Functional characterization of human variants of NFKBIA: a key regulator of immune responsiveness implicated in susceptibility to infectious and inflammatory disease

Abstract

Objective/purpose: Genetic association studies have identified several polymorphisms in genes of the innate immunity cascade that appear to influence susceptibility to asthma and other inflammatory diseases. However, most candidate genes have not been functionally characterized for their impact on human immune responsiveness. An excellent candidate for functional investigation is NFKBIA which encodes I-Ba-the major negative regulator of NF-B. Single nucleotide polymorphisms (SNPs) in the promoter region of NFKBIA have been implicated in various infectious and inflammatory diseases. Specifically, the linked promoter SNPs rs2233406, rs3138053 and rs2233409 have been implicated in sarcoidosis, trachoma, acute respiratory distress syndrome, invasive pneumococcal disease, Graves' disease and respiratory syncytial virus. We investigated the mechanistic and functional impact of the promoter variants of NFKBIA on human immune responsiveness. Methods: Using a coding SNP that was in high linkage with NFKBIA SNPs rs3138053/rs2233406/rs2233409, we designed and validated an allelespecific PCR assay that could detect subtle differences in allele ratios between the major (ACC) and minor (GTT) promoter variants of SNPs rs3138053/rs2233406/rs2233409. Peripheral blood mononuclear cells of homozygous (ACC/ACC) and heterozygous (ACC/GTT) individuals were stimulated with LPS and live cultures of Streptococcus pneumoniae (serotype 14) for 3 and 4 hours. PBMCs of NFKBIA homozygotes and heterozygotes were stimulated with various Toll-like-receptor (TLR) ligands of the innate immune cascade to assay for differences in the innate immune response.