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A cutaneous gene therapy approach to treat infection through keratinocyte-targeted overexpression of antimicrobial peptides

MARTA CARRETERO^*, MARCELA DEL RÍO^*, MARTA GARCÍA^*, MARÍA JOSÉ ESCÁMEZ^*, ISABEL MIRONES^*, LUIS RIVAS, CRISTINA BALAGUE, JOSE LUIS JORCANO^* and FERNANDO LARCHER^*,1

^* Epithelial Damage, Repair, and Tissue Engineering, Ciemat-Fundación Marcelino Botín, Madrid, Spain;
Centro de Investigaciones Biologicas (CSIC), Madrid, Spain; and
Almirall Prodesfarma, S. A., Barcelona, Spain

¹Correspondence: CIEMAT Avenida Complutense 22, Edificio 7, Madrid 28040, Spain. E-mail: fernando.larcher{at}ciemat.es

SPECIFIC AIMS

The aim of this study was to develop a novel strategy for antimicrobial treatment associated with covering burn wounds to overcome problems related to infection by resistant bacterial strains, the principal cause of skin graft failure in burn patients. Here we present an adenoviral gene transfer approach aimed at delivering human antimicrobial peptides from keratinocytes. We assessed both activity and specificity for bacteria killing action by using different in vitro settings, including cultured skin equivalents.

PRINCIPAL FINDINGS

1. Bactericidal activity of AMP transduced keratinocytes
The spontaneously immortalized human keratinocyte cell line (HaCaT) was transduced using adenoviral vectors containing the complete sequence of human antimicrobial peptides HBD-2, HBD-3 and LL-37 along with an Ires-GFP expression cassette. Infectivity was monitored by visualization of infected cells under UV light, and AMP expression was assessed either by RT-PCR using total mRNA from adenoviral transduced cells or by immuno-dot blot analyses using concentrated conditioned media from adenoviral transduced HaCaT cells. Whereas control GFP+ HaCaT cells did not express detectable levels of these AMPs, adenoviral transfer rendered secreted peptide levels consistent with those previously reported for antimicrobial activity (HBD-3 was expressed at 50 µg/mL whereas LL-37 and HBD-2 were produced at 25 µg/mL and 100 µg/mL, respectively). To address whether secreted peptides are correctly processed, concentrated supernatants from adenoviral transduced HaCaT cells were run on SDS-PAGE along with recombinant mature peptides, followed by immunoblotting with anti-LL-37 and anti-HBD-3 antibodies. At least a fraction of overexpressed HBD-3 and LL-37 antimicrobial peptides showed identical mobilities to the recombinant mature peptides (data not shown). Moreover, overexpression of these AMPs did not result in a reduction in HaCaT cell viability as observed in an XTT assay.

We evaluated the killing activity of HBD-2, HBD-3, and LL-37 antimicrobial peptides expressed in 24 h conditioned media from adenoviral transduced HaCaT cells. Concentrated supernatants were incubated with bacterial suspensions containing 2 x 10⁴ mid-logarithmic-phase cells per mL and incubated for 2 h at 37°C. Serial dilutions were then plated and the number of CFU was determined the next day (Fig. 1 ). We used concentrated supernatants in order to counteract the possible inhibitory effect of salt and FBS on peptide activity. The concentration might resemble the situation in vivo, where local concentration of AMPs is augmented by retention between keratinocytes. Reduction in Escherichia coli viability ranging from 45% to 67% was observed when HBD-3 containing supernatant was used. In these conditions HBD-2 and LL-37 yielded 12%–27% and 45%–79% reduction in bacterial counts, respectively. Coexpression of antimicrobial peptides HBD-2 and LL-37 in conditioned medium from HaCaT cells produced a strong synergistic activity against E. coli (Fig. 1A ). HBD-3 and HBD-2 co-secreted along with LL-37 also efficiently killed S. aureus in this system (49%–93% and 26%–93% inhibitory range, respectively) (Fig. 1A, B ). For P. aeruginosa LL-37 showed the most potent activity (53%–89% killing activity), although in this case we could not observe any synergistic activity for HBD-2 and LL-37 (Fig. 1A ). All AMP tested showed a significant activity against E. faecalis ranging from 74% to 65% reduction in viability in all cases. As shown for P. aeruginosa, no synergistic activity was observed for LL-37 and HBD-2. The majority of these peptides were still active even when bacterial load was increased by 10-fold (2x10⁵ cells per mL) (Fig. 1C ).

View larger version (20K): [in this window] [in a new window]   Figure 1. Antimicrobial activity from adenoviral-transduced HaCaT cell supernatants. A) Bacteria were incubated with cell free supernatants for 2 h at 37°C and inoculated onto agar plates to determine the number of CFU. Percent viability was calculated relative to control survival. Error bars indicate standard deviations from the mean for three to four separate experiments. *P < 0.05 compared with control (supernatant from Ad (GFP) transduced HaCaT cells). B) A representative experiment for each bacterial strain is shown. C) Supernatant containing killing activity after increasing bacterial load by 10x. A representative experiment for each strain is shown.

