Impact of varicella-zoster virus on dendritic cell subsets in human skin during natural infection.
ABSTRACT Varicella-zoster virus (VZV) causes varicella and herpes zoster, diseases characterized by distinct cutaneous rashes. Dendritic cells (DC) are essential for inducing antiviral immune responses; however, the contribution of DC subsets to immune control during natural cutaneous VZV infection has not been investigated. Immunostaining showed that compared to normal skin, the proportion of cells expressing DC-SIGN (a dermal DC marker) or DC-LAMP and CD83 (mature DC markers) were not significantly altered in infected skin. In contrast, the frequency of Langerhans cells was significantly decreased in VZV-infected skin, whereas there was an influx of plasmacytoid DC, a potent secretor of type I interferon (IFN). Langerhans cells and plasmacytoid DC in infected skin were closely associated with VZV antigen-positive cells, and some Langerhans cells and plasmacytoid DC were VZV antigen positive. To extend these in vivo observations, both plasmacytoid DC (PDC) isolated from human blood and Langerhans cells derived from MUTZ-3 cells were shown to be permissive to VZV infection. In VZV-infected PDC cultures, significant induction of alpha IFN (IFN-alpha) did not occur, indicating the VZV inhibits the capacity of PDC to induce expression of this host defense cytokine. This study defines changes in the response of DC which occur during cutaneous VZV infection and implicates infection of DC subtypes in VZV pathogenesis.
- SourceAvailable from: Marshall V Williams[Show abstract] [Hide abstract]
ABSTRACT: We have previously shown that Epstein-Barr virus (EBV)-encoded dUTPase can modulate innate immune responses through the activation of TLR2 and NF-κB signaling. However, whether this novel immune function of the dUTPase is specific for EBV or a common property of the Herpesviridae family is not known. In this study, we demonstrate that the purified viral dUTPases encoded by herpes simplex virus type 2 (HSV-2), human herpesvirus-6A (HHV-6A), human herpesvirus-8 (HHV-8) and varicella-zoster virus (VZV) differentially activate NF-κB through ligation of TLR2/TLR1 heterodimers. Furthermore, activation of NF-κB by the viral dUTPases was inhibited by anti-TLR2 blocking antibodies (Abs) and the over-expression of dominant-negative constructs of TLR2, lacking the TIR domain, and MyD88 in human embryonic kidney 293 cells expressing TLR2/TLR1. In addition, treatment of human dendritic cells and PBMCs with the herpesviruses-encoded dUTPases from HSV-2, HHV-6A, HHV-8, and VZV resulted in the secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, IL-10, and IFN-γ. Interestingly, blocking experiments revealed that the anti-TLR2 Ab significantly reduced the secretion of cytokines by the various herpesviruses-encoded dUTPases (p < 0.05). To our knowledge, this is the first report demonstrating that a non-structural protein encoded by herpesviruses HHV-6A, HHV-8, VZV and to a lesser extent HSV-2 is a pathogen-associated molecular pattern. Our results reveal a novel function of the virus-encoded dUTPases, which may be important to the pathophysiology of diseases caused by these viruses. More importantly, this study demonstrates that the immunomodulatory functions of dUTPases are a common property of the Herpesviridae family and thus, the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by these herpesviruses.Frontiers in microbiology. 01/2014; 5:504.
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ABSTRACT: This study is to analyze distribution of peripheral T cell subsets including regulatory T cells in patients with acute herpes zoster (AHZ) and investigate its relationship to the development of postherpetic neuralgia (PHN). T lymphocyte subsets in peripheral blood from 76 patients with AHZ and 38 normal controls were analyzed using flow cytometry. According to the visual analogue scales in the acuter phase and followed up by PHN for 3 months, patients with herpes zoster (HZ) were divided into different patient groups. The results demonstrated a significant decrease in the percentage of CD4 lymphocytes and CD4/CD8 ratios in each patient group as compared with normal controls, and the CD4+ subsets in the severe group were significantly decreased as compared with mild group and moderate group (p < 0.01). Moreover, there seemed a relationship between severity of acute pain during zoster and PHN. The proportions of regulatory T cells (Treg; CD4+CD25+FoxP3+ T cells) were significantly elevated from AHZ patients as compared with normal controls, and CD4+ T cells were inversely correlated with Treg cells (p < 0.01). Furthermore, FoxP3 expression was significantly increased by CD4+CD25(high) T cells in the severe group as compared with mild group, moderate group and normal controls. These results indicated that T cellular immunity in AHZ patients was impaired and suggested increased activation of Treg cells may suppress anti-viral CD4+ T cells immune responses, especially in the severe HZ patients. Treg cells may play an important role in the pathogenesis of AHZ and progression toward PHN.Archives for Dermatological Research 05/2013; · 2.71 Impact Factor
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ABSTRACT: Varicella zoster virus (VZV) is the causative agent of varicella (chickenpox) and zoster (shingles). Investigating VZV pathogenesis is challenging as VZV is a human-specific virus and infection does not occur, or is highly restricted, in other species. However, the use of human tissue xenografts in mice with severe combined immunodeficiency (SCID) enables the analysis of VZV infection in differentiated human cells in their typical tissue microenvironment. Xenografts of human skin, dorsal root ganglia or foetal thymus that contains T cells can be infected with mutant viruses or in the presence of inhibitors of viral or cellular functions to assess the molecular mechanisms of VZV-host interactions. In this Review, we discuss how these models have improved our understanding of VZV pathogenesis.Nature Reviews Microbiology 02/2014; · 22.49 Impact Factor
JOURNAL OF VIROLOGY, Apr. 2010, p. 4060–4072
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 8
Impact of Varicella-Zoster Virus on Dendritic Cell Subsets in Human
Skin during Natural Infection?†
Jennifer H. Huch,1,2Anthony L. Cunningham,2Ann M. Arvin,3Najla Nasr,2
Saskia J. A. M. Santegoets,4Eric Slobedman,5Barry Slobedman,2‡
and Allison Abendroth1,2*‡
Discipline of Infectious Diseases and Immunology, University of Sydney, Sydney, New South Wales 2006, Australia1; Centre For
Virus Research, Westmead Millennium Institute and University of Sydney, Westmead, New South Wales 2145, Australia2;
Departments of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, Stanford,
California 943053; Department of Pathology, VU University Medical Center, De Boelelaan 1117,
Amsterdam 1081HV, Netherlands4; and Laverty Pathology, North Ryde,
New South Wales, 2113, Australia5
Received 14 July 2009/Accepted 8 January 2010
Varicella-zoster virus (VZV) causes varicella and herpes zoster, diseases characterized by distinct cutaneous
rashes. Dendritic cells (DC) are essential for inducing antiviral immune responses; however, the contribution
of DC subsets to immune control during natural cutaneous VZV infection has not been investigated. Immu-
nostaining showed that compared to normal skin, the proportion of cells expressing DC-SIGN (a dermal DC
marker) or DC-LAMP and CD83 (mature DC markers) were not significantly altered in infected skin. In
contrast, the frequency of Langerhans cells was significantly decreased in VZV-infected skin, whereas there was
an influx of plasmacytoid DC, a potent secretor of type I interferon (IFN). Langerhans cells and plasmacytoid
DC in infected skin were closely associated with VZV antigen-positive cells, and some Langerhans cells and
plasmacytoid DC were VZV antigen positive. To extend these in vivo observations, both plasmacytoid DC (PDC)
isolated from human blood and Langerhans cells derived from MUTZ-3 cells were shown to be permissive to
VZV infection. In VZV-infected PDC cultures, significant induction of alpha IFN (IFN-?) did not occur,
indicating the VZV inhibits the capacity of PDC to induce expression of this host defense cytokine. This study
defines changes in the response of DC which occur during cutaneous VZV infection and implicates infection
of DC subtypes in VZV pathogenesis.
