human antimicrobial peptides ll-37 and human β-defensin-2 reduce viral replication in keratinocytes...

Human antimicrobial peptides LL-37 and human b-defensin-2 reduceviral replication in keratinocytes infected with varicella zoster virus

L. R. Crack, L. Jones, G. N. Malavige,* V. Patel and G. S. Ogg†

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford and NIHR Research Centre, Oxford, Oxfordshire, UK;

*Faculty of Medical Sciences, University of Sri Jayawardanapura, Sri Lanka; and †NIHR Biomedical Research Centre, Oxford, UK

doi:10.1111/j.1365-2230.2012.04305.x

Summary Background. There is mounting evidence that antimicrobial peptides have an

important role in cutaneous defence, but the expression of these antimicrobial peptides

in atopic eczema (AE) is still unclear. There are several families of antimicrobial

peptides, including cathelicidins and human b-defensins. Patients with AE are more

susceptible to severe cutaneous viral infections, including varicella zoster virus (VZV).

Aim. To characterize the functional activity of the antimicrobial peptides LL-37

(human cathelicidin) and human b-defensin (hBD)-2 keratinocytes were infected with

VZV, in a skin-infection model.

Methods. Flow-cytometry analysis was used to investigate LL-37 expression in

normal human keratinocytes, and quantitative PCR was used to determine viral loads

in infected HaCaT keratinocytes and B cells, with and without exogenous LL-37 and

hBD-2.

Results. LL-37 expression was present in keratinocytes, and both exogenous LL-37

and hBD-2 significantly reduced VZV load in infected keratinocytes and B cells. Specific

antibodies blocked the antiviral action exhibited by these antimicrobial peptides. Pre-

incubation of VZV with LL-37, but not hBD-2, further reduced VZV load.

Conclusions. Both LL-37 and hBD-2 have an antiviral effect on VZV replication in

the keratinocyte HaCaT cell line and in B cells, but their mechanism of action is

different. Evidence of the relationship between antimicrobial peptide expression and

higher susceptibility to infections in AE skin is still emerging. Developing novel

antiviral therapies based on antimicrobial peptides may provide improved treatment

options for patients with AE.

Introduction

More than 1300 antimicrobial peptides have been found

in a wide range of species and, as their name suggests,

they are important in host defence.1 These peptides are

generally cationic, and form a variety of structural

motifs that are 12–100 amino acids in length.2 It is

thought that the wide-ranging biological activities

exhibited by antimicrobial peptides are linked to their

structure. This includes not only antibacterial, antiviral

and antifungal activity, but also numerous immuno-

modulatory properties. Several antimicrobial peptides

have been isolated in human skin and identified as

having an important role in cutaneous defence, includ-

ing human b-defensin (hBD)-23 and hBD-3,4 the only

human cathelicidin LL-37,5 ribonuclease (RNase)75, the

S100 protein psoriasin,6 and the sweat gland-derived

protein dermcidin.8

LL-37 is synthesized and secreted in cells and tissues

exposed to environmental microbes, such as the skin

and mucosal epithelia, particularly on the buccal

Correspondence: Professor Graham Ogg, MRC Human Immunology Unit,

Weatherall Institute of Molecular Medicine, University of Oxford and NIHR

Research Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK

E-mail: [email protected]

Conflict of interest: none declared.

Accepted for publication 8 September 2011

Experimental dermatology • Original article CEDClinical and Experimental Dermatology

� The Author(s)

534 CED � 2012 British Association of Dermatologists • Clinical and Experimental Dermatology, 37, 534–543

mucosa of the tongue, oesophagus, cervix and vagi-

na.9,10 Cationic peptides can be constitutively expressed,

such as in eccrine sweat glands11 and airway surface

fluids,12 or their expression can be induced by inflam-

matory cytokines, such as interleukin (IL)-6 and tumour

necrosis factor (TNF)-a, or in response to pathogen-

associated molecular patterns, such as lipopolysaccha-

ride (LPS).5 In neutrophils, hCAP18 is stored in the

unprocessed form within peroxidase-negative granules,

and only upon cellular release is the C-terminal LL-37

mature peptide cleaved from the N-terminal cathelin

precursor by serine proteases in azurophilic granules.13–15

LL-37 is also expressed in salivary glands, the epididy-

mis, testes and seminal fluid.16–18 Expression of LL-37

is inducible in keratinocytes,5 and is rapidly upregulat-

ed in the epidermis as a result of injury.19 Expression

of the gene encoding LL-37 has also been detected

in natural killer cells, B cells, monocytes and cd T

cells.16

Human b-defensins are expressed in epithelial tissues,

the first line of host defence. Expression of hBD-2 has

been shown in keratinocytes, gingival mucosa and

tracheal epithelium.3,20,21 Expression of hBD-2 can be

induced by IL-1b, TNF-a and LPS, and by several types

of microbe.3,22,23 hBD-2 expression is therefore induced

in response to inflammation.20,24–26 By contrast, hBD-1

is not inducible,27 suggesting that it has a role in

antimicrobial defence in the absence of inflammation.

