Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model

Abstract

Recent studies suggest that the gene defect in cystic fibrosis (CF) leads to a breach in innate immunity. We describe a novel genetic strategy for reversing the CF-specific defect of antimicrobial activity by transferring a gene encoding a secreted cathelicidin peptide antibiotic into the airway epithelium grown in a human bronchial xenograft model. The airway surface fluid (ASF) from CF xenografts failed to kill Pseudomonas aeruginosa or Staphylococcus aureus. Partial reconstitution of CF transmembrane conductance regulator expression after adenovirus-mediated gene transfer restored the antimicrobial activity of ASF from CF xenografts to normal levels. Exposure of CF xenografts to an adenovirus expressing the human cathelicidin LL-37/hCAP-18 increased levels of this peptide in the ASF three- to fourfold above the normal concentrations, which were equivalent in ASF from CF and normal xenografts before gene transfer. The increase of LL-37 was sufficient to restore bacterial killing to normal levels. The data presented describe an alternative genetic approach to the treatment of CF based on enhanced expression of an endogenous antimicrobial peptide and provide strong evidence that expression of antimicrobial peptides indeed protects against bacterial infection.

Figure 1

Antimicrobial activity of ASF from human bronchial xenografts against Pseudomonas aeruginosa PAO1 (a) and Staphylococcus aureus ATCC 29213 (b). Xenografts were prepared with normal cells (NL) or CF cells (CF) and were partly treated with the adenoviral vectors H5.020.CBCFTR (AdCFTR), H5.020.CMVLL-37 (AdLL-37), or H5.020.CMVPG3 (AdPG3). Arrows indicate the numbers of CFU in the inoculum (open arrow) or in the NB positive control (filled arrow).

Figure 2

Expression of the antimicrobial peptide LL-37/hCAP-18 before and after adenoviral transfer of its cDNA to xenografts. (a and b) Immunohistochemical detection of LL-37 in respiratory epithelia of xenografts. Whereas CF epithelia transduced with H5.020.CBCFTR (a) show low level of expression, those transduced with the LL-37 vector (b) show a strong LL-37–specific signal. Scale bar in a and b: 10 μm. (c) Concentration of LL-37/hCAP-18 in ASF. The concentration of LL-37/hCAP-18 is not significantly different in ASF from xenografts prepared with normal cells (NL), CF cells (CF), or CF cells transduced with H5.020.CBCFTR (AdCFTR). Gene transfer of LL-37/hCAP-18 results in increased levels of LL-37 in the ASF (AdLL-37). (d) Immunoblotting of HPLC-purified proteins obtained from H5.020.CMVLL-37–transduced xenografts reveals the presence of increased amounts of mature LL-37 (37 COOH-terminal amino acids of LL-37/hCAP-18) (XG CF Ad.LL-37) compared with CF cells transduced with H5.020.CBCFTR (XG CF Ad.CFTR). Mouse lung extract (ML) was used as negative control and did not show any positive staining. Molecular weight markers are in kilodaltons.