Varicella-zoster virus (VZV) is a highly species-specific hu-
man herpesvirus that causes the diseases varicella (chicken
pox) and herpes zoster (shingles). Varicella results from the
primary phase of infection and is characterized by a diffuse
rash of vesiculopustular lesions that appear in crops and usu-
ally resolve within 1 to 2 weeks (7, 26). Primary infection is
initiated by inoculation of mucosal sites, such as the upper
respiratory tract and the conjunctiva, with infectious virus,
usually contained within respiratory droplets (3, 23). Following
inoculation, there is a 10- to 21-day incubation period during
which VZV is transported to the regional lymph nodes; how-
ever, it remains unclear which cell types are responsible for
transport of VZV during natural infection (3). It has been
hypothesized that dendritic cells (DC) of the respiratory mu-
cosa may be among the first cells to encounter VZV during
primary infection and are capable of virus transport to the
draining lymph nodes (1, 45). It is postulated that within lymph
nodes, VZV undergoes a period of replication, resulting in a
primary cell-associated viremia, during which time virus is
transported to the reticuloendothelial organs, where it under-
goes another period of replication that results in a secondary
cell-associated viremia and virus transport to the skin (3, 23).
However, VZV has recently been shown to have tropism for
human tonsillar CD4?T lymphocytes (37), and it has been
demonstrated that these T lymphocytes express skin homing
markers that may allow them to transport VZV directly from
the lymph node to the skin during primary viremia (38). Once
the virus reaches the skin, it infects cutaneous epithelial cells,
resulting in distinctive vesiculopustular lesions.
During the course of primary infection, VZV establishes a
lifelong latent infection within the sensory ganglia, from which
virus may reactivate years later to cause herpes zoster (22, 42,
53). VZV reactivation results in the production of new infec-
tious virus and a characteristic vesiculopustular rash, which
differs from that of varicella insofar as the distribution of the
lesions is typically unilateral and covers only 1 to 2 dermatomes
(8). In both primary and reactivated VZV infection of human
skin, VZV antigens are detectable in the epidermis and dermis
(2, 30, 46, 47, 49, 52), and although some studies have exam-
ined the immune infiltrate present in these lesions, most have
focused on T lymphocytes, macrophages, and NK cells (40, 48,
50, 51, 58). The role of DC subsets in VZV infection in human
skin has not been previously explored in vivo.
Our laboratory provided the first evidence that VZV could
productively infect human immature and mature monocyte-
derived dendritic cells (MDDC) in vitro (1, 45), and Hu and
Cohen (2005) showed that VZV ORF47 was critical for rep-
* Corresponding author. Mailing address: University of Sydney, Dis-
cipline of Infectious Diseases and Immunology, Blackburn Building,
Room 601, New South Wales 2006, Australia. Phone: 61-2-93516878.
Fax: 61-2-93514731. E-mail: firstname.lastname@example.org.
‡ A.A. and B.S. contributed equally to the manuscript.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 3 February 2010.
lication of virus in human immature DC but not mature DC
(29). However, whether DC become directly infected during
natural VZV skin infection and the impact VZV infection may
have on DC subsets has yet to be elucidated. The two subsets
of DC that are normally present in the skin and which may be
involved in the pathogenesis of VZV infection are the Lang-
erhans cells (LC) of the epidermis and dermal DC (DDC)
(60). LC are present in an immature state in uninfected skin
and in upper respiratory tract epithelium. Upon capture of
foreign antigens, LC have the capacity to migrate from the
periphery to the lymph nodes, where they seek interaction with
T lymphocytes (60). Although the location of cutaneous DC
suggests that they are a DC subset likely to be involved in the
pathogenesis of VZV infection, other subsets of DC, such as
the blood-derived myeloid DC (MDC) and plasmacytoid DC
(PDC), are also potentially important in the pathogenesis of
VZV infection. Of particular interest are PDC, since these
cells are important in innate antiviral immune responses due to
their ability to recruit to sites of inflammation and secrete high
levels of alpha interferon (IFN-?) (6, 18, 56). PDC also par-
ticipate in adaptive immune responses through their secretion
of cytokines and chemokines that promote activation of effec-
tor cells, including NK cells, NKT cells, B lymphocytes, and T
lymphocytes, and also through their capacity to present antigen
to T lymphocytes (9, 63). Whether PDC and LC can be in-
fected with VZV and their roles during infection have not been
In this study, we sought to identify and compare the subsets
of DC present in human skin lesions following natural VZV
infection and to assess DC permissiveness to VZV infection.
We utilized immunohistochemical (IHC) and immunofluores-
cent (IFA) staining to characterize DC subsets within the skin
of multiple patients with either varicella or herpes zoster, and
identified profound changes in the frequency of LC and PDC
as a consequence of cutaneous VZV infection. In addition,
some LC and PDC costained with a range of VZV antigens
indicative of productive infection. PDC isolated from human
blood and LC derived from the MUTZ-3 cells were shown to
be permissive to productive VZV infection in vitro. This study
defines changes in the type and distribution of DC during
natural cutaneous VZV infection and implicates infection of
specific DC subsets in VZV pathogenesis.
MATERIALS AND METHODS
Skin samples and preparation of sections for immunohistochemical and im-
munofluorescent staining. Samples from varicella skin lesions (n ? 5), herpes
zoster skin lesions (n ? 5), and uninfected skin (n ? 3) were each obtained
from separate donors by punch biopsy, fixed, and paraffin embedded. The
varicella and zoster cases ranged from 3 to 72 h and 72 h to 7 days, respec-
tively, after the first appearance of the rash (Table 1). Samples were obtained
with the approval of the University of Sydney and Sydney West Area Health
Service Human Research Ethics Committees. Sections were dewaxed, hy-
drated, and washed for 2 min in Tris-buffered saline (TBS), 50 mM Tris
(Amresco), and 150 mM NaCl (Amresco) in distilled water (dH2O), before
blocking of endogenous peroxidase with 3% H2O2(Fronine, Australia) in
dH2O for 5 min. Sections were incubated in unmasking buffer, either 0.01 M
citrate buffer (pH 6.0) (AnalaR, Australia) in dH2O or 1 mM EDTA buffer
(pH 8.0) (Gibco) in dH2O, as determined during antibody optimization, at
95°C for 15 min, after which sections were cooled in unmasking buffer for 20
min at room temperature (RT).
Antibodies. All antibodies used for immunohistochemistry, immunofluores-
cent staining, and flow cytometry and the dilutions and conditions under which
they were used are listed in Table 2.
Preparation of cell spots for immunohistochemical and immunofluorescent
staining. Cells were centrifuged and resuspended in 10 to 20 ?l of phosphate-
buffered saline (PBS) (Amresco). The cells were spotted onto Superfrost Plus
slides (Menzel-Glaser, Germany), fixed with 4% paraformaldehyde (Electron
Microscopy Sciences) for 15 min at RT, and then stored in PBS at 4°C for up to
48 h, until use. Immediately prior to staining, cell spots were permeabilized with
0.2% Triton (Sigma) in PBS for 10 min at RT.
Immunohistochemical staining. Five-micrometer paraffin-embedded sections
were washed in TBS for 2 min before blocking serum, either 10% normal goat
serum (NGS) (Sigma) in TBS, used with the IDetect super stain system (ID
Labs), or 10% normal human serum (NHS) in TBS, used with the Mach4
universal horseradish peroxidase (HRP) polymer kit (Biocare Medical), was
applied in a humidified chamber for 30 min at RT. The blocking serum was
removed before the primary antibodies were applied in a humidified chamber for
1 h at RT, following which sections were washed for 5 min in TBS. The IDetect
super stain system (HRP) was used as specified by the supplier with antibodies
against CD1a, langerin, DC-SIGN, CD83, and VZV gE. The Mach4 universal
HRP polymer kit was used as recommended by the supplier with antibodies
against CD123 and DC-LAMP. The colored enzyme substrate (Vector VIP
peroxidase substrate kit; Vector Laboratories) was applied to all sections for 10
min at RT, after which slides were washed for 10 min in TBS. The sections were
counterstained with a 1:3 solution of Mayer’s hematoxylin (Fronine, Australia) in
TBS for 10 s, after which they were washed for 5 min in lukewarm H2O.
