Antimicrobial peptide regulation by small molecules in humans and other primates

Graduation date: 2011Antimicrobial peptides (AMPs) play an important role in the innate immune system. Determining the pathways by which these proteins are regulated allows for modulation of their expression for better health. Two families of antimicrobial peptides have been studied in humans: cathelicidins and defensins. There is a single cathelicidin in humans called human cathelicidin antimicrobial peptide (CAMP). Defensins are divided into two families in humans, the alpha and beta defensins. In the beta defensin family, defensin beta 4 (DEFB4) is an inducible antimicrobial peptide. Both CAMP and DEFB4 play integral roles in maintaining barrier defenses and health.\ud The human cathelicidin antimicrobial peptide gene is regulated by a wide array of small molecules; however, there are still many untested small molecules. We proposed a high throughput screen to find additional compounds that regulate antimicrobial peptides. After screening nearly 5,500 small molecules in the NIH Clincal Compound Library and the ChemBridge DIVERSet libarary, two stilbenoids were found that regulate cathelicidin expression. When combined with 1,25 dihydroxy vitamin D₃ both stilbenoids synergistically induced cathelicidin gene expression in U937 cells.\ud DEFB4 is an antimicrobial peptide induced by inflammatory responses and during infections. Several studies observed that DEFB4 is regulated by 1,25 dihydroxy vitamin D₃ either through a vitamin D response element (VDRE) in the promoter or by an indirect pathway that activates NF-kB. It is unclear if the vitamin D receptor directly regulates the DEFB4 gene by binding to its promoter. We hypothesized that if vitamin D induces DEFB4 by the VDR binding to the promoter, then the putative VDRE would be evolutionarily and functionally conserved in humans and primates. To test this hypothesis, we obtained the promoter sequences from 11 primates and investigated the conservation of the VDRE. The sequence was conserved in primates which suggest the VDRE sequence was selected for over 50-60 million years of evolution. This supports a role for the vitamin D pathway in the regulation of the DEFB4 gene, but functional assays have\ud failed to clearly demonstrate a response of the DEFB4 gene to 1,25 dihydroxy vitamin D in tissue culture systems. Additional experiments are required to elucidate the role of the vitamin D pathway in regulating the DEFB4 gene.\ud A thorough understanding of antimicrobial peptide gene expression will lay the foundation for therapeutic approaches to strengthen the innate immune system