GWAS for Interleukin-1β levels in gingival crevicular fluid identifies IL37 variants in periodontal inflammation

Abstract

There is no agnostic GWAS evidence for the genetic control of IL-1β expression in periodontal disease. Here we report a GWAS for "high" gingival crevicular fluid IL-1β expression among 4910 European-American adults and identify association signals in the IL37 locus. rs3811046 at this locus (p = 3.3 × 10^-22) is associated with severe chronic periodontitis (OR = 1.50; 95% CI = 1.12-2.00), 10-year incident tooth loss (≥3 teeth: RR = 1.33; 95% CI = 1.09-1.62) and aggressive periodontitis (OR = 1.12; 95% CI = 1.01-1.26) in an independent sample of 4927 German/Dutch adults. The minor allele at rs3811046 is associated with increased expression of IL-1β in periodontal tissue. In RAW macrophages, PBMCs and transgenic mice, the IL37 variant increases expression of IL-1β and IL-6, inducing more severe periodontal disease, while IL-37 protein production is impaired and shows reduced cleavage by caspase-1. A second variant in the IL37 locus (rs2708943, p = 4.2 × 10^-7) associates with attenuated IL37 mRNA expression. Overall, we demonstrate that IL37 variants modulate the inflammatory cascade in periodontal disease.

Fig. 1

Data analysis workflow and Manhattan plot for GCF and serum IL-1β. a Analysis Workflow schema for GCF IL-1β QTL illustrating genomic and phenotype association of GCF-IL1β trait. b Manhattan Plot for Top Quartile of GCF IL-1β, as compared to lower 3 quartiles. All 22 chromosomes are shown, plotting the -log10 (p values) for each of the 656,292 high-quality SNPS using ProbABEL. For GCF-IL-1β the dominant locus emerges in chr2 with no other genomic regions showing SNPs that cross the significance threshold of p = 5 × 10^–8, as shown as a horizontal reference line. The insert panel represents the Manhattan plot for the serum IL-1β for the same subjects. Although no serum IL-1β SNPs reach GWA significance, the top three are identified in chr 3(rs7627767), 5(rs13188793) and 16 (rs6565189)

Fig. 2

LocusZoom Plot for IL-37 SNPs. The genomic region shown in the Manhattan plot in chr2 is magnified using LocusZoom to illustrate the individual SNPs within the dominant locus for Top Quartile of GCF IL-1β. a LocusZoom Plot shows entire IL-1 gene complex region from IL1A to IL1RN. IL37 (IL1F7, rs3811046, p = 3.3 × 10⁻²²), IL36G (IL1F9, rs11677903, p = 5.4 × 10⁻¹⁴), IL38 (IL1F10, rs1301182, p = 2.4 × 10⁻⁸), IL36A (IL1F6, rs6714534, p = 6.7 × 10⁻⁹) and IL1B (rs16944 p = 6.5 × 10⁻¹⁹). b LocusZoom Plot for IL-37 SNP-Variant 1 highlighting the lead SNP at rs3811047 (missense). c LocusZoom Plot for IL-37 SNP-Variant 2 that shows additional missense SNPS in disequilibrium with rs2708943 as the indexed SNP

Fig. 3

Association of IL-37V1 with GCF Cytokines and DC IL-1β mRNA expression. a Z-Score Standardized Log GCF IL-1β Levels by IL-37 Variant 1 Haplotype. This figure reflects the Z-score for each of the six GCF mediators assayed in a sample of 107 subjects who were genotyped for the IL-37 V1 locus by pyrosequencing, assaying six mediators within the same GCF sample for each subject and normalizing to IL-37WT. There was an overall increase in the levels of these mediators expressed as a composite z score for each subject pooling each mediator and transforming using the 1.1 allele as zero (P = 0.026) and comparing 1.1 to 1.2 + 2.2. The increased expression of IL-1β and IL-8 were significant by pairwise comparisons comparing 1.1 to 2.2 at p = 0.046 and p = 0.03. b IL-1β secretion at various times by dendritic cells from IL-37WT-1.1 individuals and homozygous IL-37V1-2.2 after LPS stimulation. Levels of IL-1β mRNA (fold expression) are shown at LPS concentration stimulation using isolated peripheral blood monocytes differentiated to DCs from subjects who were pyrosequenced for IL-37WT-1.1 (group 1) or for IL-37V1-2.2 (group 2). The insert shows and overall significant enhanced level of IL-1β mRNA among group 2 (V1 2.2) over time expressed as area under curve (AUC) with standard error bars at p < 0.05

