Regulation and characterization of antimicrobial peptides in man and mice

Abstract

The gene-encoded antimicrobial peptides have emerged as an important component of innate immunity. In mammals, antimicrobial peptides are either directly secreted from cells or found in cell granules, which can be released after stimuli. This thesis aims at investigating antimicrobial peptides stored in granules in neutrophils and in Paneth cells of the small intestine.

The condition severe congenital neutropenia (SCN) illustrates the pivotal requirement for neutrophils as primary defence cells since affected individuals die in infancy. In this thesis I show that all patients with SCN, independent of inheritance pattern, genetic mutations or G-CSF responsiveness, display a deficiency of the antimicrobial peptide LL-37 and its pro-form in plasma and in neutrophils, even after G-CSF treatment. This deficiency points at a common mechanistic denominator for all patients with SCN. The LL-37 phenotype may be used as a diagnostic method to discriminate the lifelong, and without treatment, life threatening disease SCN from the more benign forms of chronic neutropenia. Granulocytic precursors from patients with SCN were here demonstrated to produce pro-LL-37 after in vitro stimulation with the hormonal form of vitamin D3 . The induction of pro-LL-37 in plasma did not take place in vivo following oral treatment of one patient with a low dose vitamin D3.

Neutrophils and Paneth cells are strikingly similar in their repertoire of granule derived antimicrobial peptides. Paneth cells reside at the base of the crypt of the small intestine of most mammals, where they secrete high concentrations of antimicrobial substances that sterilize the crypt and shape the profile of the resident microflora. Here I show that the mouse Paneth cell arsenal is broadened by the presence of cryptdin related sequence (CRS) peptides. The antimicrobial spectrum is increased further by their ability to form both homo- and hetero dimers, which have distinct but overlapping antimicrobial spectra. In addition, different repertoires of CRS peptides in different mouse strains, suggest a highly efficient positive selection pressure. Two groups of transcripts coding for CRS peptides could be separated based on expression profile in the small intestine. One group was expressed at a consistently high level along the small intestine, while the other group increased strongly through the length of the small intestine. A similar increase, although not as prominent, has also been demonstrated by others for the enteric antimicrobial peptide human defensin 5, which is involved in the pathomechanism of Crohn´s ileitis. Elucidating the regulatory pattern in mice may thus be useful in unravelling the regulation of human enteric antimicrobial peptides.