Investigation of functionality of the haplotypes in the IL4 gene

Author(s)

Giovana Anovazzi

• Document type: Doctoral Thesis
• Press: Araraquara. 2016-05-03.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara
• Defense date: 2016-03-28
• Advisor: Raquel Mantuaneli Scarel Caminaga

Abstract

Periodontal disease (PD) is multifactorial, with the influence of factors such as the presence of periodontopathogenic micro-reaction of the immune system, genetic susceptibility of the host, smoking, stress and presence of systemic diseases. The inflamed periodontal tissue produces various cytokines, among which interleukin 4 (IL- 4). Studies by this group found that subjects carrying the -590 polymorphism (T/C) +33 (T/C) and VNTR (I/D) in the IL4 gene haplotype forming the TCI/CCI are 5 times more susceptible to PD (ORadjusted= 5.3; 95% CI = 2.2 - 12.9), while the TTD/CTI haplotype conferred protection against the development of PD (ORadjusted= 0.18; 95% CI = 0.04- 0.88). So, to check the functionality of the haplotypes in the IL4 gene, the aim of this study is to investigate possible differences in the immune response against inflammatory and periodontal bacteria stimuli in collected blood cells from patients with different haplotypes. The functionality of these haplotypes in the IL4 gene will also be investigated by means of regulation of expression of the reporter gene present in the recombinant plasmids constructed artificially (constructs). Peripheral blood was collected from 5 patients of each haplotype and stimulated with inflammatory mediators and periodontal bacteria to assess the gene expression (mRNA RT-qPCR), secreted proteins (Multiplex) and intracellular proteins featuring cellular phenotypic profile (flow cytometry). Constructs containing each haplotype were transfected into cells JM and its functionality was evaluated by GFP reporter gene expression using flow cytometry. Regardless of the type of stimulation to which the cell cultures were subjected they were detected higherlevels of mRNA and protein of IL-4 haplotype in the group CTI/TTD, which also showed higher levels of other anti-inflammatory cytokines. Cell cultures to the haplotype TCI/CCI showed the opposite result; i.e., higher levels of pro-inflammatory cytokines. It was also observed that the TCI construct showed the highest levels of GFP expression compared to the construct containing only the polymorphism in the promoter region. In conclusion the haplotypes formed by polymorphisms in the IL4 gene influenced the gene expression levels and protein. (AU)

• FAPESP's process: 14/04638-2 - Investigation of the functional role of haplotypes in the interleukin 4 gene
• Grantee: Giovana Anovazzi Medeiros
• Support Opportunities: Scholarships in Brazil - Doctorate