Following counterstaining, the slides were dehydrated and allowed to air dry.
Ultramount 4 mounting medium (Fronine, Australia) was applied to each sec-
tion before coverslips (22 by 50 mm; no. 1) (Menzel-Glaser, Germany) were
placed on the slides. Slides were then viewed under a light microscope (model
DM1000) (Leica Microsystems, Germany).
Dual immunofluorescent staining. Paraffin-embedded sections or cell spots
were washed for 2 min in TBS before blocking serum, 20% normal donkey serum
(NDS) (Sigma) in TBS, was applied in a humidified chamber for 30 min at RT.
The blocking serum was removed before the first primary antibodies were ap-
plied in a dark, humidified chamber for 1 h at 37°C, following which sections
were washed for 5 min in TBS. Appropriate secondary antibodies were applied
to sections in a dark, humidified chamber for 30 min at 37°C, followed by a 5-min
wash in TBS. Sections were blocked again, as previously described, with 20%
NDS. The blocking buffer was again removed from the slides, following which the
second primary antibodies and appropriate secondary antibodies were applied
and slides were washed as before. Following the final wash step, 3 to 6 ?l of
ProLong Gold antifade reagent with 4?,6-diamidino-2-phenylindole (DAPI)
(Molecular Probes) was applied to each section or cell spot. Coverslips were
mounted, and slides were sealed before viewing under a fluorescence microscope
(model BX51) (Olympus).
Human foreskin fibroblast and virus culture. Human foreskin fibroblasts
(HFF) were derived from normal human foreskin tissue and used to culture
VZV. HFF were grown in tissue culture medium Dulbecco’s modified Eagle
medium (DMEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (CSL, Australia) and penicillin (10,000 U/ml)-streptomycin (10,000
?g/ml) (Gibco) and incubated at 37°C with 5% CO2. HFF infected with a clinical
isolate of VZV, strain Schenke, were used to infect 80%-confluent monolayers of
TABLE 1. Uninfected skin, varicella skin lesion, and herpes zoster
skin lesion biopsy specimens used in this study
aNA, not applicable.
VOL. 84, 2010IMPACT OF VZV ON DC SUBSETS IN HUMAN SKIN4061
uninfected HFF to propagate the virus. At 2? to 3? cytopathic effect (CPE),
where 1? represented 10% and 4? represented 100% of the cell monolayer
showing plaque formation, VZV-infected monolayers were harvested. To harvest
both uninfected and VZV-infected HFF, medium was removed from the flask
and cells were washed with PBS at RT. Following aspiration of the PBS, cells
were dislodged following incubation with trypsin-EDTA (Invitrogen) for 5 min at
37°C. Following trypsinization, cells were resuspended in 5 ml of PBS and
counted using a hemocytometer.
Isolation of PBMC and PDC. Peripheral blood mononuclear cells (PBMC)
were separated from buffy coats (Australian Red Cross Blood Bank, Australia)
by density gradient sedimentation on Ficoll-Paque medium (GE Healthcare,
United Kingdom). T lymphocytes and monocytes within the PBMC population
were depleted by incubating PBMC with anti-CD3 and anti-CD14 microbeads
(Miltenyi, Germany) and then running the PBMC through two CS columns
(Miltenyi, Germany) in a magnetic field. Plasmacytoid DC (PDC) were then
positively selected by incubating the remaining PBMC with FcR blocking reagent
(Miltenyi, Germany) and anti-CD304 microbeads (Miltenyi, Germany) and run-
ning the cells through an MS column (Miltenyi, Germany) in a magnetic field.
The bound cells were removed by plunging 1 ml of MACSwash (1% AB serum
[Sigma]–5 mM EDTA [Gibco] in PBS) through the column. This positively
selected cell fraction was then run through a second MS column to increase the
purity of the isolated PDC population. Approximately 3 ? 104PDC were then
analyzed by flow cytometry to determine the purity of the isolated PDC popu-
lation. The isolated PDC sample was stained with anti-Lin-1—fluorescein iso-
thiocyanate (FITC), anti-CD123-phycoerythrin (PE), anti-HLA-DR—peridinin
chlorophyll protein (PerCP), and anti-CD11c-allophycocyanin (APC) (Table 2)
as described and analyzed on a FACSAria flow cytometer (BD Biosciences).
PDC were defined as cells staining positive for CD123 and HLA-DR and staining
negative for Lin-1 and CD11c. PDC were maintained in culture in RPMI 1640
supplemented with 10% human AB serum and 5 ng/ml interleukin 3 (IL-3)
Infection of PDC. PDC were cultured with uninfected HFF or VZV-infected
HFF at a ratio of 2 HFF:1 PDC in a volume of medium that resulted in a total
cell concentration of 1,000 cells/?l. Uninfected HFF and VZV-infected HFF
were cultured separately as controls in a volume of medium that resulted in a cell
concentration of 1,000 cells/?l. Cultures were then incubated for 24 h at 37°C in
an atmosphere of 5% CO2in air. In some experiments, the Toll-like receptor
(TLR) agonist ODN2216 (InvivoGen) was added at a concentration of 2.5 ?M.
Generation of LC from MUTZ-3 cells. MUTZ-3 is a human CD34?acute
myeloid leukemia cell line provided by S. Santegoets (VU University Medical
Center, Netherlands). MUTZ-3 cells can be induced to acquire an LC-like
phenotype and have been used in the study of other virus-LC interactions (12, 39,
43, 55). MUTZ-3 cells were initially cultured at a concentration of 0.1 ? 106
cells/ml in minimal essential medium (MEM)-alpha containing ribonucleosides
and deoxyribonucleosides (Invitrogen, Australia) and supplemented with 10%
conditioned medium from the human renal carcinoma cell line 5637, 20% heat-
inactivated FBS (CSL, Australia), penicillin (10,000 U/ml)-streptomycin (10,000
?g/ml) (Gibco), and 50 ?M ?-mercaptoethanol (Sigma) in a 12-well tissue
culture plate with each well containing 2 ml of medium. After 7 days in culture
at 37°C with 5% CO2, the MUTZ-3 cells were collected and resuspended in
MEM-alpha as described above, additionally supplemented with 100 ng/ml gran-
ulocyte-macrophage colony-stimulating factor (GM-CSF) (Invitrogen, Austra-
lia), 2.5 ng/ml tumor necrosis factor alpha (TNF-?) (R&D Systems), and 5 ng/ml
transforming growth factor ?1 (TGF-?1) (R&D Systems) at a concentration of
0.25 ? 106cells/ml in a 12-well tissue culture plate with each well containing 2 ml
of medium to differentiate the MUTZ-3 cells into LC. After 3 days in culture at
37°C with 5% CO2, medium and cytokines were replenished as per 1 ml per well,
and this was again repeated on day 7. On day 10 post-cytokine stimulation, cells
were harvested. Approximately 1 ? 105MUTZ-3-derived cells were then ana-
lyzed by flow cytometry to confirm the phenotype of the MUTZ-3-derived cells
as being LC-like. The MUTZ-3-derived LC sample was stained with anti-lange-
rin-PE (CD207), anti-DC-SIGN-FITC (CD209), anti-mannose receptor (MR)-
APC (CD206), anti-CD1a-PE-Cy5, and anti-CD83-FITC as described above and
analyzed on a FACSCanto flow cytometer (BD Biosciences) and with FlowJo
fluorescence-activated cell sorter (FACS) analysis software (BD Biosciences).
Immature MUTZ-3-derived LC were defined as cells staining positive for lan-
gerin and CD1a and negative for DC-SIGN, mannose receptor, and CD83-like
authentic immature epithelial LCs in situ.
