <i>Tannerella forsythia</i> and <i>Parvimona micra</i> in site-specific subgingival samples were detected in saliva with an AUC of 0.79 (sensitivity: 0.61, specificity: 0.94), compared to an AUC of 0.76 (sensitivity: 0.56, specificity: 0.94) in pooled subgingival samples.</p><p>Conclusions</p><p>Site-specific presence of periodontal pathogens was detected with comparable accuracy in stimulated saliva samples and pooled subgingival plaque samples. Consequently, saliva may be a reasonable surrogate for pooled subgingival samples when screening for presence of periopathogens. Future large-scale studies are needed to confirm findings from this study.</p></div