Expression of cathelicidin antimicrobial peptides in man and rat

Abstract: Endogenous antimicrobial peptides are effectors of innate immunity. They kill microorganisms by disrupting their cell membranes, but are also believed to have signaling functions in the immune system. They are present in epithelia, secretions and phagocytic cells. Major families of mammalian antimicrobial peptides are the defensins and the cathelicidins. This thesis describes different aspects of gene regulation of the human cathelicidin antimicrobial peptide hCAP18/LL-37 and characterization of rCRAMP, the homolog to LL-37 in the rat. Rat rCRAMP peptide was isolated from neutrophils. In addition, the peptide and the corresponding messenger RNA (mRNA) was found in tissues of the gastrointestinal tract, lung and thymus. Expression pattern, antimicrobial activity, and predicted alpha-helical structure were similar to mouse CRAMP and human LL-37. Contact with live bacteria was not required for rCRAMP expression since germ-free rats also expressed it. Phylogenetic analysis of cathelicidin preproregions confirmed homology of rCRAMP to other cathelicidins and illustrated that gene duplication in this family has been species- or lineage-specific. Characterization of rCRAMP enables use of the rat for studying responses related to cathelicidin expression. rCRAMP was also isolated from the central nervous system (CNS) of the rat, and determined to constitute a major part of the peptide/protein antimicrobial activity in rat CNS. The peptide was active against the neuropathogen Neisseria meningitidis. Regions of the CNS positive for rCRAMP both on the peptide/protein and mRNA levels were olfactory bulb, cerebellum, medulla oblongata and spinal cord. We hypothesize that rCRAMP is part of an ancient immune defense in rat CNS, but it may have additional functions. This is the first detailed report of cathelicidin expression in brain and spinal cord, and provides a basis from which to conduct functional studies of cathelicidins in the CNS. Gene regulation of LL-37 was studied with focus on the colonic epithelium. The colon harbors large quantities of bacteria and LL-37 is expressed in colonic epithelial cells as part of a defense barrier. Butyrate is produced by bacterial fermentation in the colon and this short chain fatty acid was found to induce LL-37 transcription via MAPK/ERK kinase. Further analysis of potential regulatory elements in the CAMP gene encoding LL-37 was conducted using a luciferase reporter system. This indicated presence of different silencer elements and an enhancer element in the promoter. Electrophoretic mobility shift assay indicated that a transcription factor of the Ets family likely binds to the enhancer element. An intron appeared to be involved in the inducing effect of butyrate, and an possible alternative upstream translation start codon was also identified. Increased knowledge regarding LL-37 transcription in the colon may lead to improved treatment of diseases caused by enteric pathogens.