Investigation of the functional role of haplotypes in the interleukin 4 and interleukin 8 genes

Abstract

Periodontal Disease (PD) is multifactorial, with the influence of factors such as the presence of microorganisms periodontopathogenic, genetic susceptibility of the host, immune system reaction, smoking, stress and systemic diseases. The inflamed periodontal tissue produces various cytokines, among which interleukin 4 (IL-4) and interleukin 8 (IL-8). Studies by this group investigated polymorphisms in IL4 and IL8 in the 500 patients with and without periodontitis, and individuals have been identified with genetic susceptibility to PD. Polymorphisms -251(T/A), +396(T/G) and +781(C/T) in the IL8 gene, forming the haplotype ATC / TTC showed 2 times higher susceptibility to PD (OR adjusted = 2,241, 95% CI= 1,10 -4,55). Regarding the IL4 gene, individuals carrying the haplotype TCI/CCI formed by polymorphisms -590(T/C), +33 (T/C) and VNTR (I/D) are 5 times more susceptible to PD (ORadjusted = 5,3; 95% CI = 2,2 - 12,9), while haplotype TTD/CTI confer protection against PD ORadjusted = 0.18 (95% CI = 0,04 - 0,88). All results remained significant after adjusting for variables such as age, gender, ethnicity and habito of smoking. Recently, it was investigated whether these haplotypes influence the levels of periodontal pathogens, the production of IL-4 and IL-8 and the response to periodontal treatment, comparing outcomes among patients carrying different haplotypes. However, despite some significant results, it was not possible to make sure that this was due to the influence of haplotypes. Therefore, it is necessary to perform in vitro studies with more controlled conditions, to verify the actual functionality of the haplotypes in the genes IL4 and IL8. The results suggested that individuals classified as genetically susceptible to periodontitis for carrying certain haplotypes in the IL8 gene respond in a more exaggerated even with low microbial challenge when compared to individuals considered genetically resistant to periodontitis. Regarding the IL4 gene haplotype (TCI / CCI), associated with susceptibility to PD, it was shown that individuals carrying this haplotype had higher levels of P. gingivalis, T. forsythia, T. denticola compared to patients genetically protected against DP (haplotype TTD / CTI).So, the objectives of this research project: 1) From cells collected of peripheral blood from patients with different haplotypes IL4 and IL8 genes, investigate possible differences in innate and adaptive immune response, 2) From artificially constructed recombinant plasmids containing alleles that make up the different haplotypes (constructs) investigate the specific role of polymorphisms in the regulation of gene expression. It must be considered that, although select patients with the haplotypes of interest, in objective 1, will influence the patient's overall genetic load, whereas the second goal, only the functionality of these polymorphisms/haplotypes in genes IL4 and IL8 is to be evaluated. Regarding the first objective: after conferring haplotype sequencing in genes IL4 and IL8 for each patient, peripheral blood will be collected to evaluate the interaction of each polymorphism/haplotype in response to antigens by the regulation of gene expression of IL-8/IL-4 (mRNA), secreted proteins (multiplex) and intracellular protein for characterization of cell phenotypic profile (flow cytometry). Regarding the second objective: the constructs containing the haplotype (some will pass through site-directed mutagenesis) will be transfected into NIH3T3 cells and the functionality of the different polymorphisms/haplotype in the promoter and/or intron will be assessed by reporter gene expression using fluorescence microscopy and flow cytometry. Intended to compare the response of the possible influence of different haplotypes between that obtained from cell types of patients (objective 1) the response obtained by insertion of the constructs in the lineage NIH3T3 (objective 2). (AU)