Culture of human renal carcinoma cell line 5637. Human renal carcinoma cell
line 5637 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
[DSMZ], Germany) was seeded at a concentration of 0.5 ? 106cells/ml in 30 ml
of tissue culture medium RPMI (Gibco) supplemented with 10% heat-inacti-
TABLE 2. Antibodies used in this study
Anti-human DLEC (BDCA-2)
Mouse IgG1 isotype control
Mouse IgG2a isotype control
Mouse IgG2b isotype control
Whole rabbit serum
Goat IgG isotype control
Goat pAb IgG
Mouse MAb mix
1:500 (IFp, IFc)
1:500 (IFp, IFc)
1:500 (IFp, IFc)
1:10 (IFp), 1:100 (IHC, IFc)
1:100 (IHC, IFp)
1:10 (IFp), 1:50 (IFc)
aHTU, high-temperature unmasking.
bpAb, polyclonal antibody.
cIFp, immunofluorescent staining on paraffin sections; IFc, immunofluorescent staining on cell spots; IHC, immunohistochemistry; pdIHC, immunohistochemistry
with polymer detection; FC, flow cytometry.
4062 HUCH ET AL.J. VIROL.
vated FBS (CSL, Australia) in a 180-cm2tissue culture flask and incubated at
37°C with 5% CO2. The cells were allowed to become confluent, following which
the medium was aspirated and replaced with fresh medium. The cells were
cultured for up to a further 48 h, after which the medium was collected to be used
as conditioned medium. To prepare the conditioned medium, the medium was
centrifuged at 1,500 rpm for 10 min, filtered through a 0.2-?m filter to remove
cell debris, and then stored at ?80°C until use.
Infection of MUTZ-3-derived LC. In a tissue culture plate, MUTZ-3-derived
LC were cultured in separate wells containing uninfected HFF or VZV-infected
HFF at a ratio of 2 HFF:1 LC in a volume of medium that resulted in a total cell
concentration of 500 cells/?l. The medium in which the HFF and LC cultures
were incubated was supplemented with GM-CSF, TNF-?, and TGF-?1 as de-
scribed previously for the generation of LC from MUTZ-3 cells. Cultures were
then incubated for 3 days at 37°C in an atmosphere of 5% CO2in air, and at the
end of the culture period the cells were harvested to make cell spots, as described
Immunostaining and flow cytometry. Cells (5 ? 105) were washed once by
adding 1 ml of FACS buffer (1% fetal calf serum [FCS], 10 mM EDTA in PBS)
(Gibco) to each tube, centrifuging at 1,500 rpm for 5 min, and then aspirating
dry. Cells were incubated with appropriate fluorochrome-conjugated antibodies
and diluted in FACS buffer in the dark at 4°C for 20 min, following which they
were washed once more. Cells were fixed with 1% paraforaldehyde (Electron
Microscopy Sciences) in PBS prior to analysis on a FACSAria flow cytometer
Cell counting methods. Quantitative evaluation of numbers of cells stained
positive by immunohistochemistry for the various immune cell markers on
uninfected, varicella, and herpes zoster skin sections was performed by ana-
lyzing 10 ?40-magnified fields of epidermis and 30 ?40 fields of dermis,
measuring 10 fields across and 3 fields deep, with a graticule. The graticule grid
covered 0.13 mm2of tissue, which was identified as epidermis or dermis based on
histology. After obtaining a total count of the number of positive cells in each
region of tissue for each marker, for each case, the frequency of cells stained
positive for each marker per square millimeter of tissue was obtained. Quanti-
tative evaluation of numbers of cells stained positive for the BDCA-2, lan-
gerin, and VZV proteins by immunofluorescence on normal, varicella, and
herpes zoster skin sections was performed by photographing 10 ?40 fields of
epidermis and 10 ?40 fields of dermis. Single-color photos were first taken
through each filter and were then merged. After obtaining a total count of the
number of BDCA-2?or langerin?cells and VZV?cells in each region of tissue
for each marker combination for each case, the frequency per square millimeter
of tissue was calculated. Quantitative evaluation of numbers of cells stained
positive for the langerin and VZV proteins and the BDCA-2 and VZV proteins
by immunofluorescence on MUTZ-3-derived LC and PDC cell spots, respec-
tively, was performed by photographing 25 magnification-?40 fields in a five-
field by five-field square. Single-color photos were first taken through each filter
and were then merged. After a total count of the number of total cells, BDCA-2?
cells, VZV?cells, and dually positive cells was obtained, the number of dually
positive cells was expressed as a percentage of BDCA-2?cells. A similar ap-
proach was used to elucidate the percentage of langerin?cells expressing VZV
ELISA. Supernatant from cultures of PDC cultured with ODN2216, unin-
fected HFF, or VZV-infected HFF was analyzed for the presence of IFN-? using
a human IFN-? serum sample enzyme-linked immunosorbent assay (ELISA) kit
(PBL Biomedical Laboratories), as per the directions of the manufacturer.
Frequency and distribution of DC subsets during natural
cutaneous VZV infection. To determine the nature and distri-
bution of different DC subsets during natural cutaneous VZV
infection, sections of punch biopsy specimens from varicella
and herpes zoster skin lesions and from uninfected skin were
immunohistochemically stained with a panel of DC markers:
CD1a and langerin (LC), DC-SIGN (dermal DC [DDC]),
CD83 and DC-LAMP (mature DC), and CD123 and BDCA-2
(PDC). Each sample of VZV-infected skin tested was also
stained with the appropriate isotype control antibodies. Mul-
tiple 5-?m sections from skin biopsy specimens from five sep-
arate varicella, five separate herpes zoster, and three unin-
fected (normal) cases were examined, and the frequency of
antigen-positive cells per mm2in both the epidermis and der-
mis was calculated (Fig. 1; see also Fig. S1 in the supplemental
material). Cells expressing CD1a or langerin were readily de-
tected in the epidermis of uninfected skin but rarely detected
in the dermis. This observation is consistent with the typical
distribution of resident LC in normal skin (60). In stark con-
trast, the number of cells expressing CD1a or langerin dropped
dramatically in the epidermis of VZV-infected skin from both
varicella and herpes zoster cases. This decrease was not due to
a general loss of cells within the epidermis, since counts of total
cells within the epidermis remained comparable between un-
infected and VZV-infected skin samples (data not shown).
However, the decrease in frequencies of CD1a and langerin-
positive cells were observed to be greatest immediately sur-
rounding the lesion, with staining for CD1a and langerin in the
histologically normal skin distant from the lesion being com-
parable to that of uninfected skin.
Cells expressing a DDC marker, DC-SIGN, were not
present in the epidermis in uninfected specimens, nor did they
appear in the epidermis of either varicella or herpes zoster
biopsy specimens. DC-SIGN-expressing cells were detected at
a low level in the dermis of normal, varicella, and herpes zoster
cases, and the frequencies of detection were comparable be-
tween these samples. Similarly, cells expressing a mature DC
marker (DC-LAMP) or CD83 were either absent or present at
very low levels in the epidermis and dermis of normal skin.
With the exception of a small increase in the number of cells
expressing CD83 in the epidermis of herpes zoster cases, the
frequency of cells expressing these markers did not appear to
change as a consequence of cutaneous VZV infection. There
was a striking influx of cells expressing the PDC-specific
marker (BDCA-2) in the dermis of VZV-infected skin from
both varicella and herpes zoster cases in comparison to find-
ings for uninfected skin. This influx was observed to a lesser
degree in the epidermis. In addition, there was an increase in
the number of cells expressing CD123, which is also expressed
by PDC, in the dermis of VZV-infected skin from both vari-
cella and herpes zoster cases in comparison to uninfected skin.
