Functional Role of Haplotypes in the Interleukin-4 Gene: Pilot Study

Objectives: The inflamed periodontal tissue produces various cytokines, among which interleukin-4 (IL-4). Previously, we identified in 250 patients, that individuals carrying the haplotype TCI/CCI formed by polymorphisms -590(T>C), +33(T>C) and VNTR (I/D) were 5 times more susceptible to chronic periodontal disease (PD) (ORadjusted=5,3; 95%CI=2,2-12,9), while haplotype TTD/CTI conferred protection against PD (ORadjusted=0.18; 95%CI=0,04-0,88). Recently, we verified in a clinical study that these haplotypes influenced the levels of periodontal pathogens, such as Porphyromonas gingivalis (P.g.), the production of IL-4 and the response to periodontal treatment. However, it is necessary to perform an in vitro study with more controlled conditions, to verify the actual functionality of the IL4 gene haplotypes.
The aim of this pilot study was to investigate the biological functionality of the different IL4 gene haplotypes in innate and adaptive immune response.
Methods: Sequencing conferred the IL4 haplotype of 6 patients and peripheral blood was collected. Neutrophils, monocytes and lymphocytes were separated, and stimulated with IL-1b(positive control), Aggregatibacter actinomycetemcomitans (A.a.) and P.g. for 4 hours. Expression of IL4, TNFA and GAPDH (housekeep gene) were investigated by RT-qPCR to evaluate the influence of each haplotype in response to antigens.
Results: Regarding expression of IL4 we observed significant difference between the haplotype groups for monocytes: A.a. stimulation (p=0.04); for lymphocytes: IL-1B, A.a. and P.g.; and for neutrophils: IL-1B, A.a., P.g. and unstimulated cells (p=0.02). Regarding expression of TNFA we observed significant difference for monocytes: IL-1 stimulation, A.a. and unstimulated cells (p=0.024); for lymphocytes: IL-1B; and for neutrophils: A.a. stimulation (p=0.02).
Conclusions: In general, we conclude that individuals who carry the TTD/CTI IL4 haplotype, which conferred protection against PD, presented higher levels of IL4 and TNFA genes both in innate and adaptive immune response.