TY - JOUR
AU - PhD, Dorothy M. Supp,
AB - Abstract Defensins are cationic peptides of the innate host defense system with antimicrobial activity against many of the microorganisms commonly found in burn units. Beta defensins are variably expressed in the epithelia of skin and other organs. Human beta defensin 4 reportedly has antimicrobial activity against Pseudomonas aeruginosa and is not normally expressed in intact skin. Genetic modification was used to ectopically express human beta defensin 4 in cultured primary epidermal keratinocytes. Keratinocytes expressing human beta defensin 4 showed significantly elevated antimicrobial activity against clinically-isolated P. aeruginosa compared with controls. These results suggest that genetic modification of keratinocytes can increase their resistance to microbial contamination. Bioengineered skin replacements containing human beta defensin 4-modified keratinocytes may be useful for transplantation to contaminated burn wounds. With any large skin injury, such as extensive burns, the recovery of the patient is dependent on the closure of open wounds. In many of these patients, there is a limited amount of uninjured donor skin for harvesting for autografts. Delays in wound closure can increase the risk for infection, which is a major cause of mortality in patients with burns.1 The demand for timely wound closure in burn patients has led to the advent of many alternatives to split-thickness skin autografting2 through tissue engineering. Cultured skin substitutes (CSS) comprising cultured keratinocytes and fibroblasts in a biopolymer matrix have been successfully applied as an adjunctive burn and chronic wound therapy.3,–6 CSS provide permanent replacement of both the dermal and epidermal layers and, once healed, CSS resemble human autograft skin. However, CSS contain only two cell types, and, therefore, limitations in performance remain. Most notably, CSS in vitro lack a vascular plexus, leading to slower vascularization compared with autografts.7 Because of this initial avascular state, CSS are highly susceptible to microbial contamination from grafting until vascularization is achieved.6 To date, contamination has been managed clinically through the administration of dressing fluids containing multiple antimicrobial drugs during the early healing period.8 Although this step protects the grafts, it also may facilitate the emergence of resistant organisms. A cultured skin model with innate resistance to microbial contamination could possibly eliminate or reduce the dependence on exogenous antibiotics. Beta defensins are small, cationic antimicrobial peptides of the innate host defense system. In vertebrates, defensins are variably expressed in phagocytes and the epithelia of multiple organs.9,–12,Pseudomonas aeruginosa and Staphylococcus aureus are among the wide variety of bacteria that are sensitive to the antimicrobial activity of beta defensins. These bacteria are associated with burn wound infection and sepsis. The mechanism of action for defensins is thought to be their ability to disrupt microbial membranes.11,–13 Defensins could become an alternative approach to infection control because of the differences between their mechanisms of action and those of standard antibiotic therapies. Defensins could even be effective against multiply drug-resistant bacteria.14,15 Six members of the human beta-defensin (HBD) family have been characterized and are primarily expressed in epithelia.9 HBD4 was identified using bioinformatics and functional genomic analysis.16 It was found at highest expression levels in the testis and the gastric antrum. Lower levels of expression were found in neutrophils and the epithelia of the thyroid gland, the lung, the uterus, and the kidneys.16 HBD4 mRNA expression also has been noted in very low levels in unstimulated primary keratinocytes.17 The bactericidal activity of HBD4 against P. aeruginosa is reportedly more formidable than any other HBD.18 Previous studies reported that HBD1, HBD2, and HBD3 were detected in CSS via immunohistochemistry and were localized to distinct epidermal regions.19 HBDs have the potential to contribute to the innate immunity of CSS, but their levels of expression may be too low to prevent contamination. The purpose of this study was to increase the expression level of HBD4 in cultured keratinocytes through genetic modification. Hypothetically, these modified keratinocytes could be included in