These analyses reveal changes in the distribution of cells
expressing different DC subset markers in response to cutane-
ous VZV infection. In particular, cells expressing markers of
LC decreased dramatically in the epidermis in both varicella
and herpes zoster, whereas cells expressing the PDC marker
BDCA-2 increased in frequency in the dermis and epidermis in
both varicella and herpes zoster.
In addition to assessing DC subsets, sections were also ex-
amined by IHC for the presence of VZV glycoprotein E (gE).
Normal skin incubated with anti-gE antibody yielded no spe-
cific staining and showed a normal histological profile, indi-
cated by a well-defined epidermis and dermis (Fig. 2C). Sec-
tions from varicella and herpes zoster skin lesions contained
vesicles which were located predominantly within the epider-
mis and which contained large numbers of VZV gE-positive
cells (Fig. 2A and data not shown). Notably, gE was detected
not only in and around the lesion but also within distinct
regions of infiltrating cells deeper within the dermis (Fig. 2A).
Consecutive sections of VZV-infected skin stained with isotype
control antibody showed no positive staining (Fig. 2B). Taken
together, these analyses revealed that VZV infection, which
occurred in regions of the epidermis and deeper within the
VOL. 84, 2010IMPACT OF VZV ON DC SUBSETS IN HUMAN SKIN 4063
dermis, was accompanied by changes in the frequency of dis-
tinct DC subsets within these regions.
Relationship between VZV antigen-positive cells and den-
dritic cell subsets in naturally infected skin. To more closely
examine the distribution of PDC and LC in the context of cells
infected with VZV, the immunostaining approach was modi-
fied to encompass a dual immunofluorescence assay (IFA)
such that the presence of both DC and VZV antigens could be
examined in the same skin biopsy section. Thus, sections were
stained for the PDC marker BDCA-2 in combination with
antibody against a VZV regulatory protein encoded by ORF4
or the LC marker langerin in combination with VZV ORF4
(Fig. 3). Each sample of VZV-infected skin tested was also
stained with the appropriate isotype control antibodies in ad-
dition to normal skin being stained with the anti-ORF4 and
In VZV-infected skin from both varicella and herpes zoster
cases subjected to IFA, the distribution of each DC type was
comparable to that observed by IHC staining. That is, lange-
rin?LC were rarely detected in the epidermis, whereas there
was a striking influx of BDCA-2?PDC in the dermis. In con-
trast, in normal skin, langerin?LC were readily detected in the
FIG. 1. Frequency and distribution of DC subsets in VZV-infected
skin. Immunohistochemical staining (purple) of sections of uninfected
skin (A, C, and E) and VZV-infected skin (B, D, and F) for the
Langerhans cell markers CD1a (A and B) and langerin (C and D) and
for the plasmacytoid DC marker CD123 (E and F). Sections were
lightly counterstained by hematoxylin. Immunofluorescent staining
(red) for the plasmacytoid DC (PDC) marker BDCA-2 on uninfected
skin (G) and VZV-infected skin (H) is shown. Sections were stained
with DAPI to reveal cell nuclei (blue). Examples of positively stained
cells are indicated by arrows, with BDCA-2-positive PDC shown in
panels F and H being deeper within the dermis. Images were captured
under magnification ?40, with scale bars representing 50 ?m. I and J
plot the mean frequency (? SEM) of cells stained positive for a variety
of DC markers in the epidermis and dermis, respectively, of uninfected
skin (3 donors; white bars), varicella skin (5 donors; light purple bars),
and herpes zoster lesions (5 donors; dark purple bars).
FIG. 2. Distribution of VZV antigen during natural cutaneous
VZV infection. Immunohistochemical staining (purple) for VZV gly-
coprotein E (gE) in skin biopsy specimens of a varicella lesion (A) or
uninfected skin (C) is shown. Infiltrating cells staining positive for
VZV gE in the dermis of skin from the varicella skin biopsy specimen
are boxed. A consecutive section from the varicella skin biopsy spec-
imen stained with an isotype control antibody is shown in panel B.
Images were captured under magnification ?10, with scale bars rep-
resenting 200 ?m. (D) Mean frequency (? SEM) of VZV gE-positive
cells in skin from uninfected (3 donors), varicella (5 donors), and
herpes zoster (5 donors) samples within the epidermis (light purple
bars) or dermis (dark purple bars) is plotted.
4064 HUCH ET AL.J. VIROL.
epidermis and BDCA-2? PDC were not observed in the der-
mis. VZV antigens were readily detected in the epidermis
surrounding the lesion and also deeper in the dermis in close
proximity to regions of infiltrating cells which included the
largest concentration of PDC (Fig. 3). No VZV staining was
observed in the normal control skin sections.
In some cases, cells were observed that were dually positive
for BDCA-2 and VZV antigen or langerin and VZV antigen.
These were observed sporadically in both varicella and herpes
zoster sections (see Fig. S2 in the supplemental material). To
determine whether these VZV antigen-positive DC repre-
sented bone fide infected DC, dual immunofluorescent stain-
ing was performed for either BDCA-2 or langerin in conjunc-
tion with a range of VZV antigens representing different
kinetic classes of genes expressed during the productive virus
replication cycle. The full replicative cycle of VZV in permis-
sive cells follows a regulated cascade of gene expression. These
viral genes can be divided into 3 temporal classes, immediate-
early (IE), early (E), and late (L) gene products, based upon
their expression kinetics (24). The VZV proteins IE62 and
ORF4 are IE proteins that are among the first proteins to be
translated during productive VZV infection. Next to be trans-
lated is the E class of proteins, of which ORF29 is a member.
VZV gE is one of the glycoproteins embedded in the viral
envelope, and as a member of the L gene kinetic class, it is
among the final viral proteins to be produced during produc-
tive VZV infection. Thus, sections were stained by IFA for (i)
BDCA-2 in combination with VZV IE62, ORF4, or glycopro-
tein E (gE) and (ii) langerin in combination with VZV IE62,
ORF4, or ORF29 (Fig. 4).
Dually positive cells were not observed for every case exam-
ined; however, for some cases, LC or PDC that had stained
positive for more than one VZV protein were present. In cases
of both varicella and herpes zoster, gE-positive staining was
most often observed in PDC, which if observed alone may
suggest that these PDC have acquired VZV antigen by uptake
of an infected cell and have not actually been productively
infected themselves. However, in cases of both varicella and
herpes zoster, LC and PDC were detected and stained positive
for IE62, ORF4, and ORF29, with the localization of the VZV
proteins being indicative of productive infection (31, 32). Nei-
ther VZV-infected nor normal control skin stained positive
when isotype control antibodies for VZV antigens and DC
markers were used. The specificity of VZV antigen detection
was further confirmed by a lack of staining when each VZV-
specific antibody was used on normal skin sections (Fig. 4).
These results suggest that both LC and PDC become produc-
tively infected with VZV during the course of natural cutane-
ous VZV infection.
Permissiveness of plasmacytoid DC and MUTZ-3-derived
LC to VZV infection in vitro. Given our detection of VZV
antigen-positive PDC and LC in human skin during natural
VZV infection, we sought to determine whether PDC and LC
were permissive to VZV infection in vitro. PDC were isolated
from fresh peripheral blood mononuclear cells (PBMC) from
healthy donors by magnetic bead separation by first depleting
CD3?and CD14?cells, followed by positive selection for
CD304?cells, and their phenotype was determined by flow
cytometry to confirm that they were CD123?HLA?DR?