2. Antibacterial activity in AMP overexpressing cultured skin equivalents
To determine whether these peptides are active when produced by the epidermal component of a cultured skin equivalent, HaCaT cells were grown to confluence on a fibroblast-containing fibrin matrix and were subsequently transduced with adenoviral vectors containing the different AMPs mentioned above. At 48 h postinfection, equivalents were raised to the air-liquid interface for an additional 5 days to achieve a certain degree of epidermal stratification that was confirmed by histological and immunohistochemical analysis (Fig. 2 A). To analyze antimicrobial activity in this system, different bacterial strains were left to adsorb on the AMP expressing gels, and after ON incubation at 37°C, viable bacteria were recovered, serially diluted, plated, and counted (Fig. 3 ). In this setting, reduction in E. coli viability ranged from 28% to 68% when seeded on LL-37 expressing equivalents compared with control (GFP+). When HBD-3 transduced HaCaT cells provided the epidermal component, a 34%–72% reduction in bacterial growth was observed. A representative experiment is shown in Fig. 2C . For S. aureus, equivalents transduced with adenoviral vectors containing LL-37 and HBD-3 also showed the best inhibitory activity (20%–45% and 20%–85%, respectively). Similarly, P. aeruginosa viability was inhibited when seeded on LL-37 (43%–53%) and HBD-3 (28%–35%) expressing equivalents (a representative experiment for each bacterial strain is shown in Fig. 2B, C ).

View larger version (37K): [in this window] [in a new window]   Figure 2. Antimicrobial activity of genetically modified HaCaT-containing cultured skin substitutes overexpressing AMPs. A) Histological appearance and immunohistochemical staining of human keratin-10 in a submerged HaCaT organotypic culture, 5 days after rising to the air-liquid interface. B) A representative experiment showing S. aureus growth after overnight incubation on the surface of adenoviral-transduced cultured skin substitutes. C) Percent viability of bacteria recovered from the surface of genetically modified skin substitutes relative to control equivalents expressing GFP. A representative experiment (out of three) for each bacterial strain is shown.

View larger version (18K): [in this window] [in a new window]   Figure 3. AMP delivery by cutaneous gene therapy: experimental setup for in vitro studies and expected beneficial therapeutic effect after grafting.

In contrast to the results obtained with conditioned media, we were unable to observe synergistic antimicrobial activity against E. coli and S. aureus when equivalents were transduced with both Ad (HBD-2) and Ad (LL-37). Expression analysis of antimicrobial peptides in these equivalents by using RT-PCR showed basal expression of HBD-2 and HBD-3 in control GFP+ expressing equivalents, and inducible ß defensin expression when HaCaT cells overexpressed LL-37 by adenoviral transfer. Thus, LL-37 overexpressing equivalents also co-expressed significant levels of HBD-2 that might synergize with cathelicidin, reaching maximum levels of killing activity against E. coli and S. aureus. This fact may explain the lack of any additional bactericidal activity against these bacterial strains when equivalents were transduced with both Ad (HBD-2) and Ad (LL-37) compared with those transduced with Ad (LL-37) alone.

CONCLUSIONS AND SIGNIFICANCE

We showed that a fibrin-based dermo-epidermal equivalent developed in our laboratory is suitable for clinical use in definitive restoration of cutaneous tissue loss after severe burns. Proper treatment of the burn wound is critical to avoid microbial multiplication and achieve an efficient tissue repair after skin grafting. Because there is an increase in the prevalence of multi-drug resistant microbes that limits the use of topical and systemic antibiotics in the majority of burn units, infection is the major cause of skin graft failure.

Here we provide evidence that a cultured skin equivalent that incorporates a genetically modified epidermal component expressing different human antimicrobial peptides exhibits microbicidal activity against pathogens frequently isolated from burn wounds. Thus, AMP delivery by cutaneous gene therapy could be useful to overcome problems concerning high cost production, continuous systemic administration, and adverse side effects of conventional antibiotics. In this sense, and in contrast to conventional antibiotics, activity of these AMPs has been evidenced in wound repair. Additional benefits are revealed in studies in which LL-37 has been shown to induce an angiogenic response by a direct action in endothelial cells. Moreover, LL-37 exhibits anti-endotoxic properties and enhances adaptive immune responses by attracting monocytes, T lymphocytes, neutrophils, and mast cells. LL-37 has been shown to be involved in dendritic cell differentiation and promotion of Th1 responses. Thus, we would expect production of AMPs in skin grafts to combat infection and contribute to an efficient tissue repair by acting at different levels in the wound healing process (Fig. 3) .

We and others have demonstrated a synergistic activity against E. coli and S. aureus of AMPs that belong to different structural classes such as LL-37 and HBD-2. Here we show that HBD-2 and HBD-3 expression is induced in cultured skin equivalents in which the epidermal component is provided by HaCaT cells overexpressing LL-37 by adenoviral transfer. Thus, a combined therapy between AMPs could be achieved by the use of a single gene therapy approach.

For these reasons, LL-37 may be a good candidate for a transient antimicrobial skin gene therapy not only in temporary coverage allowing control of infection but also in definitive coverage, exerting several biological activities that may cooperatively contribute to efficient tissue repair.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1515fje;

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