Several studies have investigated the expression of

various antimicrobial peptides in atopic eczema (AE). It

has been shown that, compared with psoriatic skin,

atopic skin displays reduced induction of LL-37, hBD-2,

hBD-3 and dermcidin.28–30 Most of these earlier studies

have compared atopic eczema (AE) with psoriatic rather

than with healthy skin. Recently, enhanced expression

of hBD-2, hBD-3, RNase 7 and psoriasis has been

reported in AE skin compared with healthy skin.31

Patients with AE have been shown to be more

susceptible to a number of different viral infections.

Severe infections of herpes simplex virus (HSV)-1

(eczema herpeticum),32 vaccinia virus (VV) (eczema

vaccinatum)33 and molluscum contagiosum virus

(eczema mollusculatum)34 occur more often in patients

with AE than in healthy people. Skin taken from atopic

patients with eczema herpeticum exhibit reduced cath-

elicidin expression, and higher levels of HSV-2 replica-

tion are seen in cathelicidin-deficient mice.32 Patients

with AE are also more susceptible to severe varicella

zoster virus (VZV) infections.35

We sought to determine the effect of LL-37 and hBD-2

on VZV loads in keratinocytes and B-cell lines, and to

elucidate the possible mechanism mediating these effects.

Methods

Cell culture

Keratinocyte lines (kind gift of Dr Antony Black) were

cultured at 37 �C in 5% CO2 in tissue-culture flasks

(3524; Corning Inc., Corning, New York, USA) con-

taining DMEM (D5546; Sigma Aldrich, Gillingham, UK)

supplemented with 50 lg ⁄ mL penicillin (DE17-603E;

BioWhittaker, Lonza Group Ltd., Basel, Switzerland),

50 lg ⁄ mL streptomycin (DE17-602E; BioWhittaker,

Lonza Group Ltd.), 2 mM L-glutamine (BE17-605E;

BioWhittaker, Lonza Group Ltd.) and 10% fetal calf

serum (FCS) (F9665; Sigma Aldrich). Cells were split on

reaching confluence (approximately every 4–5 days) by

removing the medium and washing the adherent

cells with trypsin–EDTA (BE17–161E; Sigma-Aldrich),

warmed to 37 �C, to remove any remaining medium, as

trypsinization is blocked by the presence of FCS.

Trypsin–EDTA (2 mL) was added to the flask for

5 min (kept at 37 �C in 5% CO2) so that all adherent

cells were suspended in the trypsin–EDTA, applying

gentle tapping if necessary. The trypsin–EDTA was

diluted with five times its volume of warm supplemented

DMEM. Cells were then spun in a centrifuge at 350 g

for 5 min, and the supernatant was discarded. The

pellet was resuspended in warm supplemented DMEM

and split appropriately into flasks.

B-cell lines were cultured in tissue-culture flasks in

RPMI (R8758; Sigma-Aldrich) supplemented with strep-

tomycin, penicillin, L-glutamine and 10% FCS, and split

every 2–3 days.

Cellular expression of LL-37

To confirm expression of LL-37 in the skin,36 normal

human keratinocytes (NHKs) at a concentration of

2 · 105 ⁄ mL were stimulated with either medium or

with a mixture of 50 ng ⁄ mL phorbol-12-myristate-13-

acetate (PMA; 79346; Sigma-Aldrich) plus 250 ng ⁄ mL

ionomycin (I3090; Sigma-Aldrich), and incubated for

1.5–2 h at 37 �C in 5% CO2 in 5 mL polypropylene

round-bottom tubes (352053; Becton Dickinson, Frank-

lin Lakes, NJ, USA). Cells were stained with anti-LL-37

(PA-LL-37–100; Innovagen AB, Lund, Sweden) or

isotype control, and incubated for 20 min at 4 �C in

the dark. The cells were again washed twice with PBS

via centrifugation at 300 g. for 5 min. Cells were fixed

with 0.5% formaldehyde, and analysed with a nine-

colour flow cytometer (CyAn; Dako, Glostrup, Denmark)

and FlowJo software (version 8; Tree Star Inc, Ashland,

OR, USA) within 48 h.

� The Author(s)

CED � 2012 British Association of Dermatologists • Clinical and Experimental Dermatology, 37, 534–543 535

Antimicrobial peptides reduce VZV replication in infected keratinocytes • L. R. Crack et al.

