Inefficient autologous tissue recovery in skin wounds increases the susceptibility of patients to infections caused by multidrug resistant microorganisms, resulting in a high mortality rate. Genetic modification of skin cells has become an important field of study because it could lead to the construction of more functional skin grafts, through the overexpression of antimicrobial peptides that would prevent early contamination and infection with bacteria. In this study, we produce and evaluate human skin equivalents (HSEs) containing transfected human primary fibroblasts and keratinocytes by polyplexes to express the antimicrobial peptide LL-37. The effect of LL-37 on the metabolic activity of normal HSEs was evaluated before the construction of the transfected HSEs, and the antimicrobial efficacy against Pseudomonas aeruginosa and Staphylococcus aureus was evaluated. Subsequently, the levels of LL-37 in the culture supernatants of transfected HSEs, as well as the local expression, were determined. It was found that LL-37 treatment significantly promoted the cellular proliferation of HSEs. Furthermore, HSEs that express elevated levels of LL-37 were shown to possess histological characteristics close to the normal skin and display enhanced antimicrobial activity against S. aureus in vitro. These findings demonstrate that HSEs expressing LL-37 through nonviral modification of skin cells are a promising approach for the prevention of bacterial colonization in wounds.
Get full access to this article
View all access options for this article.
References
1.
ShoresJT, GabrielA, GuptaS. Skin Substitutes and Alternatives. Adv Skin Wound Care, 2007; 20:493–508.
MelstromKA, SmithJW, GamelliRL, et al.New Perspectives for a new century: implications of pathogen responses for the future of antimicrobial therapy. J Burn Care Res, 2006; 27:251–264.
4.
PercivalSL, McCartySM, LipskyB. Biofilms and wounds: an overview of the evidence. Adv Wound Care, 2015; 4:373–381.
5.
WysockiAB.Evaluating and managing open skin wounds: colonization versus infection. AACN Clin Issues Adv Pract Acute Crit Care, 2002; 13:382–397.
6.
ChaneySB, GaneshK., Mathew-SteinerS, et al.Histopathological comparisons of Staphylococcus aureus and Pseudomonas aeruginosa experimental infected porcine burn wounds. Wound Repair Regen, 2017; 25:541–549.
7.
TottyJP, BuaN, SmithGE, et al.Dialkylcarbamoyl chloride (DACC)-coated dressings in the management and prevention of wound infection: a systematic review. J Wound Care, 2017; 26:107–114.
8.
SoodA, GranickMS, TomaselliNL. Wound dressings and comparative effectiveness data. Adv Wound Care, 2014; 3:511–529.
AndreadisST. Gene-modified tissue-engineered skin: the next generation of skin substitutes. In: LeeK, KaplanD, eds. Advances in Biochemical Engineering/Biotechnology. Berlin, Germany: Springer Berlin Heidelberg, 2007:241–274.
11.
MacNeilS.Progress and opportunities for tissue-engineered skin. Nature, 2007; 445:874–880.
12.
van HartenR, van WoudenberghE, van DijkA, et al.Cathelicidins: immunomodulatory antimicrobials. Vaccines, 2018; 6:63.
13.
DuplantierAJ, van HoekML. The human cathelicidin antimicrobial peptide LL-37 as a potential treatment for polymicrobial infected wounds. Front Immunol, 2013; 4:1–14.
NeshaniA, ZareH, EidgahiMR, et al.LL-37: review of antimicrobial profile against sensitive and antibiotic-resistant human bacterial pathogens. Gene Rep, 2019; 17:100519.
16.
VandammeD, LanduytB, LuytenW, et al.A comprehensive summary of LL-37, the factoctum human cathelicidin peptide. Cell Immunol, 2012; 280:22–35.
17.
BommariusB, JenssenH, ElliottM, et al.Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides, 2010; 31:1957–1965.
18.
KorolevaA, GittardS, SchlieS, et al.Fabrication of fibrin scaffolds with controlled microscale architecture by a two-photon polymerization–micromolding technique. Biofabrication, 2012; 4:015001.