Lin-1?CD11c?PDC. LC were derived from the human acute
myeloid leukemia cell line MUTZ-3 using GM-CSF, TNF-?,
and TGF-?1, following which their phenotype was confirmed
by flow cytometry as being positive for the LC markers langerin
and CD1a and negative for DC-SIGN, mannose receptor, and
FIG. 3. Detection of VZV antigens and DC subsets in skin during
natural cutaneous VZV infection. Dually immunofluorescently stained
sections of varicella skin lesion (A and B, cases V2 and V1, respectively)
and herpes zoster skin lesion (C and D, cases HZ2 and HZ1, respectively)
for combinations of DC markers and VZV antigen are shown. Panels A
and C show staining for the Langerhans cell marker langerin (red) in
D show staining for the plasmacytoid DC marker BDCA-2 (red) in com-
bination with staining for the VZV (green) in the dermis, with panel B
showing an area closer to the lesion and panel D showing an area deeper
within the dermis. Panels E and F show isotype control stained sections
from varicella and herpes zoster skin samples, respectively. Panels G and
H show staining of uninfected skin for langerin and VZV in the epidermis
(G) and for BDCA-2 and VZV in the dermis (H). All sections were
magnification ?40, with scale bars representing 50 ?m. Examples of
langerin and BDCA-2-positive cells are indicated by white arrows, and
examples of VZV-positive cells are indicated by orange arrows. The
location of a VZV lesion (L) is indicated when it was present within the
field of view.
VOL. 84, 2010IMPACT OF VZV ON DC SUBSETS IN HUMAN SKIN 4065
FIG. 4. VZV antigens detected in LC and PDC during natural cutaneous VZV infection. Sections of varicella and herpes zoster skin lesions
were dually immunofluorescently stained for combinations of DC markers and VZV antigens, showing the presence of dually positive cells. (A to
C) Sections stained for the plasmacytoid DC marker BDCA-2 (red) in combination with staining for VZV antigens IE62 (A) (case HZ1), ORF4
(B) (case HZ3), or gE (C) (case HZ3) (green). (D to F) Sections stained for the Langerhans cell marker langerin (red) in combination with staining
for VZV antigens IE62 (D) (case V2), ORF4 (E) (case V2), or ORF29 (F) (case V2) (green) are shown. (G to I) Staining of uninfected skin for
langerin and IE62 (G), langerin and ORF29 (H), or BDCA-2 and gE (I) is shown. All sections were stained with DAPI to mark cell nuclei (blue).
The main images were captured under magnification ?40, with scale bars representing 50 ?m. Examples of dually positive cells are boxed and
shown at higher magnification as insets for each staining combination.
4066 HUCH ET AL.J. VIROL.
CD83. We assessed the susceptibility of these PDC and
MUTZ-3-derived LC to VZV infection. VZV is highly cell
associated in cell culture, and high-titer cell-free stocks cannot
be generated (5, 21, 25, 61, 62). Thus, we infected PDC and
MUTZ-3-derived LC with cell-associated VZV by culturing
the cells in the presence of VZV-infected human foreskin
fibroblasts (HFF) or uninfected HFF. We have previously used
this method of infection to demonstrate productive infection of
monocyte-derived DC (1, 45). After 24 h, the cells were col-
lected and were used to make cell spots, which were then
costained by IFA for the VZV markers IE62, ORF4, ORF29
and gE in combination with BDCA-2 for the PDC (Fig. 5) and
langerin for the MUTZ-3-derived LC (Fig. 6).
VZV antigens were readily detected in both BDCA-2?PDC
and langerin?MUTZ-3-derived LC inoculated with VZV-in-
fected HFF. Representative images of dually positive cells
were captured by confocal microscopy (Fig. 5A and 6A). The
immediate-early VZV proteins IE62 and ORF4 localized to
the nuclei and cytoplasms, respectively, of BDCA-2?PDC and
langerin?MUTZ-3-derived LC. The early gene product
ORF29 showed nuclear localization, and the late viral antigen
gE localized to the cytoplasm and cell surface of BDCA-2?
PDC and langerin?MUTZ-3-derived LC. The subcellular lo-
calization of the viral gene products we observed in PDC and
langerin?MUTZ-3-derived LC was consistent with that pre-
viously reported for productive infection of permissive cells,
such as human fibroblasts and human immature and mature
MDDC (1, 45). Although not a direct measure of infectious
virus production, the detection of viral antigens from all three
kinetic classes is indicative of replicating virus, with this ap-
proach having been used previously to define replicating VZV
in other cell types infected with cell-associated virus (1, 28, 45).
Neither VZV-infected nor mock-infected PDC nor langerin?
MUTZ-3-derived LC populations stained positive when iso-
type control antibodies for BDCA-2 or langerin and VZV
antigens were used in parallel. The proportion of BDCA-2?
PDC expressing viral antigens was determined from four in-
dependent experiments using four different blood donors, and
the proportion of langerin?MUTZ-3-derived LC expressing
viral antigens was determined from three independent exper-
iments (Fig. 5B and 6B). This analysis demonstrated that a
significant proportion of both PDC and MUTZ-3-derived LC
became viral antigen positive and that viral antigens from all
three kinetic classes were represented in these cells. Further-
more, there was no significant difference between the percent-
ages of cells positive for any of the four VZV antigens detected
in PDC or MUTZ-3-derived LC. It was concluded that human
PDC and MUTZ-3-derived LC are permissive to VZV infec-
tion and are likely to support the full virus replicative cycle.
IFN-? production by PDC cultured with VZV-infected HFF.
The most distinctive functional characteristic of PDC is their
ability to synthesize IFN-? (6, 9, 18, 56, 63). Thus, we assessed
the impact of VZV infection of PDC on IFN-? synthesis. PDC
were cultured for 24 h with mock-infected or VZV-infected
HFF before supernatants were collected and the amount of
FIG. 5. PDC in vitro are permissive to VZV infection. (A) Dual
immunofluorescent staining of plasmacytoid DC exposed to VZV for
the plasmacytoid DC marker BDCA-2 (red) and VZV antigen IE62,
ORF4, ORF29, or gE (green). Staining with isotype control antibodies
and mock-infected plasmacytoid DC stained for BDCA-2 and VZV
IE62 are also shown. Scale bars, 20 ?m. (B) The percentage (? SEM)
of BDCA-2?cells costaining for each VZV antigen from 4 indepen-
dent replicate experiments.
FIG. 6. MUTZ-3-derived LC are permissive to VZV infection.
(A) Dual immunofluorescent staining of MUTZ-3-derived LC exposed
to VZV for the LC marker langerin (red) and VZV antigen IE62,
ORF4, ORF29, or gE (green). Staining with isotype control antibodies
and mock-infected MUTZ-3 LC stained for langerin and VZV IE62
are also shown. Scale bars, 20 ?m. (B) The percentage (? SEM) of
langerin?cells costaining for each VZV antigen from 3 independent
VOL. 84, 2010IMPACT OF VZV ON DC SUBSETS IN HUMAN SKIN 4067
IFN-? measured by ELISA. In two independent replicate ex-
periments, cultures of PDC infected with VZV showed little or
no change in the amount of secreted IFN-?, which remained at
levels comparable to those of mock-infected PDC cultures
(Fig. 7). Assessment of PDC cultures at 8, 12, and 36 h postin-
fection did not yield any higher levels of IFN-? secretion (data
not shown). As a positive control for capacity to induce IFN-?,
mock-infected PDC were also cultured for 24 h with 2.5 ?M
ODN2216, a TLR9 agonist which stimulates IFN-? production
by PDC (36). This treatment induced high levels of secreted
IFN-? by mock-infected PDC cultures. In contrast, VZV-in-
fected PDC cultures yielded little IFN-? when treated with
ODN2216 (Fig. 7).
In addition to assessing IFN-? production by PDC, culture
supernatants from VZV-infected HFF (which were used for
inoculating PDC) were assessed for IFN-? production by
ELISA. In nine independent replicate experiments examining
VZV-infected HFF, six experiments showed no detectable
IFN-? production and three experiments showed extremely
low levels (10 to 30 pg/ml) (data not shown). Thus, VZV-
infected HFF produce negligible levels of IFN-?. At 24 h
postinfection, there was no significant difference in PDC via-
bility between mock and infected cultures as determined by
flow cytometric analysis of propidium iodide (PI) and annexin
V staining on gated PDC (data not shown). Taken together,
these results demonstrate that VZV is capable of infecting
PDC but that infection does not induce significant IFN-? pro-
duction and infected PDC cultures remain refractory to IFN-?
induction even when stimulated with ODN2216.
