IL10 promoter rs6667202 polymorphism is functional in health but not in grade c periodontitis patients: A pilot study

Abstract

Background and objective:Previous studies have demonstrated an association between the IL10 promoter rs6667202 (C > A) single-nucleotide polymorphism (SNP) and grade C, stage 3 or 4 periodontitis (Perio4C) in the Brazilian population, where the altered A allele was detected more frequently in these patients. However, no functional analysis of this variation has yet been performed. Thus, the objective of this preliminary study was to evaluate the functionality of rs6667202 in gingival fibroblasts (GFs) of individuals with Perio4C and with periodontal health (PH) stimulated with Aggregatibacter actinomycetencomitans protein extract (AaPE).Methods: Patients with PH and Perio4C were segregated according to their genotype (AA, AC, or CC), and a biopsy was performed to establish the culture of the GFs.After GFs exposure to AaPE at 5 µg/ml for 1.5 h, RNA was extracted to analyze IL10 expression by qPCR. Aliquots of the cell's supernatant were subjected to immunoenzymatic analysis (MAGpix) to detect interleukin-10 (IL-10). Results:In PH, the genotypes AA and AC are related to less expression of IL10 (p = 0.027 and p < 0.0001) and less production of IL-10 (p = 0.002 and p = 0.001), when compared to CC. In Perio4C, there was no statistical difference between the genotypes (p > 0.05), although a lower IL-10 expression and release compared with PH CC was seen (p = 0.033 and p < 0.001). Conclusion:The rs6667202 SNP is functional in PH, as it decreases the expression and production of IL-10. In Perio4C, other factors may be masking its action by altering the IL-10's response.

Risk Genetic Variants (IL-10) for Osteoporosis in Han Population from Northwest China

“…This determines that IL-10 has important and broad clinical application prospects in autoimmune and gene therapy of inflammatory diseases. In recent years, a correlation between IL-10 genetic polymorphism and various disease risks has been reported, including tuberculosis, 21 periodontitis, 22 and bacterial sepsis. 23 In addition, the correlation between IL-10 genetic polymorphism and bone mineral density and OP risk has also been reported in many populations, including South Korea, 19 Turkey, 16 Taiwan, 18 and Chinese postmenopausal women.…”

Immune response characterization of primary gingival fibroblasts from Grade C periodontitis patients

“…Our research group previously obtained the cell populations used in this study, and the complete patient's inclusion and exclusion criteria, as well as the method used to obtain the primary culture of HGFs, are fully described in Stolf et al. (2002) 16 …”

“…Cell viability of HGFs incubated with Aa PE was determined in a previous study using one population of Perio4C group (virtually most susceptible to Aa aggression) and a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay after 3 and 7 days of incubation 16 . In the same study, a time course and dose–response experiment were performed to evaluate which concentration of Aa PE, a lower of 5 μg/ml and a higher of 20 μg/ml, and which period of exposure, 1.5, 3, 6, 12, or 24 h, promotes maximum gene expression by the HGFs 16 .…”

“…Cell viability of HGFs incubated with AaPE was determined in a previous study using one population of Perio4C group (virtually most susceptible to Aa aggression) and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after 3 and 7 days of incubation. 16 In the same study, a time course and dose-response experiment were performed to evaluate which concentration of AaPE, a lower of 5 μg/ml and a higher of 20 μg/ml, and which period of exposure, 1.5, 3, 6, 12, or 24 h, promotes maximum gene expression by the HGFs. 16 Based on Stolf et al, 2022 results, the cells were then exposed to 5 and 20 μg/ml of AaPE and also to the combination of 12-myristate 13phorbol acetate and ionomycin -calcium salt ** (PMA), a potent inducer of inflammation, at concentrations of 1 and 100 ng/ml, respectively, 18 during 1.5 and 3 h. The cells were seeded in 6-well cell culture plates at a concentration of 15 × 10 4 cells per well and grown in a standard culture medium for 24 h in a 37 • C, 5% CO 2 , and 98% humidity incubator.…”

“…16 In the same study, a time course and dose-response experiment were performed to evaluate which concentration of AaPE, a lower of 5 μg/ml and a higher of 20 μg/ml, and which period of exposure, 1.5, 3, 6, 12, or 24 h, promotes maximum gene expression by the HGFs. 16 Based on Stolf et al, 2022 results, the cells were then exposed to 5 and 20 μg/ml of AaPE and also to the combination of 12-myristate 13phorbol acetate and ionomycin -calcium salt ** (PMA), a potent inducer of inflammation, at concentrations of 1 and 100 ng/ml, respectively, 18 during 1.5 and 3 h. The cells were seeded in 6-well cell culture plates at a concentration of 15 × 10 4 cells per well and grown in a standard culture medium for 24 h in a 37 • C, 5% CO 2 , and 98% humidity incubator. Then, the standard medium was replaced for medium containing AaPE at 5 and 20 μg/ml, and for PMA, the negative control was represented by unstimulated cells.…”