19.
HokugoA, TakamotoT, TabataY. Preparation of hybrid scaffold from fibrin and biodegradable polymer fiber. Biomaterials, 2006; 27:61–67.
20.
Thomas-VirnigCL, CentanniJM, JohnstonCE, et al.Inhibition of multidrug-resistant Acinetobacter baumannii by nonviral expression of hCAP-18 in a bioengineered human skin tissue. Mol Ther, 2009; 17:562–569.
21.
YiY, HahmS, LeeK. Retroviral gene therapy: safety issues and possible solutions. Curr Gene Ther, 2005; 5:25–35.
22.
KottermanMA, ChalbergTW, SchafferDV. Viral vectors for gene therapy: translational and clinical outlook. Annu Rev Biomed Eng, 2015; 17:63–89.
23.
HsuCY, UludağH. A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethylenimine. Nat Protoc, 2012; 7:935–945.
24.
LaU, WagnerE. Nucleic acid therapeutics using polyplexes: a journey of 50 years (and beyond). Chem Rev, 2015; 115:11043–11078.
25.
DixitS, BaganiziDR, SahuR, et al.Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin. J Biol Eng, 2017; 11:49.
26.
MoralesM, PérezD, CorreaL, et al.Evaluation of fibrin-based dermal-epidermal organotypic cultures for in vitro skin corrosion and irritation testing of chemicals according to OECD TG 431 and 439. Toxicol In Vitro, 2016; 36:89–96.
27.
Patiño VargasMI, Mesa CadavidM, Arenas GómezCM, et al.Polyplexes system to enhance the LL-37 antimicrobial peptide expression in human skin cells. Tissue Eng Part A, 2020; 26:400–410.
28.
Becerra ColoradoNY, Arenas GómezCM, Patiño VargasMI, et al.Polyplexes system vs nucleofection for human skin cells transfection and effect of IRES sequence. Tissue Eng Part C Methods, 2018; 24:233–241.
29.
Gutiérrez-HuanteM, MartínezH, BustamanteVH, et al.Bicarbonate enhances the in vitro antibiotic activity of kanamycin in Escherichia coli. Lett Appl Microbiol, 2015; 60:440–446.
30.
FarhaMA, FrenchS, StokesJM, et al.Bicarbonate alters bacterial susceptibility to antibiotics by targeting the proton motive force. ACS Infect Dis, 2018; 4:382–390.
31.
BraffMH, Di NardoA, GalloRL. Keratinocytes store the antimicrobial peptide cathelicidin in lamellar bodies. J Invest Dermatol, 2005; 124:394–400.
32.
GooJ, JiJH, JeonH, et al.Expression of antimicrobial peptides such as LL-37 and hBD-2 in nonlesional skin of atopic individuals. Pediatr Dermatol, 2010; 27:341–348.
33.
ReschelT, Koňák Č, OupickýD, et al.Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations. J Control Release, 2002; 81:201–217.
34.
GeitaniR, Ayoub MoubareckC, TouquiL, et al.Cationic antimicrobial peptides: alternatives and/or adjuvants to antibiotics active against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. BMC Microbiol, 2019; 19:54.
35.
MataraciE, DoslerS. In vitro activities of antibiotics and antimicrobial cationic peptides alone and in combination against methicillin-resistant Staphylococcus aureus biofilms. Antimicrob Agents Chemother, 2012; 56:6366–6371.
36.
GrassiL, MaisettaG, EsinS, et al.Combination strategies to enhance the efficacy of antimicrobial peptides against bacterial biofilms. Front Microbiol, 2017; 8:2409.
37.
ZharkovaMS, OrlovDS, GolubevaOY, et al.Application of antimicrobial peptides of the innate immune system in combination with conventional antibiotics—a novel way to combat antibiotic resistance?. Front Cell Infect Microbiol, 2019; 9:128.
38.
BowdishDME, DavidsonDJ, LauYE, et al.Impact of LL-37 on anti-infective immunity. J Leukoc Biol, 2005; 77:451–459.