Fig. 4

Expression and localization of IL-37 in human gingival tissue. a The sketch of five IL-37 isoforms. b PCR products amplified with specific primer pairs to different isoforms of IL-37 cDNA. c The relative mRNA levels of different isoforms of IL-37 in human gingival tissues from sites with periodontitis were quantitated using real time PCR with isoform-specific primers. d The relative mRNA levels of IL-37b (isoform1) in gingival tissues between health and periodontitis (n ≥ 5/group). Data show the mean ± SD. ***p < 0.0001 (unpaired two-tailed Student’s t-test). e The representative level of IL-37 positive staining in epithelial and connective tissue of human gingival tissue from healthy and periodontitis patient using IHC. Scale bar is 200 µm. f The localization of IL-37b and CD138 were determined in serial slides of gingival tissue from periodontitis patient. The localization of IL-37 in human gingival tissue was mainly associated with epithelial and infiltrated plasma cells. The black circles with red number indicate representative region of IL-37 positive staining. A, B represent the low magnification of IL-37 and CD138 staining in human gingival tissue with Scale bar 200 µm. C–F represent the high magnification of two immune cells infiltrated regions in the connective tissue with Scale bar 20 µm. g, h represent the high magnification of epithelial cells layer with Scale bar 20 µm. g IL-37b and CD138 were co-localized in human gingival tissue of periodontitis patient by immunofluorescence using confocal microscope. The results represent CD138 (green), IL-37 (red), DAPI (blue), and bright field (white). The scale bar is 20 µm. Data shown here are representative of three independent experiments

Fig. 5

rhIL-37bWT and variants suppress RAW and PBMC response to LPS in vitro. a The analysis workflow for role of IL-37 and IL-37 variants in murine and human cell responses and murine periodontitis model. b The protein sketch of pro-IL-37b including cleavage site (amino acid 20) and variant sites (green: V1, red: V2). c Pro and mature rhIL-37b extracted from E. coli were detected on a 12% SDS–PAGE and analyzed by Coomassie blue and western blot using anti-IL-37 antibody. d IL-6 production in supernatant of RAW cells treated with gradient concentrations of pro and mature rhIL-37b following LPS (50 ng/ml) stimulation. Data show the mean±SD. *p < 0.05 (One-way ANOVA and Dunnett’s multiple comparisons tests, compare the mean of each group with the mean of LPS alone stimulation group). (+) or (−) indicate the cells with or without LPS stimulation. Results are representative of three independent experiments. e IL-6 production in supernatant of RAW cells treated with different concentrations of mature form of WT, V1, V2, and V1V2 rHIL-37b following LPS (50 ng/ml) stimulation. Data show the mean ± SD. *p < 0.05 (One-way ANOVA and Dunnett’s multiple comparisons tests, compare the mean of each group with the mean of LPS alone stimulation group). Results are representative of three independent experiments. f IL-6 production in supernatant of human PBMC cells treated with different concentrations of mature form of WT, V1, V2, and V1V2 rHIL-37b following LPS (50 ng/ml) stimulation. Data show the mean ± SD. *p < 0.05 (One-way ANOVA and Dunnett’s multiple comparisons tests, compare the mean of each group with the mean of LPS alone stimulation group). Results are representative of three independent experiments