Assessment of exogenous antimicrobial peptides on

varicella zoster virus load in human keratinocytes or

B cells

To investigate the effect of the antimicrobial peptides

LL-37 and hBD-2 on the VZV load in the human

keratinocyte HaCaT cell line, dose–time response

experiments were conducted. Human keratinocytes

(HaCaT cells) or B cells (2 mL of culture containing

5 · 105 ⁄ mL) were placed into 24-well plates, and

either infected with live attenuated VZV vaccine

[Varilrix� (VZV); 091505, GlaxoSmithKline, Brentford,

Middlesex UK] or left uninfected. Either LL-37

(HC4013; HyCult Biotechnology, Uden, The Nether-

lands) or hBD-2 (CYT-571; ProSpec-Tany Technogene

Ltd., East Brunswick, NJ, USA) was then added to the

infected cells at varying concentrations (a 10-fold

dilution series in five steps from 0.4 lg ⁄ mL down to

0.00004 lg ⁄ mL in phosphate-buffered saline). The

starting concentration was determined, based on

previous reports of the unstimulated physiological

concentrations reported for these antimicrobial pep-

tides.

In blocking experiments, anti-LL-37 (PA-LL-37-100;

Innovagen, Sweden) and anti-hBD-2 (AHP849; AbD

Serotec, Oxford, UK) were added to the cells. In pre-

incubation experiments, VZV vaccine was incubated for

24 h with 0.4 lg ⁄ mL LL-37 or hBD-2, and subse-

quently used to infect HaCaT cells.

DNA was extracted on days 3, 7 or 10 after infection,

and viral load determined by quantitative (q)PCR

assays. DNA was extracted (DNA Blood Mini Kit,

51306; Qiagen, Hilden, Germany) in accordance with

the manufacturer’s instructions, and stored at 4 �C.

The ORF29 plasmid used to generate the standard

curve was diluted in a stock solution of 1 · 106

plasmids ⁄ 5 lL. The 106 plasmid stock solution was

diluted in salmon-sperm DNA (AM9680; Applied Bio-

systems, Warrington, Cheshire, UK) (0.3 pg ⁄ lL) to give

concentrations of 1, 5, 50,102, 103, 104, and 105

copies ⁄ 5 lL. The qPCR master mix components are

listed in Table 1 (for 100 samples, and in the order in

which they were added). Samples and standards (5 lL)

were added to a 96-well reaction plate (4346907;

MicroAmp Fast Reaction Plate; Applied Biosystems)

using sterile filter tips (TF-20-L-R-S; Thistle Scientific,

Glasgow, UK). Distilled water was used as a negative

control, and salmon-sperm DNA as a nontemplate

control. Subsequently, 20 lL of the qPCR master mix

were then added to each well. The plate was sealed with

optical adhesive film (4311971, MicroAmp; Applied

Biosystems), thoroughly mixed by vortexing, and briefly

spun in a centrifuge at 300 g. The plate was loaded

into the PCR analyser (7700 Fast Real-time PCR

System; Applied Biosystems) and the results analysed

(7500 Fast System SDS Software; Applied Biosciences,

Carlsbad, CA, USA). The reaction conditions were

50 �C for 2 min, followed by 95 �C for 10 min, then

60 cycles of 95 �C for 15 s, and a final step at 60 �C for

60 s.

The sequence of the ORF29-specific VZV probe used

was 5¢-(FAM)CCCGTGGAGCGCGTCGAAA(TAMRA)-3¢.Data were analysed using the Fast 7700 software

(Applied Biosystems), with the real-time fluorescence

values measured by the quantity of the reporter dye,

FAM, released during amplification.

Blocking of antimicrobial peptides with antibodies

Experiments were carried out to determine whether the

effects of LL-37 or hBD-2 could be blocked with specific

antibodies. Infected HaCaT cells were incubated with

LL-37 or hBD-2 with or without the corresponding

specific antibody.

Table 1 Components of the varicella zoster viral load quantitative PCR master mix for 100 samples.

Component Volume, lL Concentration Source Catalogue no.

Distilled water 1300 N ⁄ A N ⁄ A N ⁄ A10x buffer* 250 Contains 15 mmol ⁄ L MgCl2 HotStar Taq DNA polymerase kit 203205

MgCl2* 50 25 mmol ⁄ L HotStar Taq DNA polymerase kit 203205

DNTPs† 75 100 mmol ⁄ L DNTP set 10297018

Forward primer† 100 1 lg ⁄ lL N ⁄ A N ⁄ A§

Reverse primer 100 1 lg ⁄ lL N ⁄ A N ⁄ A§

HotStarTaq† 25 5 U ⁄ lL HotStar Taq DNA polymerase kit 203205

Probe‡ 100 5 pmol ⁄ lL N ⁄ A N ⁄ A§

Total 2000 – – –

N ⁄ A, not applicable. *Qiagen, Hilden, Germany; �Invitrogen Europe Holdings, Paisley, UK; �Applied Biosystems, Warrington, Cheshire, UK;

§custom-made.

� The Author(s)



Incubation of virus with the antimicrobial peptides

before infection

To investigate whether the antimicrobial peptides might

have a direct effect on the virus, VZV was incubated

with LL-37 or hBD-2 for 24 h before the keratinocytes

were infected with the VZV solution. After 10 days, the

cell DNA was extracted, and viral loads were determined

by qPCR.