CSS and ultimately aid in the protection against microbial contamination. MATERIALS AND METHODS Genetic Modification of Keratinocytes With HBD4 A cDNA containing the coding sequence for HBD4 was isolated by reverse-transcription polymerase chain reaction (RT-PCR) from human epididymis RNA (BD Biosciences/Clontech, Palo Alto, CA) using the Titan One-Tube RT-PCR Kit (Roche Applied Science, Indianapolis, IN). The primers sequences used for RT-PCR were synthesized by the University of Cincinnati College of Medicine DNA Core Facility. The sequences were: 5′- CCAGCATTATGCAGAGACTTG-3′ (forward primer) and 5′- CAGCACTACTGCGTTTCAGGG-3′ (reverse primer). The resulting 242 base pair HBD4 cDNA was cloned into the MIEG3 replication incompetent retroviral vector (Courtesy of Dr. DA Williams, Cincinnati Children's Hospital Research Foundation). This vector (Figure 1A) contains a constitutive viral promoter, an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) coding sequence. Figure 1. View largeDownload slide Genetic modification of keratinocytes for HBD4 expression. A. Diagram of the HBD4 gene transfer vector. A cDNA fragment (double gray line) was isolated by reverse transcription polymerase chain reaction using gene-specific primers (arrows) and was cloned into the MIEG3 retroviral vector (top). An internal ribosome entry site sequence between the HBD4 gene and the enhanced green fluorescent protein coding sequence allows two proteins to be made (bottom) from one bicistronic mRNA transcript (middle). B. Example of microscopy used to visualize EGFP fluorescence (green) in keratinocytes that have incorporated the HBD4 or empty vector. Cell nuclei were counterstained using propidium iodide (red). Figure 1. View largeDownload slide Genetic modification of keratinocytes for HBD4 expression. A. Diagram of the HBD4 gene transfer vector. A cDNA fragment (double gray line) was isolated by reverse transcription polymerase chain reaction using gene-specific primers (arrows) and was cloned into the MIEG3 retroviral vector (top). An internal ribosome entry site sequence between the HBD4 gene and the enhanced green fluorescent protein coding sequence allows two proteins to be made (bottom) from one bicistronic mRNA transcript (middle). B. Example of microscopy used to visualize EGFP fluorescence (green) in keratinocytes that have incorporated the HBD4 or empty vector. Cell nuclei were counterstained using propidium iodide (red). Epidermal keratinocytes were isolated from a human skin sample obtained from a 29-year-old female donor undergoing reduction mammoplasty, as previously described.20,21 Primary keratinocytes were transduced with the HBD4 retrovirus or with the MIEG3 empty vector (negative control). Infectious retrovirus stocks were collected from transiently transfected packaging cells at the Viral Vector Core Facility at Cincinnati Children's Hospital Medical Center. Culture medium containing either the HBD4 or control empty virus was preincubated for 6 hours at 37°C on RetroNectin™ dishes (Takara Mirus Bio Corp., Madison, WI). Keratinocytes were then inoculated onto the dishes and allowed to incubate with the virus overnight. The next day, a secondary incubation with virus-containing medium was allowed for 2 hours at 37°C. The plates were then incubated in keratinocyte medium for an additional 1 to 2 days until cells reached 90% confluence. The keratinocytes were subcultured to expand the populations for experiments. A portion of the culture was inoculated onto glass chamber slides to monitor EGFP fluorescence. Aliquots of cells were snap- frozen in liquid nitrogen and stored at −70°C for further analysis. Chamber slides were rinsed with phosphate-buffered saline, fixed with 4% paraformaldehyde solution (Electron Microscopy Sciences, Hatfield, PA), and counterstained with Vectastain Mounting Medium with propidium iodide (Vector Laboratories, Inc., Burlingame, CA). Slides were examined using a MicrophotFXA microscope (Nikon, Melville, NY) equipped with epifluorescent illumination. To estimate the transduction efficiency (% cells EGFP-positive), samples were photographed using Spot-Jr. digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Pictures were then subjected to color-threshold analysis using Metamorph Image Analysis software to determine percent positive cells within a population. Analysis of HBD4 Expression Expression of HBD4 mRNA was analyzed by Northern blot hybridization using the Amersham Alk-Phos Direct Labeling and Detection System with CDP-Star (Amersham Biosciences, Piscataway, NJ). Total cellular RNA was isolated using the RNeasy Mini Kit (QIAGEN, Santa Clarita, CA) and separated on a 1% agarose gel using NorthernMax buffers (Ambion, Inc, Austin, TX). The gel was transferred overnight onto Brightstar-Plus Membranes (Ambion, Inc.) using the Turboblotter system (Schleicher & Schuell, Keene, NH). The HBD4 cDNA fragment (see Figure 1A) was used as the probe. In addition, RNA samples isolated from HBD4- and control-modified cells were subjected to RT-PCR using the Titan One-Tube RT-PCR Kit. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) primers (BD Biosciences/Clontech, San Diego, CA) were used for positive control reactions. Batches were set up for each RNA sample with all reagents except for the primers. Each batch was equally divided into separate tubes; gene-specific or G3PDH primers were added, and amplifications were performed simultaneously using a GeneAmp 9600 PCR System (Perkin-Elmer, Boston, MA). We used 0.5 µg of total RNA per reaction, following the manufacturer's protocol (35 cycles total) with an annealing temperature of 56°C. RNA was replaced with water for the negative control reactions. Reaction products were visualized by electrophoresis on 1.5% agarose gels. For Western blot analysis, snap-frozen cells were lysed and cellular protein was isolated using the Mammalian Cell Lysis kit (Sigma Chemical Co., St. Louis, MO). Total protein was assayed using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Proteins were then separated on duplicate 12% acrylamide gels with MES running buffer (Invitrogen Corporation, Carlsbad, CA) under reducing conditions. One gel was then electroblotted onto a 0.1-μm nitrocellulose membrane (Schleicher & Schuell) and subjected to WesternBreeze Chromogenic detection kit (Invitrogen). The primary antibody used was a mouse anti-human beta defensin-4 antibody (Abcam, Cambridge, MA). Primary antibody (1:1000 dilution; 1 µg/ml) incubation was performed for 1 hour at room temperature. The duplicate gel was stained using the Colloidal Blue Staining Kit (Invitrogen) to demonstrate equivalence of protein loading. As a positive control, synthetic HBD4 protein (0.02 μg; Peptides International, Louisville, KY) was added to an adjacent lane on the gel; however, this amount of protein was too low to be visualized by Colloidal Blue staining. Antimicrobial Assays Assay 1. Snap frozen cells (6 × 106 per tube, n = 10 per group) were mechanically lysed in phosphate-buffered saline using a 1.5-ml Pellet Pestle (Nalge Nunc International, Rochester, NY) with a Pellet Pestle Motor (Kontes Glass Co., Vineland, NJ). A clinical isolate of P. aeruginosa, cultured from a pediatric burn patient, was added to each sample at a concentration of 104/ml. The tubes were allowed to incubate for 3 hours in a humidified chamber at 37°C. Serial dilutions from each tube were plated onto Mueller Hinton agar plates and incubated overnight at 37°C. The next day the colonies on each plate were counted. Assay 2. HBD4-modified keratinocytes and MIEG-3 empty vector-modified keratinocytes were cultured in 24-well multiwell plates until cells reached 90% confluence. Cells were allowed to condition the media (1 ml per well) for 48 hours, and then 1 × 104P. aeruginosa were added to each well. Bacteria were incubated with media/keratinocytes for 4 hours at 37°C, and aliquots were plated onto TSA plates for quantitation. Statistical Analysis Comparisons between bacterial colony counts were performed with the t test using SigmaStat Statistical Software Version 2.0 (SSPS Inc., Chicago, IL). RESULTS An HBD4 gene transfer vector was constructed using a replication incompetent retrovirus vector, MIEG3 (Figure 1A). Coexpression of HBD4 and EGFP from a single bicistronic mRNA containing the IRES sequence permits the translation of separate HBD4 and EGFP proteins, which facilitates the evaluation of transduction by EGFP fluorescence (Figure 1B) without possible negative affects that may result from a fusion protein. Image analysis indicated that gene transfer efficiencies for this study ranged from 10% to 19%; thus, the populations consisted of both modified and unmodified cells. Expression of HBD4 was detected in the cells modified with the HBD4 retroviral vector via Northern blot detection (Figure 2A). HBD4 was not detected in cells modified with an empty vector. Expression of HBD4 mRNA was further confirmed using RT-PCR (Figure 2B). The presence of HBD4 protein was demonstrated using Western blot analysis with an antibody specific for HBD4 (Figure 2C). Figure 2. View largeDownload slide Expression analysis of HBD4-modified keratinocytes. A. Northern blot analysis (top) of total RNA isolated from HBD-4-modified keratinocytes (HK+) and empty vector-modified keratinocytes (HK−). The cDNA probe was specific to the human HBD-4 gene. The ethidium bromide-stained ribosomal RNA bands (bottom) are shown as loading controls. B. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of HBD4 mRNA expression (top). G3PDH was used as a positive control (bottom). Lanes contain RT-PCR products from: HK+, HBD4-modified keratinocytes; HK−, keratinocytes modified with an empty vector; HK, unmodified (nontransduced) keratinocytes; HF, human fibroblasts. Water was included as a negative control. C. Western blot analysis of HBD4 protein expression (top) in the HBD4-modified keratinocytes (HK+); no specific band was seen in empty vector-modified keratinocytes (HK−). The Colloidal blue-stained gel is shown (bottom). Synthetic HBD4 peptide was loaded (right lane) as a positive control. Figure 2. View largeDownload slide Expression analysis of HBD4-modified keratinocytes. A. Northern blot analysis (top) of total RNA isolated from HBD-4-modified keratinocytes (HK+) and empty vector-modified keratinocytes (HK−). The cDNA probe was specific to the human HBD-4 gene. The ethidium bromide-stained ribosomal RNA bands (bottom) are shown as loading controls. B. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of HBD4 mRNA expression (top). G3PDH was used as a positive control (bottom). Lanes contain RT-PCR products from: HK+, HBD4-modified keratinocytes; HK−, keratinocytes modified with an empty vector; HK, unmodified (nontransduced) keratinocytes; HF, human fibroblasts. Water was included as a negative control. C. Western blot analysis of HBD4 protein expression (top) in the HBD4-modified keratinocytes (HK+); no specific band was seen in empty vector-modified keratinocytes (HK−). The Colloidal blue-stained gel is shown (bottom). Synthetic HBD4 peptide was loaded (right lane) as a positive control. Antimicrobial activity of the HBD4-modified cells was evaluated using two separate assays. In assay 1, lysates of HBD4-modified or empty vector-modified keratinocytes were challenged with P. aeruginosa. The HBD4-modified keratinocytes showed a statistically significant (P ≤ .03) 25% reduction in P. aeruginosa colony formation compared with the empty vector modified cells (Figure 3A). In assay 2, live cells in conditioned media were incubated in the presence of P. aeruginosa. In this evaluation, the HBD4-modified cells showed a statistically significant (P ≤ .001) 29% reduction in P. aeruginosa colony formation as compared with empty vector-modified cells (Figure 3B). Figure 3. View largeDownload slide Increased expression of HBD4 correlates with significantly increased antimicrobial activity against P. aeruginosa. A. Results of Assay 1, in which lysed empty-vector modified (gray) or HBD4-modified (black) keratinocytes were incubated with bacteria. B. Results of Assay 2, in which live keratinocytes in conditioned media were incubated with bacteria. Figure 3. View largeDownload slide Increased expression of HBD4 correlates with significantly increased antimicrobial activity against P. aeruginosa. A. Results of Assay 1, in which lysed empty-vector modified (gray) or HBD4-modified (black) keratinocytes were incubated with bacteria. B. Results of Assay 2, in which live keratinocytes in conditioned media were incubated with bacteria. DISCUSSION Patients with burn injury are notoriously susceptible to infection, because of both the nature of the burn wound and the accompanying immunosuppression.22,23 With the reported absence of HBD2 in burn blister fluid and reduced expression in burn wounds, it has been speculated that the lower levels of defensin expression could promote the growth of microorganisms.24,25 Although the mechanism for reduced defensin expression remains unclear, burn injury does result in the release of multiple inflammatory mediators.26 Many of these cytokines that are elevated in burn patients, including interleukin-1 (IL-1), IL-6, interferon gamma, and tumor necrosis factor-α, have been hypothesized to help regulate defensin expression in vitro.9,15,27,–29 These mediators may therefore play a role in the