Capacity of human β-defensin expression in gene-transduced and cytokine-induced cells

Abstract

The purpose of this study was to determine the capacity of cells transduced with human β-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9-99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabe-Neo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFα. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction. KeywordsHBD-2; HBD-3; Lentiviral vectors; Retroviral vectors; IL-1; TNFα; Gingival epithelial cells Natural antimicrobial peptides have been considered as an alternative option for infection control due to the increasing resistance of microbes to conventional antibiotics. Because some potent antimicrobial peptides are encoded by single genes, the possibility of artificially delivering these genes into tissues/organs to augment innate immunity against infection or creating transgenic animals to produce antimicrobial peptides has been explored [1][2][3][4][5]. Subsequently, the term antimicrobial gene therapy has emerged [6] and engineered skin NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript models have been used to test the concept of antimicrobial gene therapy both in vitro and in vivo [7,8]. Human β-defensins (HBDs or DEFB) and cathelicidin LL37 are being actively used as study models for their potent ability to kill a broad spectrum of bacteria and fungi [6,7,[9][10][11][12][13], as well as to inhibit viral infections [14,15]. HBDs are expressed mostly in epithelia [11,12,[16][17][18][19][20][21][22][23][24]. Besides the cloned, sequenced, and well-characterized HBD-1, -2, -3, and -4, there appears to be at least 28 more HBDs existing based on the genomic surveys using computational search strategy [25][26][27]. Fourteen HBDs or DEFB gene transcripts (DEFB-1, -4, and DEFB-103 to -114) were investigated using reverse transcriptionpolymerase chain reaction (RT-PCR) to detect their expression in gingival keratinocytes and a selected few have been classified as constitutive or inducible [27].HBDs are considered to play an important role in epithelial defense mechanism and the regulation of their expression in diseased conditions is being actively investigated. Bacteria and cytokines IL-1 and TNFα upregulate HBD-2 expression in lung and skin epithelial cells [10,28] and induction of HBDs by these stimuli is...

Mouse salivary glands and human β-defensin-2 as a study model for antimicrobial gene therapy: technical considerations

“…Chen, UCLA [30]. The SIN18-E vector containing the enhanced green fluorescent protein (EGFP) reporter cDNA was released and inserted with hBD-2 cDNA yielding SIN18-hBD-2 as described previously [17].…”

“…The viral supernatant was collected on Days 2 and 3 after transfection, filtered (0.45 μm) and concentrated ca. 100-fold by ultracentrifugation as described previously [17]. Viral concentration was measured against known concentrations of viral protein p24 and virus titration was performed using 293T cells with various dilutions of the viral stock and analysed for EGFP expression by fluorescence microscopy and flow cytometry on Day 3 post infection.…”

“…The amount of secreted hBD-2 was determined by ELISA as described previously [9,17] using monoclonal anti-human hBD-2 antibodies (1:5000 dilution) as anchoring antibodies, polyclonal rabbit anti-human hBD-2 antibodies (1:2000 dilution) as detecting antibodies and horseradish peroxidase (HRPO)-labelled polyclonal goat anti-rabbit immunoglobulin G (Pierce, Rockford, IL) as a second-step antibody. Bound HRPO was visualised with fresh developing buffer containing a substrate of optimal concentrations of o-phenylenediamine dihydrochloride (Sigma), 0.01% H 2 O 2 and 20 mM sodium citrate pH 4.0.…”

“…Fourteen hBD or DEFB gene transcripts (DEFB-1, -4 and -103-114) were investigated using reverse transcription polymerase chain reaction (RT-PCR) to detect their expression in gingival keratinocytes [16]. Transduction of epithelial cells in skin or oral mucosa with hBDs has been tested for its potential to enhance infection control [8,12,17]. Primary fibroblasts have also been proposed as target cells to express hBDs, but the amount of antimicrobial peptide secreted may be insufficient to exert an effect [17,18].…”

“…Transduction of epithelial cells in skin or oral mucosa with hBDs has been tested for its potential to enhance infection control [8,12,17]. Primary fibroblasts have also been proposed as target cells to express hBDs, but the amount of antimicrobial peptide secreted may be insufficient to exert an effect [17,18].…”