Abstract:
Lipopolysaccharides (LPS), when released from the outer membrane of Gram-negative bacteria, bind to macrophages, inducing production of pro-inflammatory cytokines and mediator molecules. In addition, associated overproduction of free radicals causes oxidative stress and is a major factor for triggering local inflammation causing tissue and cellular damage. Several cationic antimicrobial peptides (AMPs), besides antimicrobial activity, also possess anti-inflammatory and antioxidant properties. The advantage of this multifunctionality is that while the infection is cleared, inflammation and associated free radical cellular and tissue damage is reduced.
In this study the potential anti-inflammatory and antioxidant properties of synthetic AMPs, Os and Os-C, derived from a defensin (OsDef2) previously identified in the tick, Ornithodoros savignyi, were investigated. Os is derived from the carboxy-terminal of OsDef2, whereas Os-C is an analogue of Os from which the Cys residues were omitted. Both Os and Os-C were found to bind LPS in vitro by measuring their ability to inhibit the LPS-induced activation of Limulus amoebocyte lysate (LAL) enzyme. The binding of free LPS caused the loss of both Os and Os-C bactericidal activity against Escherichia coli (E.coli) confirming the affinity of both peptides for LPS. Like glutathione (GSH), Os scavenged nitric oxide (NO), which is possibly due to the presence of three Cys residues and the formation of nitrosothiols with NO. Melittin (control) and Os-C, peptides lacking Cys residues, were found to be poor NO scavengers.
RAW 264.7 cells exposed simultaneously to LPS/IFN-γ and Os or Os-C showed that both peptides attenuated the LPS-induced production of NO, as well as TNF-α, in a dosage-dependant manner. In contrast, a 90 minute pre-incubation of RAW 264.7 cells with peptides, followed by LPS/IFN-γ stimulation, showed that both peptides, had a lesser effect on the inhibition of NO production when compared to controls. Although the control peptides, melittin and polymyxin B, had higher NO inhibition activity, both were associated with a loss of cellular viability. The results of the pre-incubation experiments indicated that Os and Os-C, besides binding to LPS, might have specific intracellular sites of action that allow for inhibition of LPS mediated NO formation. In contrast to both melittin and polymyxin B, Os and Os-C were found to be non-toxic towards the RAW 264.7 cells even at peptide concentrations 132 and 57 times higher than the minimum bactericidal concentration (MBC), respectively. Os was found to possess higher cellular antioxidant activity compared to Os-C, which may be attributed to the presence of antioxidant amino acids. This study identifies the multifunctional properties of Os and Os-C, however the specific mode of action of each peptide needs to be further elucidated.