Aciclovir treatment

As a positive control for the system, infected keratino-

cytes were treated with the antiviral agent aciclovir, at

varying doses and over the same 10-day time course

used for the antimicrobial peptides.

Assessment of early apoptosis and cell death in

keratinocytes

Keratinocytes were stained with an apoptosis marker

(annexin V) and a marker of dead cells (Viaprobe) on

days 3, 7 and 10 postinfection, and the percentage of

annexin V-positive or Viaprobe-positive cells was

assessed in uninfected, infected and infected cells treated

or not with exogenous LL-37 or hBD-2. The uninfected

control and infected HaCaT cells, with and without

exogenous antimicrobial peptides, had equivalent pro-

portions of dead and dying cells, suggesting that the

reduction in viral load seen in infected keratinocytes

incubated with antimicrobial peptides is not simply a

result of reduced cell numbers for VZV virions to infect.

Statistical analysis

The unpaired t-test was performed as appropriate using

Prism software (version 4.0 for Windows; GraphPad,

San Diego, CA, USA). The D’Agostino–Pearson Omnibus

test was used to test the normality of data distribution.

In all cases, normally distributed data resulted in

parametric methods being used.

Results

Cellular expression of LL-37

Expression of LL-37 in NHKs in response to PMA ⁄ ion-

omycin stimulation was compared with an isotype

control (Fig. 1). As expected, LL-37 was strongly

expressed in these keratinocytes.

Effect of exogenous antimicrobial peptides on varicella

zoster virus load in human keratinocytes

The changes in VZV load over time were assessed for

varying doses of LL-37 (Fig. 2a) and exogenous hBD-2.

There was little difference between the infected

HaCaT control cells and the HaCaT cells incubated with

LL-37 or hBD-2. On day 7, the viral load was signifi-

cantly lower in the cells treated with higher concentra-

tions of LL-37, but there was no difference in the cells

treated with hBD-2. On day 10, the viral load was

significantly lower in all cells treated with LL-37,

regardless of dose, and was also significantly lower in

all cells treated with hBD-2, except for the lowest

concentration of 4 · 10)5 lg ⁄ mL.

Effect of exogenous antimicrobial peptides on varicella

zoster virus load in human B cells

To see if this antiviral effect could be replicated in other

cells, human B cells either were left uninfected, were

infected with VZV, or were treated with additional

Figure 1 Flow-cytometry analysis of LL-37 expression in human keratinocytes. (a) Cells were gated on live keratinocytes; (b) keratinocyte

expression of LL-37 (blue), compared with the isotype control (red).

� The Author(s)



exogenous LL-37 or hBD-2 (at 0.4 lg ⁄ mL). B cells have

been shown to express LL-37 and hBD-2 mRNA, and

are readily infected by VZV.

On day 3, a slight but insignificant difference in viral

load between the infected control cells and the infected

cells incubated with LL-37 was seen. On day 7, there

was a decrease in the VZV load, which was of borderline

significance (P = 0.05, unpaired t-test), and reached

significance by day 10 (P < 0.05, unpaired t-test). By

contrast, there was little difference in viral load between

infected control B cells and infected B cells incubated

with hBD-2 seen on either day 3 or day 7 (Fig. 2d), but

the difference was significant on day 10 (P < 0.05,

unpaired t-test).

Effect on varicella zoster virus load of blocking

antimicrobial peptides with antibodies

Having determined that both antimicrobial peptides

could reduce VZV load in keratinocytes, we sought to

characterize whether this response could be blocked

with specific antibodies.

As shown previously, there was a reduction in viral

load in infected HaCaT cells incubated with LL-37

(Fig. 3a) or hBD-2 (Fig. 3b) compared with the infected

control. Addition of anti-LL-37 or anti-hBD-2 alone had

no effect on VZV load. The VZV load was significantly

higher after addition of the antimicrobial peptide plus its

antibody together, compared with the addition of the

peptide alone (P < 0.01, unpaired t-test)

These data suggest that the addition of specific

antibodies blocks the antiviral effect of LL-37 and

hBD-2.

Investigating the potential antiviral mechanisms of

LL-37 and hBD-2

Addition of exogenous LL-37 reduced the viral load in

infected keratinocytes (Fig. 4a). Keratinocytes infected

Figure 2 Effect of exogenous antimicrobial peptides on varicella zoster virus (VZV) load in human keratinocytes and B cells. VZV-infected

(a,b) HaCaT cells or (c,d) B cells were incubated with 0.4, 0.04, 4 · 10)3, 4 · 10)4 and 4 · 10)5 lg ⁄ mL of (a,c) LL-37 or (b,d)

hBD-2. DNA was extracted on days 3, 7 and 10 postinfection, and the viral load determined by quantitative PCR. *P < 0.05 compared

with the infected control (unpaired t-test).