Fig. 6

rhIL-37bWT and variants suppress the pathogenesis of periodontitis model in vivo. a The sketch to test the role of human recombinant IL-37b in murine periodontitis model. b Representative three-dimensional maxillary molars of pro, mature IL-37b and PBS injection groups. The distances from the cemento-enamel junction and alveolar bone crest (CEJ–ABC) of first molar root in buccal side of no-ligature and ligature sites are indicated by blue arrows. Scale bar is 0.5 mm. c The quantitative data of the distance from CEJ-ABC of first molar root in buccal side for each group. The results are expressed as the mean ± SD (n = 5/group). *p < 0.05 (One-way ANOVA and Tukey multiple comparisons tests). d Representative HE and MPO staining of pro, mature IL-37b and PBS injection groups. Scale bar is 200um. MPO positive cell are indicated by red arrowheads. E Epithelial layer. C Connective tissue. R Root of teeth. B Alveolar bone. e The quantitative data of the MPO positive cells between the root of first and second molar for each group. The results are expressed as the mean ± SD (n = 5/group). *p < 0.05, **p < 0.01 (One-way ANOVA and Tukey multiple comparisons tests). f The IL-1β expression level in the gingival tissues of ligature induced periodontitis model between PBS and mature IL-37bWT injection groups. The results are expressed as the mean ± SD (n = 6/group). **p < 0.01 (unpaired two tailed Student’s t test). g Representative IL-1β staining of PBS and mature IL-37b injection groups. IL-1β positive cell are indicated by red arrowheads. E (Epithelial layer). C (Connective tissue). R (Root of teeth). B (Alveolar bone). Scale bar is 200 μm for I and II. 500 μm for III and IV. h Representative three-dimensional maxillary molars of IL-37b V1, V2, V1V2, and PBS injection groups. Scale bar is 0.5 mm. i The quantitative data of the distance from CEJ-ABC of first molar root in buccal side for each group. The results are expressed as the mean ± SD (n ≥ 5/group). **p < 0.01 (One-way ANOVA and Tukey multiple comparisons tests)

Fig. 7

IL-37b expression in HEK293T cells transfected with IL-37bWT and Variants. a Representative picture of RFP positive HEK293T cells after transfection. Scale bar is 400 µm. b Western blot to detect IL-37b production in the cell lysates of HEK293T cells transient transfected with IL-37b WT and Variants plasmid. c IL-37b mRNA level in the cell lysates of HEK293T cells transient transfected with IL-37b WT and Variants plasmid. d IL-37b production in the supernatant and lysate of HEK293T cells co-transfected IL-37b (WT or V1) and caspase-1 plasmid. e The sketch of Transwell system to detect the suppressive activity of IL-37b WT and V1. f After LPS stimulation, IL-1β expression level in the supernatant of co-culturing THP-1 cells with HEK293T cells transfected by IL-37b WT or V1 plasmid. The results are expressed as the mean ± SD. **p < 0.01 (unpaired two-tailed Student’s t-test). Results are representative of three independent experiments

Fig. 8

IL-37b expression among IL-37bWT and Variants in IL-37b transgenic mice. a The agarose gel confirms the IL-37b insert in IL-37WT, V1 and V1V2 transgenic mice which is absent in the background (control) WT strain. b IL-37b production in the blood cell lysates of tgIL-37WT, tgIL-37V1 and control mice. c Semi-quantitative band density analysis of IL-37b from panel b normalized to β-actin. Data are means ± SD (n = 3/group). ***p < 0.001(unpaired two-tailed Student’s t-test). d IL-37b production in the blood cell lysates of tgIL-37WT and tgIL-37V1V2 mice (n = 3/group). e IL-6 expression level in the supernatant of BMDM (Bone marrow-derived macrophage) from tgIL-37WT and tgIL-37V1 mice at various LPS concentrations. The results are expressed as the mean ± SD. **p < 0.01, ***p < 0.001 (One-way ANOVA and Sidak multiple comparisons test). Results are representative of three independent experiments. f BMDM IL-1β expression by tgIL-37WT and tgIL-37V1 mice at various LPS concentrations in presence of ATP. The results are expressed as the mean ± SD. *p < 0.05, ***p < 0.001 (One-way ANOVA and Sidak multiple comparisons test). (+) indicates the ATP stimulation for BMDM. Results are representative of three independent experiments. g The IL-1β expression level in the gingival tissues of ligature induced periodontitis model between IL-37WT and IL-37V1 transgenic mice group. The results are expressed as the mean ± SD (n ≥ 6/group). *p < 0.05 (unpaired two tailed Student’s t test). h Representative three-dimensional maxillary molars of IL-37WT and V1 transgenic mice in ligature induced periodontitis model. Scale bar is 0.5 mm. i The quantitative data of the distance from CEJ-ABC of first and second molar root in buccal side for each group. The results are expressed as the mean ± SD (n ≥ 5/group). (unpaired two tailed Student’s t test). *p < 0.05