altered defensin expression in burn wounds. Avascular skin substitutes are more susceptible to microbial contamination than split-thickness skin autograft. However, skin substitutes are becoming increasingly important for closure of massive burn wounds because of the lack of available donor sites from the patient for autograft. In a previous study, we examined the expression of HBD genes in CSS. Although the CSS expressed HBD1, HBD 2, and HBD3 to some varying degrees, it was speculated that the levels of defensins present may be insufficient to combat microbial growth in CSS in clinical settings because of the high incidence of infection in burn patients.19 Antimicrobial peptides are structurally and mechanistically different from conventional antibiotics. Therefore, they have the potential to be used as novel therapeutic agents for treating infection. We established expression of HBD4 in cultured keratinocyte populations using a retroviral vector and challenged these cells with an isolate of P. aeruginosa that was derived from a burn patient to assess antimicrobial activity. We observed a 25% to 29% reduction in P. aeruginosa colony formation in the cell population modified with HBD4 compared with the sham-modified cells. Therefore, we conclude that increased expression of HBD4 in cultured keratinocytes results in enhanced antimicrobial activity. In future studies, we will enrich for the modified cells by selecting for EGFP fluorescence using flow sorting, which should result in a corresponding increase in antimicrobial activity. We will incorporate these modified cells into CSS in an attempt to increase their innate immunity. Although it is technically possible to add the synthetic peptide to the irrigation fluid currently in use to combat infection, it would be cost-prohibitive, adding approximately $200,000 per surgery. Genetic modification of the cells in CSS offers a feasible alternative that provides stable local production of the peptide by the modified cells. Emerging drug-resistant strains of bacteria have asserted the need for the development of novel therapeutic options. Natural and synthetic antimicrobial peptides have been shown to be effective at reducing microbial levels in a number of preclinical studies.30,–34 Hypothetically, incorporating HBD4-modified keratinocytes into CSS may lead to improved resistance to microbial contamination after grafting. This would be expected to improve engraftment and would also have the additional benefit of reduced dependence on topical antibiotics. Supported by a medical research grant from the Shriners Hospitals for Children. ACKNOWLEDGMENTS We appreciate the support of Dr. Steven Boyce at the Cincinnati Shriners Hospital and thank Jodi Miller and Sherry Liles in Dr. Boyce's laboratory for media preparation. We are grateful to Dr. David A. Williams in the Division of Experimental Hematology at Cincinnati Children's Hospital Research Foundation for generously providing the MIEG3 retroviral vector used in this study, and we thank the Viral Vector Core Facility at Cincinnati Children's Hospital for retrovirus production. REFERENCES 1. Berthod F, Damour O In vitro reconstructed skin models for wound coverage in deep burns. Br J Dermatol  1997; 136: 809– 16. Google Scholar CrossRef Search ADS PubMed  2. Supp DM, Boyce ST Engineered skin substitutes: practices and potentials. Clinics Dermatol  2005; 23: 403– 12. Google Scholar CrossRef Search ADS   3. Boyce ST, Greenhalgh DG, Kagan RJ, et al.   Skin anatomy and antigen expression after burn wound closure with composite grafts of cultured skin cells and biopolymers. Plast Reconstr Surg  1993; 91: 632– 41. Google Scholar CrossRef Search ADS PubMed  4. 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Google Scholar CrossRef Search ADS PubMed  Footnotes 2 Presented at the 38th Annual Meeting of the American Burn Association, April 4–7, 2006, Las Vegas, Nevada. 3 Awarded First Place in the Poster Competition at the meeting of the American Burn Association, Las Vegas, Nevada April 4 to 7, 2006. Copyright © 2007 by the American Burn Association
TI - Expression of Human Beta Defensin 4 in Genetically Modified Keratinocytes Enhances Antimicrobial Activity
JF - Journal of Burn Care & Research
DO - 10.1097/BCR.0b013E31802C88FD
DA - 2007-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/expression-of-human-beta-defensin-4-in-genetically-modified-2ZhehY4J1m
SP - 127
EP - 132
VL - 28
IS - 1
DP - DeepDyve
ER -