� The Author(s)



with VZV pre-incubated with peptide had a significant

further reduction in viral load compared with the

infected control and with infected keratinocytes incu-

bated with LL-37 (P < 0.05, unpaired t-test). Interest-

ingly, this effect was not seen for hBD-2 (Fig. 4b),

suggesting that LL-37 and hBD-2 utilize different

antiviral mechanisms.

Effect of the antiviral agent aciclovir on varicella zoster

replication in keratinocytes

At day 3, aciclovir reduced the viral load in a dose-

dependent manner, with a higher VZV load at the lower

concentrations (Fig. 5). However, at days 7 and 10, the

VZV loads were almost completely abolished by aciclo-

vir, at all concentrations.

Levels of early apoptosis and cell death in keratinocytes

To exclude a possible role of cell death in the observed

antiviral effects of LL-37 and hBD-2, keratinocytes were

stained with an apoptosis marker, annexin V, and a

dead-cell marker, Viaprobe, on days 3, 7 and 10

postinfection. Fi.g 6a shows the percentage of annexin

V-positive cells in uninfected, infected, and infected plus

exogenous LL-37 or hBD-2 after 3, 7 and 10 days

(Fig. 6b). The uninfected control and infected HaCaT

cells, with or without exogenous antimicrobial peptides,

had equivalent proportions of dead and dying cells,

suggesting that the reduction in viral load seen in the

infected keratinocytes incubated with antimicrobial

peptides is not simply a result of reduced cell numbers

for VZV virions to infect.

Discussion

To investigate the effect of LL-37, a functional model

using human keratinocyte (HaCAT) cells was used. The

cells were infected with VZV, and incubated with

varying concentrations of exogenous LL-37 or hBD-2.

It has previously been reported that HaCaT cells do not

express LL-37 mRNA in response to either LPS or

ultraviolet stimulation.36 Therefore, HaCaT cells were

used, as they are devoid of endogenous LL-37. Because

LL-37 is upregulated in keratinocytes in response to

infectious agents,5 the lack of endogenous LL-37 was

essential to establish the concentration of LL-37

required to exert any possible antiviral effect. The same

experiments were carried out with B cells, which have

been shown to express LL-37 and hBD-2 mRNA, and

are readily infected by VZV. The viral load of VZV was

determined by qPCR.

Both peptides reduced the VZV load in human

keratinocytes; this reduction was significant 10 days

postinfection for both peptides at physiologically rele-

vant concentrations, and for higher concentrations of

LL-37 at 7 days postinfection. This pattern of antiviral

Figure 3 Effect of blocking antibodies on viral load. Infected HaCaT

cells were treated with (a) LL-37 (0.4 lg ⁄ mL), anti-LL-37, or both;

and (b) hBD-2 (0.4 lg ⁄ mL), anti-hBD-2, or both. DNA was

extracted after 10 days, and the viral load determined by quanti-

tative PCR. (a) P < 0.01, (b) P < 0.001 (unpaired t-test).

Figure 4 Effect of pre-incubation of varicella zoster virus (VZV)

with antimicrobial peptides before infection. VZV was incubated for

24 h with 0.4 lg ⁄ mL (a) LL-37 or (b) hBD-2, then HaCaT cells

were infected with VZV alone or pre-incubated VZV, and incubated

with the relevant exogenous peptide for 10 days postinfection.

(a) P < 0.05, (b) non-significant.

� The Author(s)



activity was replicated in B cells. Having determined

that both antimicrobial peptides can reduce VZV load in

keratinocytes, we sought to characterize whether this

response could be blocked with specific antibodies,

which indeed was found to be the case, further

indicating the antiviral activity of these peptides. Pre-

incubation of VZV with LL-37 but not hBD-2 further

reduced the viral load after 10 days.

As a positive control for the system, infected kera-

tinocytes were treated with the antiviral agent

aciclovir, at varying doses and over the same 10-day

time course. Aciclovir is a potent inhibitor of VZV, but

has little effect on host cells.37–39 We found that

aciclovir reduced the viral load in a dose-dependent

fashion at 3 days postincubation, and had completely

abolished the viral load by day 7. The percentages of

dead or dying cells increased during the time course of

incubation; however, this increase was proportional in

all treatments.

Although it was previously reported that LL-37 has

little effect on HSV-1 and 2,40 more recent evidence

suggests that in fact, LL-37 exhibits significant anti-

viral activity against HSV.33 This antiviral activity

Figure 5 Effect of the antiviral agent aciclovir on varicella zoster virus (VZV) load in keratinocytes: dose–time response. VZV-infected B cells

10, 5, 2, 1 or 0.5 lmol ⁄ L aciclovir, and DNA was extracted on days (a) 3, (b) 7 and (c) 10 postinfection,and the viral load determined by

quantitative PCR. Statistical significance compared with the infected control was determined by unpaired t-test with Welch correction.

