Increased Sphingosine-1-Phosphate Serum Concentrations in Subjects with Periodontitis: A Matter of Inflammation

Figures & data

Table 1 Baseline Characteristics of Study Subjects; Study of Health in Pomerania (SHIP-Trend-0; 2008–2012)

	N	Participants Aged 20–83 Years	N	Participants Aged 20–83 Years with ≥12 Periodontal Measurement Sites
Age, years	3371	50.1 ± 14.8	3231	49.4 ± 14.6
Male gender	3371	1632 (48.4%)	3231	1562 (48.3%)
School education	3371		3231
<10 years		620 (18.4%)		555 (17.2%)
10 years		1823 (54.1%)		1767 (54.7%)
>10 years		928 (27.5%)		909 (28.1%)
Smoking status	3371		3231
Never		1243 (36.9%)		1196 (37.0%)
Former with <20 pack years		990 (29.4%)		949 (29.4%)
Former with ≥20 pack years		235 (7.0%)		216 (6.7%)
Current with <20 pack years		617 (18.3%)		613 (19.0%)
Current with ≥20 pack years		286 (8.5%)		257 (7.9%)
Known diabetes mellitus, yes	3371	290 (8.6%)	3231	250 (7.7%)
HbA1c, %	3371	5.3 ± 0.8	3231	5.3 ± 0.8
Body mass index, kg/m^2	3371	27.9 ± 5.1	3231	27.7 ± 5.0
Waist-to-hip-ratio	3371	0.88 ± 0.09	3231	0.88 ± 0.09
Medication, yes	3371		3231
Statins		364 (10.8%)		314 (9.7%)
Acetylsalicylic acid as platelet  aggregation inhibitor		283 (8.4%)		242 (7.5%)
Cortisone		25 (0.7%)		20 (0.6%)
Systemic antibiotics		36 (1.1%)		34 (1.1%)
Mean PD, mm	3371	2.6 ± 0.7 2.4 (2.1; 2.8)	3231	2.6 ± 0.7 2.4 (2.1; 2.8)
Percentage of sites with PD 3+mm, %	3371	43.0 ± 23.4 40.0 (25.0; 58.3)	3231	42.2 ± 22.7 39.6 (25.0; 57.1)
Percentage of sites with PD 4+mm, %	3371	14.4 ± 19.3 6.3 (0; 20.0)	3231	13.8 ± 18.6 6.3 (0; 19.2)
Mean CAL, mm	3198	2.5 ± 1.7 2.1 (1.6; 3.3)	2983	2.3 ± 1.5 1.9 (1.2; 3.1)
Percentage of sites with CAL 3+mm, %	3198	39.8 ± 34.7 31.3 (7.1; 70.0)	2983	36.7 ± 33.1 27.5 (5.8; 64.3)
Percentage of sites with CAL 4+mm, %	3198	24.7 ± 30.9 9.4 (0; 40.4)	2983	21.6 ± 27.9 8.1 (0; 33.3)
CDC/AAP case definition	3120		2983
No or mild		1511 (48.4%)		1476 (49.5%)
Moderate		1088 (34.9%)		1027 (34.4%)
Severe		521 (16.7%)		480 (16.1%)
Number of teeth	4042		–
28		677 (16.8%)		–
20–27		2032 (50.3%)		–
10–19		664 (16.4%)		–
1–9		405 (10.0%)		–
0		264 (6.5%)		–
Number of decayed or filled surfaces	3421	16.6 ± 10.3 16.0 (9.0; 24.0)	–	–
Percentage of decayed or filled surfaces, %	3421	38.1 ± 25.3 34.4 (18.6; 53.1)	–	–
Sphingosine-1-phosphate, nmol/L	3371	811.7 ± 177.1 800.1 (684.3; 919.8)	3231	813.6 ± 175.8 801.7 (686.7; 920.3)
High-sensitivity C-reactive protein, mg/L	3198	2.5 ± 5.0 1.3 (0.7; 2.8)	3066	2.5 ± 5.0 1.2 (0.7; 2.7)
High-sensitivity C-reactive protein	3283		3150
≤2 mg/L		2180 (66.4%)		2124 (67.4%)
>2 mg/L and ≤3 mg/L		355 (10.8%)		339 (10.8%)
>3 mg/L		748 (22.8%)		687 (21.8%)

Notes: Data are presented as mean ± standard deviation (SD), median (25%; 75% quantile) or absolute frequencies (percentages). Medication intake was defined for regularly taken statins (ATC code C10AA), corticosteroids (ATC codes H02A and H02B), acetylsalicylic acid as platelet aggregation inhibitor (B01AC06) or systemic antibiotics (ATC code J01).

Abbreviations: PD, probing depth; CAL, clinical attachment loss; CDC/AAP, Centers for Disease Control and Prevention/American Academy of Periodontology.

Table 2 Results from Linear Regression Models Analyzing the Association Between Periodontitis Variables and Sphingosine-1-Phosphate Serum Levels (Continuously Modelled) in All Subjects (Aged 20–83 Years)

