Actions of Antimicrobial Peptides and Bacterial Components in Inflammation

Detta är en avhandling från Faculty of Medicine, Lund University

Sammanfattning: Antimicrobial peptides are evolutionally ancient parts of the innate immune system and their primary role is to protect us from infections. The human cathelicidin-derived antimicrobial peptide, LL-37, not only possesses broad spectrum antimicrobial activities but is also able to bind and neutralize bacterial lipopolysaccharide (LPS), an important trigger of the widespread inflammatory response contributing to septic shock. LL-37 has been studied as an alternative to conventional antibiotics but clinical trials have been hampered by indications of its toxic effect on mammalian cells and evidence that its antimicrobial effects are inhibited by serum. It has been proposed that the cytotoxicity of LL-37 is due to hydrophobic amino acids. We were able, by removing hydrophobic amino acids from the N-terminal of LL-37, to generate less cytotoxic peptides with retained antimicrobial and LPS–neutralizing actions in serum. By using computer simulation we identified an active domain of LL-37, a 21 amino acid fragment, GKE, displaying similar antimicrobial and LPS-binding activity in vitro as native LL-37 but less toxic and therefore holding promise as a template for the development of peptide antibiotics for treating sepsis. Sepsis due to Gram-positive bacteria is becoming increasingly prevalent. The Gram-positive bacterium Streptococcus pyogenes, carrying a surface protein named M1 being fundamental for its virulence, is the major cause of severe streptococcal infections like streptococcal toxic shock syndrome and necrotizing fasciitis. We found that M1 protein is able to induce vascular nitric oxide (NO) production, which in turn relaxes smooth muscle cells and could thereby contribute to the severe hypotension seen in septic patients. This we confirmed by showing that M1 protein caused hyporesponsiveness to the vasoconstrictor, phenylephrine, in rat aorta. Bacterial compounds are able to activate Toll-like receptors generating an inflammatory response. Experiments using wild type and knockout mice revealed that M1 protein is able to attach to both TLR2 and TLR4 (TLR) in mice, only activating the latter. M1 protein only attached to TLR2 in human blood vessels. LL-37 possesses immunomodulatory effects. In order to explore potential modulatory effects of GKE on vascular nitric oxide production we used the well identified proinflammatory compounds interleukin-1? (IL-1?), M1 protein from Streptococcus pyogenes and lipoteichoic acid (LTA). All three induced vascular NO production in rat aorta. GKE at low concentration inhibited IL-1?-induced NO production, but synergistically increased it at higher concentrations. GKE did not affect the M1 protein induced NO production while GKE inhibited LTA induced NO production. Thus, GKE seems to have complex modulatory effects on vascular nitric oxide production depending on the inflammatory compound used.