39.
RamosR, SilvaJP, RodriguesAC, et al.Wound healing activity of the human antimicrobial peptide LL37. Peptides, 2011; 32:1469–1476.
40.
PerronGG, ZasloffM, BellG. Experimental evolution of resistance to an antimicrobial peptide. Proc R Soc B Biol Sci, 2006; 273:251–256.
41.
MercerDK, TorresMDT, DuaySS, et al.Antimicrobial susceptibility testing of antimicrobial peptides to better predict efficacy. Front Cell Infect Microbiol, 2020; 10:326.
42.
Ibáñez de AldecoaAL, ZafraO, González-PastorJE. Mechanisms and regulation of extracellular DNA release and its biological roles in microbial communities. Front Microbiol, 2017; 8:1390.
43.
McPheeJB, BainsM, WinsorG, et al.Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa. J Bacteriol, 2006; 188:3995–4006.
44.
LewenzaS.Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa. Front Microbiol, 2013; 4:21.
45.
NajmiZ, KumarA, ScaliaAC, et al.Evaluation of Nisin and LL-37 antimicrobial peptides as tool to preserve articular cartilage healing in a septic environment. Front Bioeng Biotechnol, 2020; 8:561.
46.
WangG, HankeML, MishraB, et al.Transformation of human cathelicidin LL-37 into selective, stable, and potent antimicrobial compounds. ACS Chem Biol, 2014; 9:1997–2002.
47.
HancockREW, HaneyEF, GillEE. The immunology of host defence peptides: beyond antimicrobial activity. Nat Rev Immunol, 2016; 16:321–334.
48.
MilhanNVM, de BarrosPP, de Lima ZutinEA, et al.The antimicrobial peptide LL-37 as a possible adjunct for the proliferation and differentiation of dental pulp stem cells. J Endod, 2017; 43:2048–2053.
49.
TokumaruS, SayamaK, ShirakataY, et al.Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37. J Immunol, 2005; 175:4662–4668.
50.
AdaseCA, BorkowskiAW, ZhangLJ, et al.Non-coding double-stranded RNA and antimicrobial peptide LL-37 induce growth factor expression from keratinocytes and endothelial cells. J Biol Chem, 2016; 291:11635–11646.
51.
CurrieLJ, SharpeJR, MartinR. The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review. Plast Reconstr Surg, 2001; 108:1713–1726.
52.
LeiY, RahimM, NgQ, et al.Hyaluronic acid and fibrin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery. J Control Release, 2011; 153:255–261.
53.
YoungbloodRL, TruongNF, SeguraT, et al.It's all in the delivery: designing hydrogels for cell and non-viral gene therapies. Mol Ther, 2018; 26:2087–2106.
54.
BeckerS, FrankelMB, SchneewindO, et al.Release of protein A from the cell wall of Staphylococcus aureus. Proc Natl Acad Sci, 111:1574–1579
55.
AkiyamaT, NiyonsabaF, KiatsurayanonC, et al.The human cathelicidin LL-37 host defense peptide upregulates tight junction-related proteins and increases human epidermal keratinocyte barrier function. J Innate Immun, 2014; 6:739–753.
56.
HeilbornJD, NilssonMF, KratzG, et al.The cathelicidin anti-microbial peptide LL-37 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium. J Invest Dermatol, 2003; 120:379–389.
57.
JacobsenF, MittlerD, HirschT, et al.Transient cutaneous adenoviral gene therapy with human host defense peptide hCAP-18/LL-37 is effective for the treatment of burn wound infections. Gene Ther, 2005; 12:1494–1502.
58.
HornefMW, WickMJ, RhenM, et al.Bacterial strategies for overcoming host innate and adaptive immune responses. Nat Immunol, 2002; 3:1033–1040.
59.
ShaykhievR, BeisswengerC, KändlerK, et al.Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure. Am J Physiol Cell Mol Physiol, 2005; 289:L842–L848.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
