The dual functionality of antimicrobial peptides Os and Os-C in human leukocytes

Antimicrobial peptides (AMPs), Os and Os-C, have been identified as multifunctional peptides with antibacterial, antiendotoxin, and anti-inflammatory properties. For further development of Os and Os-C as therapeutic peptides, it is essential to evaluate these effects in human mononuclear (MN) and polymorphonuclear (PMN) leukocytes. The cytotoxicity and the effects of both peptides on MN and PMN morphology were determined with the Alamar-Blue assay and scanning electron microscopy, respectively. The ability of Os and Os-C to induce reactive oxygen species (ROS) and to protect against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative damage in both cell populations was evaluated using 2',7'-dichlorofluorescin diacetate (DCFH-DA). Using fluorescently labeled peptides, the ability of the peptides to cross the cell membranes of MN and PMN was also evaluated. At the minimum bactericidal concentrations of Os and Os-C, neither peptide was cytotoxic. Os caused morphological features of toxicity at 100 μM, entered MN cells, and also protected these cells against oxidative damage. Os-C caused MN and PMN leukocyte activation associated with ROS formation and was unable to penetrate cell membranes, indicating extracellular membrane interactions. This study confirms that both Os and Os-C at less than 100 μM are not cytotoxic. The MN-specific uptake of Os identifies it as a cell-specific cargo-carrier peptide, with additional anti-inflammatory properties. In contrast, the ability of Os-C to activate MN and PMN cells implies that this peptide should be further evaluated as an AMP, which, in addition to its ability to eradicate infection, can further enhance host immunity. These novel characteristics of Os and Os-C indicate that these AMPs as peptides can be further developed for specific applications.