Figure 6 Cell death in varicella zoster virus (VZV)-infected

keratinocytes. Keratinocytes were infected with VZV and incubated

with no peptide, 0.4 lg ⁄ mL LL-37, or 0.4 lg ⁄ mL hBD-2. Cells

were harvested on days 3, 7 and 10 postinfection, and stained with

anti-annexin V and a marker of dead cells (Viaprobe). Percentage

of (a) annexin V-positive and (b) Viaprobe-positive cells.

� The Author(s)



was supported by another study, in which LL-37

exhibited anti-HSV activity in corneal and conjunctival

epithelia.41 LL-37 has also been shown to inhibit

vaccinia virus in vitro and in vivo,42 and to inhibit

human immunodeficiency virus (HIV)-1 replication in

CD4+ T cells.43 In response to viral infection early in

the innate immune response, some cells exhibit

upregulation of human b-defensins. In human oral

epithelial cells, HIV-1 has been shown to upregulate

the mRNA of hBD-2 and hBD-3, even in the absence of

HIV-1 replication.44 These cells lack the HIV-1 entry

receptors CXCR4 and CCR5, so whether interaction

between the cells and the virus is responsible for this

response is unclear. A similar response was seen in

human bronchial epithelial cells to human rhinovirus,

although unlike HIV-1, this response was dependent

on active viral replication.45,46 Evidently, little is

understood about the antiviral mechanisms used by

antimicrobial peptides, and whether their structural

differences influence their antiviral activity. Our data

suggests that although both antimicrobial peptides

exert anti-VZV activity, their mechanism of action

differs. Because VZV has an incubation period of 10–

21 days this finding may be relevant to disease

pathogenesis.47

A crucial difference between the anti-VZV actions

exhibited by these two antimicrobial peptides is that pre-

incubation of VZV with LL-37 but not hBD-2 further

reduced the viral load at 10 days postinfection. This

variation could perhaps be explained by differences in

their mechanism of action. Current models indicate that

antimicrobial peptides can either disrupt the envelope or

interact with viral glycoproteins. Previous work has

shown that LL-37 alters the morphology of VV.43

Exposing VZV to the antimicrobial peptides and observ-

ing any morphological changes may elucidate whether

these peptides directly disrupt the viral membrane; these

experiments are underway. It is of interest that signif-

icant effects of the antimicrobial peptides were not seen

in this study until day 10; it may be that this contributes

to the clinical observation that the VZV rash may take

several days to resolve.

The course of infection in was found to differ between

B cells and keratinocytes, which could be an important

finding. Apart from actual viral load, the pattern of

infection differed between the cell types over time. This

might be attributable to the inherent lack of anti-

microbial peptides expressed by HaCaT cells. Expression

of LL-37 mRNA has been investigated previously in B

cells.16 Further work is required to assess the viral life

cycle in these cell types and the effects of antimicrobial

peptides on VZV.

Although cell death reached approximate levels of

50% dying and 30% dead, the levels were consistent in

both uninfected and infected controls, and in cells

treated with LL-37 or hBD-2. Thus, it is not likely that

cell death plays a role in the reduction in viral loads

seen in keratinocytes treated with these antimicrobial

peptides.

Conclusion

In summary, these data suggest that both LL-37 and

hBD-2 have an antiviral effect on VZV replication in

keratinocytes (and B cells), but their mechanism of

action is different. Children with AE have more severe

symptoms associated with VZV compared with their

nonatopic counterparts.35 Developing novel antiviral

treatments based on antimicrobial peptides may

provide improved therapy options for patients with

AE.

What is already known about thistopic?

• Little is known about the antiviral mecha-

nisms of antimicrobial peptides (antimicrobial

peptides).

• Compared with their nonatopic counterparts,

children with AE may have more severe symptoms

associated with VZV.

• Evidence of the role of antimicrobial peptides in

AE is mounting.

• AE skin has been shown to express lower levels

of certain antimicrobial peptides when compared

with psoriatic skin, but more recent evidence

suggests that higher levels of some antimicrobial

peptides occur in AE compared with normal skin.

What does this study add?

• The antimicrobial peptides LL-37 and hBD-2

have an antiviral effect on VZV replication in

keratinocytes (and B cells), but their mechanism of

action is different.

• Developing novel antimicrobial peptide-based

antiviral treatments may offer potential improve-

ments in treatment options for patients with AE.

� The Author(s)



Acknowledgements

We are most grateful to the Medical Research Council

and Oxford NIHR Biomedical Research Centre Pro-

gramme for support, and to all patient and control

donors who participated in this research.

References

1 Wang G, Li X, Wang Z. APD2. the updated antimicrobial

peptide database and its application in peptide design. Nucl

Acids Res 2009; 37: D933–D937.

2 Zasloff M. Antimicrobial peptides of multicellular organ-

isms. Nature 2002; 415: 389–395.

