The ATC/TTC haplotype in the Interleukin 8 gene in response to Gram-negative bacteria: A pilot study.

OBJECTIVE: The aim of this study was to investigate the functionality of ATC/TTC (Hap-1) and ATT/TTC (Hap-2) Interleukin (IL) 8 gene haplotypes in the response of neutrophils to Gram-negative bacteria associated with periodontitis. DESIGN: Neutrophils were isolated by gradient centrifugation from whole peripheral blood of systemically healthy individuals presenting the two IL8 gene haplotypes. Neutrophils were stimulated with P. gingivalis, A. actinomycetemcomitans and PMA/ionomycin. Cytokine gene expression (RT-qPCR) and migration/chemotaxis (boyden chamber assay) were compared according to the presence of Hap-1 or Hap-2 haplotypes. Protein production was also evaluted in the multiplex assay using the mixed population of leukocytes present in the whole blood from the same individuals. The influence of these two haplotypes on the IL8 promoter activity was assessed in gene-reporter experiments. RESULTS: Hap-1 haplotype in neutrophils and leukocytes exacerbated the response to stimulation with Gram-negative bacteria, with higher levels of TNF-α (mRNA and protein), IL-1ß, IL-2R and IFN-γ (protein) and with increased chemotaxis. Presence of the T allele at the rs4071 polymorphism (alias -251) was associated with increased activity of IL8 proximal promoter. CONCLUSIONS: Neutrophils and leukocytes carrying the Hap-1 haplotype (ATC/TTC) in the IL8 gene present an enhanced response to stimulation with Gram-negative bacteria associated with periodontitis. Presence of the T allele (rs4073) in the IL8 proximal promoter increases transcription activity.

Functionality of the Interleukin 8 haplotypes in lymphocytes and macrophages in response to gram-negative periodontopathogens.

Individuals carrying the ATC/TTC haplotype (Hap-1) in the interleukin 8 (IL8) gene were reported as more susceptible to chronic periodontitis (CP), an infectious disease associated with Gram-negative bacteria, in comparison to patients with the ATT/TTC haplotype (Hap-2). This study investigated the functionality of the IL8 haplotypes in lymphocytes and monocytes of individuals carrying the Hap-1 or Hap-2 IL8 haplotypes in the response to CP-associated Gram-negative bacteria (periodontopathogens). Peripheral blood was collected from 6 subjects carrying each haplotype, and their immune cells were challenged with periodontopathogens or phorbol 12-myristate 13-acetate (PMA) plus Ionomycin. Depending on the immune cell type (lymphocytes or monocyte-derived macrophages) the assessed outcomes were: phenotypical polarization, gene expression, phagocytic activity, chemotaxis and production of reactive oxygen species (ROS). Subjects carrying the Hap-1 haplotype showed increased expression of IL8 and TNFA and significantly skewing towards pro-inflammatory Th1/M1/Th17 phenotypes. There was increased percentage of ROS-producing monocyte-derived macrophages from individuals carrying the Hap-1 haplotype. Cells from individuals presenting the Hap-2 haplotype had an overall attenuated response to periodontopathogens, with a significant shift towards the Treg phenotype. In conclusion, the IL8 haplotypes showed to be functional both in monocyte-derived macrophages and lymphocytes. The Hap-1 haplotype previously associated with increased susceptibility to CP demonstrated greater skewing to pro-inflammatory Th1/M1/Th17 phenotypes and production of ROS.