		Model 1, Adjusted for Age and Gender		Model 2, Fully Adjusted
Periodontitis variables	N	B (95% CI)	P value	B (95% CI)	P value
Mean PD, mm	3371	12.89 (3.83; 21.95)	0.005	10.12 (0.73; 19.51)	0.04
Percentage of sites with PD 3+mm, 10%	3371	4.52 (1.83; 7.23)	0.001	3.83 (1.02; 6.64)	0.008
Percentage of sites with PD 4+mm, 10%	3371	4.94 (1.72; 8.16)	0.003	3.83 (0.48; 7.19)	0.03
Mean CAL, mm	3198	6.72 (2.20; 11.24)	0.004	5.14 (0.44; 9.83)	0.03
Percentage of sites with CAL 3+mm, 10%	3198	4.13 (1.84; 6.42)	<0.001	3.38 (1.00; 5.76)	0.005
Percentage of sites with CAL 4+mm, 10%	3198	3.27 (0.84; 5.71)	0.008	2.68 (0.15; 5.21)	0.04
CDC/AAP definition	3120
No or mild	1511	0.00 (Ref.)		0.00 (Ref.)
Moderate	1088	18.81 (3.74; 33.88)	0.01	14.75 (−0.42; 29.92)	0.06
Severe	521	25.50 (6.03; 44.97)	0.01	20.02 (0.10; 39.94)	0.049
		Ptrend=0.006		Ptrend=0.03
Number of teeth	4042
28	677	0.00 (Ref.)		0.00 (Ref.)
20–27	2032	21.16 (3.91; 38.40)	0.02	20.56 (3.35; 37.78)	0.02
10–19	664	25.77 (3.16; 48.37)	0.03	22.51 (−0.31; 45.34)	0.05
1–9	405	13.64 (−12.11; 39.39)	0.30	12.36 (−13.92; 38.64)	0.40
0	264	24.56 (−4.42; 53.55)	0.10	20.10 (−9.88; 50.09)	0.20
		Ptrend=0.32		Ptrend=0.51
Number of teeth (only subjects with PD assessments)	3370
28	630	0.00 (Ref.)		0.00 (Ref.)
20–27	1850	17.93 (−0.32; 36.17)	0.05	16.98 (−1.21; 35.17)	0.07
10–19	583	25.82 (1.74; 49.89)	0.04	21.34 (−2.96; 45.63)	0.09
1–9	307	12.44 (−16.09; 40.96)	0.40	12.02 (−17.04; 41.08)	0.40
		Ptrend=0.29		Ptrend=0.38

Notes: Model 2 was adjusted for age (cubic splines with 3 knots), gender, school education, smoking status combined with pack-years, waist-to-hip-ratio, known diabetes mellitus and HbA1c.

Abbreviations: PD, probing depth; CAL, clinical attachment loss; CDC/AAP, Centers for Disease Control and Prevention/American Academy of Periodontology; N, number; B, linear regression coefficient; CI, confidence interval.

Figure 1 Model predicted mean (with 95% confidence bands) of sphingosine-1-phosphate serum concentrations for varying levels of (A) mean probing depth and (B) mean clinical attachment loss.

Table 3 Results from Linear Regression Models Analyzing the Association Between Periodontitis Variables and Sphingosine-1-Phosphate Serum Levels (Continuously Modelled) in Subjects Aged 20–83 Years with at Least 12 Measurement Sites for Assessments of Probing Depth or Clinical Attachment Loss

		Model 1, Adjusted for Age and Gender		Model 2, Fully Adjusted
Periodontitis variables	N	B (95% CI)	P value	B (95% CI)	P value
Mean PD, mm	3231	12.61 (3.06; 22.17)	0.01	9.30 (−0.63; 19.23)	0.07
Percentage of sites with PD 3+mm, 10%	3231	4.43 (1.61; 7.24)	0.002	3.51 (0.57; 6.45)	0.02
Percentage of sites with PD 4+mm, 10%	3231	5.23 (1.82; 8.63)	0.003	3.96 (0.40; 7.51)	0.03
Mean CAL, mm	2983	8.13 (2.84; 13.42)	0.003	6.07 (0.59; 11.55)	0.03
Percentage of sites with CAL 3+mm, 10%	2983	4.75 (2.31; 7.20)	<0.001	3.82 (1.28; 6.35)	0.003
Percentage of sites with CAL 4+mm, 10%	2983	4.02 (1.27; 6.77)	0.03	3.16 (0.31; 6.01)	0.004
CDC/AAP case definition	2983
No or mild	1476	0.00 (Ref.)		0.00 (Ref.)
Moderate	1027	20.02 (4.63; 35.40)	0.01	15.22 (−0.27; 30.71)	0.054
Severe	480	29.22 (9.16; 49.27)	0.004	22.79 (2.24; 43.35)	0.03
		Ptrend=0.002		Ptrend=0.02

Notes: Model 2 was adjusted for age (cubic splines with 3 knots), gender, school education, smoking status combined with pack-years, waist-to-hip-ratio, known diabetes mellitus and HbA1c. PD, probing depth.

Abbreviations: CAL, clinical attachment loss; CDC/AAP, Centers for Disease Control and Prevention/American Academy of Periodontology; N, number; B, linear regression coefficient; CI, confidence interval.

Figure 2 Model predicted mean (with 95% confidence bands) of sphingosine-1-phosphate serum concentrations for varying levels of high-sensitivity C-reactive protein (logarithmised). Estimates for effects of logarithmised CRP levels on S1P were B=24.5 (standard error 6.7, p<0.001) for the first spline and B=−15.4 (standard error 7.9, p=0.053) for the second spline according to the fully adjusted linear regression model.

Figure 3 Representative immunofluorescence staining of human gingival tissue (A–F). (A–C) represent paraffin-embedded sections of normal gingival tissue, whereas (D–F) show paraffin-embedded sections of inflamed gingival tissue. Both DAPI staining of nuclei (blue) and staining of the sphingosine-1-phosphate (S1P)-generating enzyme sphingosine kinase 1 (SphK1; red) were performed for all sections (A–F). Staining of the S1P-degrading enzyme sphingosine-1-phosphate lyase (Lyase; green) was conducted in section C+F. Fluorescence micrographs were taken using a laser scanning microscope (Zeiss LSM780) and a 63x/1.4 objective (bar = 10 µm).