Source: http://grantome.com/grant/NIH/ZIA-HL000659-22
Timestamp: 2019-04-20 20:20:26+00:00

Document:
Explanation The airway epithelium plays a critical role in host defense against infection. The repair process of the injured epithelium involves cell migration and proliferation, processes that increase neutrophil defensins. As reported previously, the proliferative and cytotoxic effects of HNP-1 on human lung fibroblasts and epithelial and endothelial cells are dose dependent. Low concentrations of HNP-1 have a proliferative effect on normal human lung fibroblasts (NHLF) and A549 epithelial cells. However, at concentrations greater than 20g/ml, HNP is cytotoxic for NHLF and A549 cells. To determine the effect of the HNP analogs on eukaryotic cells, lung epithelial cells (A549) or normal human lung fibroblasts (NHLF) were incubated with increasing amounts of HNP or the three HNP-ornithine analogs for 24h in serum-free media. HNP and HNP-(R14,24orn) at 10 ug/ml and HNP-(R14orn) and HNP-(R24orn) at 25 ug/ml increased the number of A549 cells (as measured by a tetrazolium reduction assay). HNP and the three analogs were toxic for NHLF at concentrations less than 10 ug/ml. HNP-(R14orn) and HNP-(R24orn) were toxic for A549 cells at concentrations above 40 ug/ml. In contrast, replacement of arginines with ornithines at amino acids 14 and 24 abolished the cytotoxic effect of HNP on A549 cells at concentrations to 100 ug/ml. The toxicity of HNP and ornithine analogs (50 ug/ml) was also tested on NCI-H441 and SAEC cells and compared to that seen with A549 and NHLF cells. The HNP analogs showed similar or less toxicity than HNP. IL-8, a chemoattractant, modulates the inflammatory response by recruiting neutrophils to the lung. Higher levels of IL-8 in BALF and plasma were found in IPF patients compared to healthy volunteers. HNP-1 has been reported to induce release of IL-8 and other chemokines and cytokines from epithelial cells and fibroblasts. To determine the effects of HNP and the ornithine analogs on IL-8 release by A549 cells, cells were incubated with the ornithine analogs and IL-8 in the medium was measured by immunoassay. HNP with a single ornithine substitution at concentrations up to 20 ug/ml increased IL-8 release as did HNP-(R14,24orn), up to 100 ug/ml, a range of concentrations not cytotoxic for A549 cells. HNP-1 increased TGF-b1 release by fibroblasts but reduced TGF-b1 production by A549 cells. A549 cells were incubated with increasing amounts of HNP analogs and TGF-b1 release was determined by immunoassay. HNP-(R24orn) and HNP-(R14,24orn) inhibited release of less TGF-b1 than did HNP and HNP-(R14orn) at equivalent concentrations. HNP1-4, released from neutrophil azurophilic granules, act as natural antibiotics, killing a variety of microorganisms. Toxicities of the HNP-ornithine analogs for E.coli-TG1 and P.aeruginosa were compared to HNP. After one hour incubation, colony-forming units (CFU) were determined by plate assay. HNP-(R14,24orn) was toxic for P.aeruginosa and E.coli-TG1. HNP-(R24orn) was not toxic for E.coli-TG1 or P.aeruginosa. HNP-(R14orn) was toxic for P.aeruginosa at 3 ug/ml but inactive against E.coli-TG1. Surprisingly, the mono-substituted analogs did not exhibit HNP-1 antibacterial activity but were toxic for epithelial cells;antibacterial activity was observed, however, with ornithine at positions 14 and 24. HNP (3 ug) was not toxic for either E. coli (ATCC 25922) or S. aureus (ATCC 29213). To evaluate how well the ornithine analogs serve as substrates for ART1, nicotinamide release concurrent with ADP-ribose transfer from NAD to the acceptor was measured after incubation of the peptides with ART1 and NAD;HNP was compared to agmatine, a decarboxylated form of arginine which is a known ADP-ribose acceptor used by ART1. HNP and HNP-(R14orn) were better acceptors of ADP-ribose than agmatine. A small amount of nicotinamide release was observed in the presence of ART1, NAD and HNP-(R14,24orn), indicating that under these assay conditions, arginine 5 and arginine 15 are poor ADP-ribose acceptors in the ART1-catalyzed reaction. To identify the products of the reaction of the ornithine analogs, ART1 and NAD, the products were separated by HPLC, purified and analyzed by MS. The early peaks in the HPLC separations were identified as ADP-ribosylated-HNP-(R14orn), or ADP-ribosylated-HNP-(R24orn), whereas the later peaks were identified as the unmodified forms. In contrast, three products were separated from the reaction of HNP-1 with NAD and ART1. Compared to HNP-1, the unmodified analogs were found at an earlier HPLC elution time, suggesting that they are less hydrophobic than HNP. ADP-ribosylated-HNP-(R24orn) eluted at a significantly earlier time, suggesting the product is as hydrophobic as the di-modified HNP-1. ADP-ribosylarginine 5 or 15 HNP were not identified in the reaction products or in HNP-(R14,24orn). Increased ADP-ribosylation of HNP-1 and HNP-(R14,24orn) was observed after reduction of the disulfide bridges with dithiothreitol. These data suggest that the quaternary structure of HNP and the analogs limit the accessibility of arginines for ADP-ribosylation by ART1. As observed by confocal laser microscopy, HNP-1 at high concentrations (10-20 ug/ml) rapidly entered A549 cells, co-localized with the ER and stimulated apoptotic cell death. The reduced cytotoxicity of HNP after di-ornithine substitution could result from changes to HNP folding that may affect its capacity to enter the cell or localize to the ER. Substitution of ornithines for two arginines in oncocin, a 19 amino acid peptide sequence derived from the insect Oncopeltus peptide 4, resulted in a peptide that was active against E.coli, but non-toxic to HeLa cells and stable in serum. Conversion of HNP-1 to the di-ornithine form resulted in reduction of injury to the epithelial cells in the airway and preservation of the proliferative response believed to be involved in wound repair. Arginines or ornithines at positions 14 and 24 in the HNP primary sequence are critical for antibacterial activity. Although lysine, arginine and ornithine have equivalent charge, the replacement of arginine by lysine affected the antimicrobial activity of alpha-defensins. The virtual lethal dose for 90% of bacteria (vLD90) increased when ornithine replaced arginine or lysine at residues 14, 15, and 24. HNP-(R14,24orn) with arginine at residue 15, however, retained significant antibacterial activity against P. aeruginosa and E. coli. suggesting that ornithine 15 had an effect other than charge. Moreover, in contrast to the toxicity of replacing arginine 14 and 24 as in HNP-(R14,24orn), the replacement of arginine 24 with ornithine abolished the antibacterial activity against P. aeruginosa and E. coli, demonstrating that the activity of HNP-1 is affected by the number and position of arginine residues replaced by ornithine. In summary, ADP-ribosylation of arginines at residues 14 and 24 by ART1 regulates HNP-1 biological activities. Site-specific ADP-ribosylation by ART1, abolishes HNP-1 antimicrobial and cytotoxic activities. HNP with ADP-ribosylated arginines 14 and 24 non-enzymatically converted to ornithines is not cytotoxic for epithelial cells but the antibacterial activity is preserved. HNP-(R14,24 orn) is a promising novel candidate;a stable peptide antibiotic that shows diminished toxicity for the epithelial cells that line the airway.

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