Interleukin 4 Haplotypes Associated With Chronic Periodontitis: Functional Analysis

Objectives: Polymorphisms -590, +33 and VNTR in the Interleukin 4 (IL4) gene forming the TCI/CCI haplotype were 5 times more susceptible to chronic periodontitis (CP) (IL4+), while patients carrying the TTD/CTI haplotype seemed to be protected against CP (IL4-) and were associated with higher levels of IL-4 protein after periodontal treatment. The aim of this study was to investigate in vitro the functionality of these haplotypes by the reporter gene (GFP=green fluorescent protein) expression regulation to understand the influence of this genetic load in the immune response related to CP.
Methods: Plasmid GFP reporter vectors were constructed containing TCI or TCD haplotypes by GenScript. Using the TCI plasmid, two site-directed mutageneses were performed to obtain the CCI and CTI constructs. The PCR for site-directed mutagenesis was performed and the PCR product was transformed in competent E. coli DH5α by thermal shock. The plasmid was purified and the product was confirmed by sequencing. Plasmid containing each TCI, TTD, CCI, CTI haplotypes or only the promoter sequence of the IL4 gene (-590T allele) were transiently transfected by combination of electroporation/cationic reagents into the JM cells (human T lymphocytes). The cells were stimulated with PMA+Ionomycin, IL-1β, and IL-4 with anti-IL-12 (to induce polarization towards the Th2 phenotype), and after 24h GFP expression was assessed by flow cytometry.
Results: In general, the IL4 promoter sequence produced the lowest expression of GFP fluorescence for all stimuli, while the CTI haplotype demonstrated the statistically significant highest transcriptional activity of the GFP reporter gene.
Conclusions: In conclusion, the CTI haplotype, previously associated with protection against CP, showed increased transcriptional activity, suggesting higher IL4 gene expression. This might biologically explain the finding that patients carrying the IL4 TTD/CTI haplotype have higher levels of anti-inflammatory IL-4, being more protected against the development of CP.