To determine whether a factor secreted from infected PDC
renders uninfected bystander PDC defective in their capacity
to secrete IFN-?, we removed culture supernatants from VZV-
infected PDC cultures and incubated these supernatants with
uninfected PDC cultures for 24 h before measuring secreted
IFN-? by ELISA. Analysis of three independent replicates
revealed only very low levels of IFN-?, demonstrating that
VZV-infected PDC culture supernatant did not induce signif-
icant levels of IFN-? secretion by uninfected PDC (Fig. 8).
When treated with ODN2216, both uninfected PDC and un-
infected PDC incubated with supernatant from infected PDC
cultures secreted high levels of IFN-?, indicating that incuba-
tion of uninfected PDC with the cell supernatant from infected
PDC cultures did not block their capacity to respond to
DC are potent antigen-presenting cells found throughout
the body, particularly at sites of pathogen entry, such as mu-
cosal and skin surfaces, where they play a critical role in anti-
viral immunity (33). However, little is known about the role of
different DC subsets in the pathogenesis of VZV and other
viruses. Assessment of naturally infected human tissues is chal-
lenging, and there is a paucity of studies examining DC in
intact tissues during human infection with any virus. This study
defines the type and distribution of changes in DC subsets
which occur in the skin of individuals suffering from varicella
or herpes zoster. We demonstrated a significant decrease in
the frequency of LC concomitant with an influx of PDC in
VZV-infected skin compared to findings for uninfected skin.
We also observed sporadic VZV antigen-positive LC in the
epidermis and VZV antigen-positive PDC within regions of
cellular infiltrate in the dermis. The type of VZV antigen
staining of these cells was consistent with replicating virus,
suggesting that some of these DC become infected in vivo. We
extended these analyses to demonstrate that both PDC and
MUTZ-3-derived LC are permissive to VZV infection, but
surprisingly, PDC do not respond to infection by secreting high
FIG. 7. VZV infection of PDC in vitro impacts IFN-? synthesis.
Measurement by ELISA of IFN-? secreted from plasmacytoid DC
cultures of either mock-infected PDC (white bars) or VZV-infected
PDC (gray bars) infected for 24 h or infected for 12 h and then treated
with a 2.5 ?M concentration of the TLR agonist ODN2216 (ODN) to
induce IFN-? production is shown. Panels A and B show results from
2 independent replicate experiments. Error bars show the range of
values from duplicate samples for each replicate.
FIG. 8. IFN-? synthesis by uninfected PDC exposed to supernatant
from VZV-infected PDC cultures. Measurement by ELISA of IFN-?
secreted from uninfected PDC or from uninfected PDC treated with
supernatant from VZV-infected PDC after treatment with (gray bars)
or without (white bars) 2.5 ?M ODN2216 is shown. Treatment of
uninfected PDC with supernatant from three independent VZV-in-
fected PDC cultures is shown. The graph shows the level of IFN-?
produced by each culture (in pg/ml) along the y axis against each
culture condition along the x axis.
4068 HUCH ET AL.J. VIROL.
levels of IFN-?. This study defines the repertoire of changes in
the response of DC and provides evidence for the infection of
specific DC subtypes during natural cutaneous VZV infection.
The frequency of CD1a?langerin?LC was strikingly re-
duced in VZV-infected skin epidermis compared to that in
uninfected skin, extending an earlier case report of CD1a ex-
pression in VZV-infected skin (51). This apparent reduction in
the number of LC during VZV infection may be a consequence
of (i) loss of expression of CD1a and langerin by LC which
remain in the skin, (ii) cell death, or (iii) rapid migration of
these cells out of the skin. In this study, we were able to show
the presence of LC that stained positive for VZV antigens
indicative of virus replication. On these cells, langerin staining
remained abundant compared to nearby uninfected LC. In
addition, staining of VZV-infected MUTZ-3-derived LC for
langerin showed that the expression of this protein remained
comparable to that in mock-infected cells (data not shown).
Taken together, these data indicated that VZV infection of LC in
vivo and MUTZ-3-derived LC in vitro did not adversely affect the
expression of langerin. Terminal deoxynucleotidyltransferase-me-
damaged DNA of apoptotic cells performed on VZV-infected
MUTZ-3-derived LC did not induce any significant level of apop-
tosis (data not shown). Taken together with our previous dem-
onstration that MDDC do not undergo apoptosis following infec-
tion with VZV (1), it suggests that the reduced LC frequency in
the epidermis was not due to apoptosis. Our results are therefore
consistent with the reduction in LC numbers in VZV-infected
skin being a consequence of LC emigration to distal sites, such as
lymph nodes, where additional T-lymphocyte priming may occur.
In the context of this hypothesis, the almost complete loss of LC
from the skin suggests that bystander LC capture VZV antigen in
the skin, stimulating LC emigration, and/or that LC carrying no
viral antigen are stimulated to emigrate. In addition, our finding
that LC are permissive to VZV replication in vivo raises the
intriguing possibility that those LC which become infected with
VZV (as opposed to those which capture viral antigen) may play
a role in virus spread. These possible outcomes highlight the
complexity of VZV-LC interactions in the skin. The establish-
ment of a fresh human skin explant model of VZV infection, such
as that described by Taylor and Moffat (57), could potentially be
adapted to enable capture and characterization of any cells emi-
grating from the epidermis to further investigate the conse-
quences of cutaneous VZV infection for LC function.
These findings implicate LC in VZV pathogenesis at the
skin and also raise the prospect that LC present in the respi-
ratory mucosa may be involved in the early stages of VZV
pathogenesis. Mucosal membranes have been shown to con-
tain LC that survey the mucosa for foreign antigens (27), and
our hypothesis is that LC present in the respiratory mucosa are
the first cells to become infected with VZV and travel to the
lymph nodes, where they then infect T lymphocytes with VZV.
VZV-infected T lymphocytes then migrate to the skin as part
of the inflammatory infiltrate (40, 51, 58), where they are
among the cells responsible for spreading VZV to cutaneous
cells. The development of lesions in the typical course of vari-
cella occurs in crops over several days (7), and our findings in
the skin of infected patients support the notion that following
the initial infiltration of VZV-infected immune cells, cutane-
ous LC become infected with VZV and emigrate to distal sites,
such as draining lymph nodes, to infect additional T lympho-
cytes that then migrate to the skin to cause additional skin
Our characterization of the infiltrating cells in VZV skin
lesions also revealed a prominent increase in the frequency of
CD123?cells as determined by IHC and of BDCA-2?cells as
determined by IFA in VZV infected-skin compared to findings
for uninfected skin. CD123 and BDCA-2 are surface markers
of PDC (15, 41), and although CD123 is also expressed by
other cell types (41, 54, 59), BDCA-2 is exclusively expressed
on PDC (15, 16). We observed similar frequencies and distri-
bution of staining for these two markers in sections of VZV-
infected skin lesions, which suggests that the majority of the
CD123?cells identified by immunohistochemistry were PDC.
We showed by dual immunofluorescent staining a small pro-
portion of VZV antigen?PDC that stained positive for VZV
antigens from all three VZV kinetic classes, indicating com-
plete replication of VZV in PDC.
Despite only sporadic detection of VZV-infected PDC and
LC during natural cutaneous infection, this finding is probably
important when considered in the context of the frequency of
infection of other cell types which were subsequently shown to
play crucial roles in the course of natural VZV infection. For
example, the proportion of VZV-infected lymphocytes in pe-
ripheral blood during natural VZV infection is very small, with
estimates in the range of 1 in 100,000 PBMCs from healthy
varicella patients becoming infected, yet the role of peripheral
blood T cells in transporting virus to distal sites is regarded as
a critical step in VZV pathogenesis (34, 35).