3 Harder J, Bartels J, Christophers E, Schroder JM. A peptide

antibiotic from human skin. Nature 1997; 387: 861.

4 Harder J, Bartels J, Christophers E, Schroder JM. Isolation

and characterization of human-beta defensin-3, a novel

human inducible peptide antibiotic. J Biol Chem 2001;

276: 5707–5713.

5 Frohm M, Agerberth B, Ahangari G et al. The expression of

the gene coding for the anti-microbial peptide LL-37 is

induced in human keratinocytes during inflammatory

disorders. J Biol Chem 1997; 272: 15258–15263.

6 Harder J, Schroder JM. RNase7, a novel innate immune

defense antimicrobial protein of healthy human skin. J Biol

Chem 2002; 277: 46779–46784.

7 Glaser R, Harder J, Bartels J et al. Antimicrobial psoriasin

(S100A7) protect human skin from Escherichia coli infec-

tion. Nat Immunol 2005; 6: 57–64.

8 Schittek B, Hipfel R, Sauer B et al. Dermcidin: a novel hu-

man antibiotic peptide secreted by sweat glands. Nat Im-

munol 2001; 2: 1133–1137.

9 Frohm Nilsson M, Sandstedt B, Sørensen O et al. The hu-

man cationic antimicrobial protein (hCAP18), a peptide

antibiotic, is widely expressed in human squamous epi-

thelia and colocalizes with interleukin-6. Infect Immun

1999; 67: 2561–2566.

10 Murakami M, Ohtake T, Dorschner RA, Gallo RL. Cathe-

licidin antimicrobial peptides are expressed in salivary

glands and saliva. J Dent Res 2002; 81: 845–850.

11 Murakami M, Ohtake T, Dorschener RA et al. Cathelicidin

anti-microbial peptide expression in sweat, an innate de-

fense system for the skin. J Invest Dermatol 2002; 119:

1090–1095.

12 Bals R, Wang X, Zasloff M, Wilson JM. The peptide anti-

biotic LL-37 ⁄ hCAP-18 is expressed in epithelia of the

human lung where it has broad antimicrobial activity at

the airway surface. Proc Natl Acad Sci 1998; 95:

9541–9546.

13 Cowland JB, Johnsen AH, Borregaard N. hCAP-18, a ca-

thelin ⁄ pro-bactenecin-like protein of human neutrophil

specific granules. FEBS Lett 1995; 368: 173–176.

14 Oren Z, Lerman JC, Gudmundsson GH et al. Structure and

organization of the human antimicrobial peptide LL-37 in

phospholipid membranes: relevance to the molecular basis

for its non-cell-selective activity. Biochem J 1999; 341:

501–513.

15 Lehrer RI, Ganz T. Cathelicidins: a family of endogenous

antimicrobial peptides. Curr Opin Hematol 2002; 9: 18–22.

16 Agerberth B, Charo J, Werr J et al. The human anti-

microbial and chemotactic peptides LL-37 and alpha-de-

fensins are expressed by specific lymphocyte and monocyte

populations. Blood 2000; 96: 3086–3093.

17 Malm J, Sorensen O, Persson T et al. The human cationic

antimicrobial protein (hCAP-18) is expressed in the epi-

thelium of human epididymis, is present in seminal plasma

at high concentrations, and is attached to spermatozoa.

Infect Immun 2000; 68: 4297–4302.

18 Andersson E, Sorensen OE, Frohm B et al. Isolation of hu-

man cationic antimicrobial protein-18 from seminal plas-

ma and its association with prostasomes. Hum Reprod

2002; 17: 2529–2534.

19 Dorschner RA, Pestonjamasp VK, Tamakuwala S et al.

Cutaneous injury induces the release of cathelicidin anti-

microbial peptides active against group A streptococcus. J

Invest Dermatol 2001; 117: 91–97.

20 Hiratsuka T, Nakazato M, Date Y et al. Identification of

human beta-defensin-2 in respiratory tract and plasma and

its increase in bacterial pneumonia. Biochem Biophys Res

Commun 1998; 249: 943–947.

21 Mathews M, Jia HP, Guthmiller JM et al. Production of

beta-defensin antimicrobial peptides by the oral mucosa

and salivary glands. Infect Immun 1999; 67: 2740–2745.

22 Chadebech P, Goidin D, Jacquet C et al. Use of human re-

constructed epidermis to analyze the regulation of beta-

defensin hBD-1, hBD-2, and hBD-3 expression in response

to LPS. Cell Biol Toxicol 2003; 19: 313–324.

23 Donnarumma G, Paoletti I, Buommino E et al. Malassezia

furfur induces the expression of beta-defensin-2 in human

keratinocytes in a protein kinase C-dependent manner.

Arch Dermatol Res 2004; 295: 474–481.

24 Schonwetter BS, Stolzenberg ED, Zasloff MA. Epithelial

antibiotics induced at sites of inflammation. Science 1995;

267: 1645–1648.

