Antimicrobial peptides and proteins in innate immunity : emphasis on isolation, characterization and gene regulation

Abstract: Antimicrobial peptides are endogenous antibiotics and effector molecules of Innate immunity. These peptides are mainly localised to epithelial linings and circulating neutrophils. They kill microbes by disruption of their membranes. The two main families of mammalian antimicrobial peptides are defensins and cathelicidins. In this thesis, the presence of antimicrobial peptides and proteins in human colon mucosa and Vernix caseosa (vernix) has been demonstrated. In addition, the gene regulation of the human cathelicidin LL-37 has been investigated in a colon epithelial cell line. A rat model for cathelicidins has also been developed. A peptide/protein extract was prepared from human colon mucosa and was found to exhibit antibacterial activity against both Gram-positive and Gram-negative bacteria, in addition to antifungal activity. Several antimicrobial peptides and proteins were identified in this extract. One of them was ubiquicidin, which has not previously been isolated from human tissue. The colon was thus shown to be protected by a complex mixture of antimicrobial peptides and proteins, which together exert potent antimicrobial activity. Vernix is a creamy substance covering the skin of the foetus during the last trimester. Peptide/protein extracts of vernix displayed potent antimicrobial activity. A multitude of antimicrobial peptides, proteins and lipids were isolated and characterized in vernix. Interestingly, nearly all of the most abundant proteins in vernix are considered to be involved in innate immunity. Vernix thereby forms a potent surface defence barrier, protecting the foetus and the newborn against bacteria, fungi and parasites. In addition, vernix components also exhibit protease inhibition and opsonizing features. The rat cathelicidin rCRAMP has been isolated and characterized as a 43-residue peptide, which is processed differently than the mouse cathelicidin CRAMP, despite identical primary structure at the processing sites. rCRAMP was shown to have an antimicrobial activity and expression pattern similar to that of LL-37. Therefore, responses related to cathelicidin expression in health and disease can now be studied in the rat. The gene regulation of LL-37 in a human colon epithelial cell line was investigated, utilizing a luciferase reporter system. The promoter was found to contain at least one enhancer and two silencer elements. The enhancer element was demonstrated to be a functional Ets binding site and one of the silencers is probably regulated by Vitamin D. In addition, the second intron was found in our system to enhance the transcriptional activity in the presence of butyrate and in cooperation with the 3' end of the promoter. Thereby the gene regulation of LL-37 is beginning to be unveiled.