The skin biopsy specimens utilized for this study were ob-
tained from different patients at different times after the ap-
pearance of either varicella or zoster rash. While it remains
possible that changes to DC subtypes may be linked to the
kinetics of the development of the lesion as virus replicates in
the skin, we did not observe any significant temporal differ-
ences in the frequencies of DC subsets in the skin biopsy
specimens that we examined. Correlation between timing of
lesion formation and various DC subsets is complicated by the
asynchronous appearance of lesions during varicella and zoster
(2). That is, the age of an individual lesion within the rash may
differ from the timing of the first appearance of the rash. A
definitive answer to this question would require sequential
biopsies of the same lesion area taken at multiple time points
from the same donor starting from the initial appearance of
the lesion, samples that would be difficult to obtain under
In a case study of a single varicella patient, a large number
of infiltrating PDC were observed within the dermis of the
varicella lesions, as determined by IHC staining for CD123 and
BDCA-2, which coincided with reduced circulating PDC dur-
ing the acute illness compared to levels after recovery (19).
This implies that circulating PDC are recruited to the skin
during varicella, presumably to contribute to the immune re-
sponse against VZV. In this varicella case, large numbers of
cells staining positive for MxA, an interferon-inducible protein
and surrogate marker of IFN-? production, were observed in
the epidermis and dermis, and since these cells were distrib-
uted in a manner similar to PDC distribution, it was concluded
that the MxA?cells were PDC (19). However, these sections
were not dually stained for BDCA-2 or CD123 and MxA, and
VOL. 84, 2010 IMPACT OF VZV ON DC SUBSETS IN HUMAN SKIN4069
thus, it remains to be conclusively established whether the
observed PDC are MxA?. In addition, MxA is a protein that is
induced by exogenous and endogenous IFN-? (1a, 52a, 60a),
and although IFN-?-producing cells can respond to their own
secreted IFN-? in an autocrine manner, MxA is also expressed
by cells only responding to and not necessarily producing
IFN-? (1a, 52a, 60a). Furthermore, this varicella case study did
not determine whether VZV-infected PDC secreted IFN-?,
since the presence of VZV antigen was not assessed. However,
the observation of PDC in a varicella lesion does suggest some
involvement of PDC in varicella. A more recent study has
demonstrated CD14?monocytes within varicella lesions that
express T-lymphocyte costimulatory molecules (20), and it was
shown in vitro that monocytes can be induced by IFN-? to
express T-lymphocyte costimulatory molecules and can then
present VZV antigen to T lymphocytes (20). Given the previ-
ous observation of PDC and MxA?cells in varicella lesions
(19), it was suggested that PDC in varicella lesions may secrete
IFN-? that results in expression of T-lymphocyte costimulatory
molecules by monocytes and subsequent presentation of VZV
antigens to T lymphocytes (20). Our results show that PDC are
recruited to the skin during cutaneous VZV infection, where a
small number are infected. This influx of PDC was not accom-
panied by an increase in the number of cells expressing the
mature DC marker (CD83), suggesting that PDC may not
mature in VZV-infected skin. Our in vitro data support per-
missiveness of PDC to VZV and demonstrate that PDC do not
respond to VZV infection by secreting significant amounts of
IFN-?. Analysis of skin biopsy specimens from additional pa-
tients using dual-staining techniques will be an important goal
of future studies to examine the maturation state of PDC and
to determine the role of bystander and infected PDC in secre-
tion of IFN-? during natural cutaneous infection. Further-
more, assessment of whether infiltrating PDC interact with T
lymphocytes within VZV-infected skin will be important in
establishing whether PDC may drive the immune response via
interactions with T lymphocytes.
An influx of PDC has also been reported by us in recurrent
genital herpes lesions caused by the closely related alphaher-
pesvirus herpes simplex virus type 2 (HSV-2) (14). PDC in
genital herpes lesions were located in the dermis and at the
dermo-epidermal junction in close proximity to T lymphocytes,
particularly CD69?activated T lymphocytes. In vitro analysis
found that despite expressing HSV glycoprotein D entry re-
ceptors, PDC do not become infected with HSV-2 and PDC
exposed to HSV-2 were able to stimulate virus-specific autol-
ogous T-lymphocyte proliferation (14). Our finding that VZV
could infect PDC in vivo and in vitro (with 40 to 70% of cells
infected by 24 h postinfection) contrasts with findings for
HSV-1 and HSV-2, which do not infect PDC (14, 44). In
addition, while we found that VZV-infected PDC did not se-
crete significant amounts of IFN-?, we and others have re-
ported that PDC exposed to HSV-1 or HSV-2 respond with
abundant production of IFN-? (11, 14, 17). These findings
point to fundamental differences between human alphaherpes-
viruses that replicate in the skin in the context of the permis-
siveness and response of PDC and indicate that VZV and HSV
have evolved different strategies to cause disease in the human
PDC have also been shown to become infected with HIV,
but these cells are still able to produce high levels of IFN-?
(53–56). This suggests that general viral infection does not
render PDC incapable of IFN-? production and that our ob-
servations of inhibited IFN-? production in VZV-inoculated
PDC cultures may be due to a specific immune evasion mech-
anism encoded by VZV. In this respect, a study of IFN-?
production in varicella skin lesions that used pSTAT as an
IFN-? marker found that the VZV-infected cells did not ex-
press pSTAT but that adjacent uninfected cells did (38). Al-
though this particular study focused on IFN-? production by
epidermal cells, which occurs by a different mechanism than in
PDC since TLR9 is not expressed in the epidermis (4), it
suggests that VZV can inhibit the IFN-? response in VZV-
infected cells. Our finding that VZV-infected PDC cultures,
which contained both infected and uninfected cells, produced
little IFN-? even when treated with ODN2216 suggests that
the uninfected PDC in these cultures may be rendered resis-
tant to the effects of ODN2216, perhaps as a consequence of a
factor(s) secreted from VZV-infected PDC in the same cul-
ture. However, when supernatants were collected from in-
fected PDC cultures and then added to uninfected PDC, these
uninfected PDC retained the capacity to respond to ODN2216
and upregulate IFN-?. These observations raise the intriguing
possibility that direct contact between VZV-infected and un-
infected cells in the same culture may affect the capacity of
uninfected PDC to express IFN-? in response to ODN2216. In
this respect, we note two recent papers which report that PDC
function was dependent on cell-to-cell contact in addition to
secreted factors (10, 13). These reports did not look specifically
at IFN-?, so examination of the control of this cytokine during
VZV infection of PDC will be an important component of
future work to define the nature of any direct functional inter-
action between VZV-infected PDC and uninfected bystander
PDC. Likewise, it will be important to further define VZV-
encoded modulation of IFN-? production by PDC to identify
any viral gene which encodes this function. Together with the
definition of changes which occur in the distribution of multi-
ple DC subsets in the skin of individuals suffering from primary
and recurrent VZV disease and the identification of LC and
PDC as subsets most affected during infection, this study im-
plicates the importance of VZV-mediated control of DC func-
tion in VZV pathogenesis.
We thank Paul Kinchington from the University of Pittsburgh for
VZV antigen-specific antibodies. We also thank Anthony Henwood of
the Children’s Hospital, Westmead, Australia, and Mark Wallace of
the Infectious Diseases Division, Naval Medical Centre, San Diego,
CA, for supplying skin samples, Rik Scheper of the Department of
Pathology, VU University Medical Center, Amsterdam, Netherlands,
for assistance with MUTZ-3-derived Langerhans cell culture, and
Heather Donaghy of the Centre for Virus Research, Westmead Mil-
lennium Institute, for assistance with plasmacytoid dendritic cell cul-
This work was supported by Australian National Health and Med-
ical Research Council Project Grant 457356. J.H.H. was the recipient
of an Australian Postgraduate Award and a Westmead Millennium
Institute Stipend Enhancement Award.
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