25 Singh PK, Jia HP, Wiles K et al. Production of beta-de-

fensins by human airway epithelia. Proc Natl Acad Sci USA

1998; 95: 14961–14966.

26 Ali RS, Falconer A, Ikram M et al. Expression of the peptide

antibiotics human beta defensin-1 and human beta de-

fensin-2 in normal human skin. J Invest Dermatol 2001;

117: 106–111.

27 Schroeder BO, Wu Z, Nuding S et al. Reduction of dis-

ulphide bonds unmasks potent antimicrobial activity of

human b-defensin 1. Nature 2011; 469: 419–423.

28 Ong PY, Ohtake T, Brandt C et al. Endogenous anti-

microbial peptides and skin infections in atopic dermatitis.

N Engl J Med 2002; 347: 1151–1160.

29 Nomura I, Goleva E, Howell MD et al. Cytokine milieu of

atopic dermatitis; as compared to psoriasis, skin prevents

induction of innate immune response genes. J Immunol

2003; 171: 3262–3269.

� The Author(s)



30 de Jongh GJ, Zeeuwen PL, Kucharekova M et al. High ex-

pression levels of keratinocyte antimicrobial proteins in

psoriasis compared with atopic dermatitis. J Invest Dermatol

2005; 125: 1163–1173.

31 Harder J, Dressel S, Wittersheim M et al. Enhanced ex-

pression and secretion of antimicrobial peptides in atopic

dermatitis and after superficial skin injury. J Invest Dermatol

2010; 130: 1355–1364.

32 Kaposi M. Pathologie und Therapie der Hautkrankheiten, 5th

edn. Berlin: Urban und Schwarzenberg, 1887.

33 Howell MD, Gallo RL, Boguniewicz M et al. Cytokine milieu

of atopic dermatitis skin subverts the innate immune re-

sponse to vaccinia virus. Immunity 2006; 24: 341–348.

34 Soloman L, Telner P. Eruptive molluscum contagiosum in

atopic dermatitis. Can Med Assoc J 1966; 95: 978–979.

35 Verbov J, Hart A. Severe varicella in a child with atopic

eczema and ichthyosis. Practitioner 1986; 230: 15–16.

36 Kim JE, Kim BJ, Jeong MS et al. Expression and modulation

of LL-37 in normal human keratinocytes, HaCaT cells, and

inflammatory skin diseases. J Korean Med Sci 2005; 20:

649–654.

37 Elion GB. Mechanism of action and selectivity of acyclovir.

Am J Med 1982; 73: 7–13.

38 Elion GB, Furman PA, Fyfe JA et al. Selectivity of action of

an antiherpetic agent, 9-(2-hydroxyethoxymethyl) gua-

nine. Proc Natl Acad Sci USA 1977; 74: 5716–5720.

39 Schaeffer HJ, Beauchamp L, de Miranda P et al. 9-(2-hy-

droxyethoxymethyl) guanine activity against viruses of the

herpes group. Nature 1978; 272: 583–585.

40 Yasin B, Pang M, Turner JS et al. Evaluation of the in-

activation of infectious Herpes simplex virus by host-de-

fense peptides. Eur J Clin Microbiol Infect Dis 2000; 19:

187–194.

41 Gordon YJ, Huang LC, Romanowski EG et al. Human

cathelicidin (LL-37), a multifunctional peptide, is

expressed by ocular surface epithelia and has potent

antibacterial and antiviral activity. Curr Eye Res 2005; 30:

385–394.

42 Howell MD, Jones JF, Kisich KO et al. Selective killing of

vaccinia virus by LL-37: implications for eczema vaccina-

tum. J Immunol 2004; 172: 1763–1767.

43 Bergman P, Walter-Jallow K, Broliden B et al. The anti-

microbial peptide LL-37 inhibits HIV-1 replication. Curr

HIV Res 2007; 5: 410–415.

44 Quinones-Mateu ME, Ball SC, Marozsan AJ et al. Human

epithelial beta-defensins 2 and 3 inhibit HIV-1 replication.

AIDS 2003; 17: F39–F48.

45 Duits LA, Nibbering PH, van Strijen E et al. Rhinovirus

increases human beta-defensin-2 and -3 mRNA expression

in cultured bronchial epithelial cells. FEMS Immunol Med

Microbiol 2003; 38: 59–64.

46 Proud D, Sanders SP, Wiehler S. Human rhinovirus in-

fection induces airway epithelial cell production of human

beta-defensin 2 both in vitro and in vivo. J Immunol 2004;

172: 4637–4645.

47 Whitley RJ. Varicella-Zoster Virus Infections. Antiviral Agents

and Viral Diseases of Man. Galasso RJWGJ, Merigan TC. New

York: Raven Press, 1990: 235–263.

� The Author(s)



human antimicrobial peptides ll-37 and human β-defensin-2 reduce viral replication in keratinocytes...

Documents