Inflammatory Cytokines Break Down
Intrinsic Immunological Tolerance of Human
Primary Keratinocytes to Cytosolic DNA
This information is current as
of June 18, 2017.
Srikanth Chiliveru, Stine H. Rahbek, Simon K. Jensen, Sofie
E. Jørgensen, Sara K. Nissen, Stig H. Christiansen, Trine H.
Mogensen, Martin R. Jakobsen, Lars Iversen, Claus
Johansen and Søren R. Paludan
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Copyright © 2014 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 192:2395-2404; Prepublished online 31
January 2014;
doi: 10.4049/jimmunol.1302120
http://www.jimmunol.org/content/192/5/2395
The Journal of Immunology
Inflammatory Cytokines Break Down Intrinsic
Immunological Tolerance of Human Primary Keratinocytes
to Cytosolic DNA
Srikanth Chiliveru,*,†,1 Stine H. Rahbek,*,†,1 Simon K. Jensen,* Sofie E. Jørgensen,*,†
Sara K. Nissen,‡ Stig H. Christiansen,* Trine H. Mogensen,†,‡ Martin R. Jakobsen,*,†
Lars Iversen,x Claus Johansen,x and Søren R. Paludan*,†
T
he skin responds to a variety of environmental, chemical,
and physical stimuli by expressing a plethora of membrane
and soluble proinflammatory molecules. Epidermal keratinocytes play an active role in the initiation and persistence of
skin inflammation (1). Nevertheless, strong inflammatory reactions
affecting the skin can be linked to diseases, as observed in inflammatory conditions such as erysipelas, atopic dermatitis, and
markedly in psoriasis. Psoriasis is a common immune-mediated
inflammatory disease affecting the skin and joints. There is ample
evidence of a pathogenic role of both the innate and adaptive
immune system in psoriasis (2). Most notably, psoriasis is characterized by infiltration of Th17 cells, which stimulate the path-
*Department of Biomedicine, Aarhus University, Aarhus DK-8000, Denmark;
†
Aarhus Research Center for Innate Immunology, Aarhus University, Aarhus DK8000, Denmark; ‡Department of Infectious Diseases, Aarhus University HospitalSkejby, Aarhus DK-8000, Denmark; and xDepartment of Dermatology, Aarhus University Hospital, Aarhus DK-8000, Denmark
1
S.C. and S.H.R. contributed equally to this work.
Received for publication August 9, 2013. Accepted for publication January 5, 2014.
This work was supported by grants from The Danish Medical Research Council (09072636 and 12-124330), The Lundbeck Foundation (R83-A7598 and R100-A9403),
The Novo Nordisk Foundation, the Kathrine og Vivo Skovgaards Fond, the Aase og
Ejnar Danielsens Fond, and the Aarhus University Research Foundation. S.C. is
a recipient of a Ph.D. scholarship from the Faculty of Health Sciences, Aarhus
University.
Address correspondence and reprint requests to Dr. Søren R. Paludan, Department
of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, DK-8000 Aarhus C,
Denmark. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: cGAS, cyclic GMP-AMP synthase; DAMP, dangerassociated molecular pattern; IFI16, IFN-inducible protein 16; IKK, IkB kinase; IRF,
IFN regulatory factor; MDM, monocyte-derived macrophage; pDC, plasmacytoid dendritic cell; RT-qPCR, quantitative RT-PCR; siRNA, small interfering RNA; STING,
stimulator of IFN genes; TBK1, TANK-binding kinase 1.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302120
ogenic development of inflammation (2, 3). Moreover, expression
of type I IFN (IFN-a and -b) and the IFN-producing plasmacytoid
dendritic cells (pDCs) are increased and activated in early psoriatic lesions (4).
DNA is a potent inducer of innate immune responses and has
been proposed to be involved in the pathogenesis of psoriasis (5, 6).
For instance, pDCs can be activated through DNA, hence stimulating TLR9-driven IFN-a production (5). In psoriasis, there is
extensive DNA replication in keratinocytes, which is associated
with accumulation of dsDNA breaks (7). It has previously been
reported that DNA replication byproducts as a result of uncontrolled DNA replication end up in the cytoplasm and stimulate
DNA-driven immune stimulation (8). In support of a role for cytoplasmic DNA in psoriasis pathogenesis, one report (6) has
demonstrated that cytoplasmic DNA stimulates inflammasome
activation and IL-1b production in keratinocytes. With respect to
the cellular systems sensing intracellular DNA and stimulating
immune responses, several have been identified (9). This includes
IFN-inducible protein 16 (IFI16), DDX41, cyclic GMP-AMP synthase (cGAS), and DNA-dependent protein kinase (10–13). Common for these DNA sensors is the need for the molecule stimulator
of IFN genes (STING) to transduce signals downstream to the transcription machinery (14).
Antimicrobial peptides are produced at damaged epithelial
surfaces, where they prevent microbial invasion of the host by
directly killing pathogens. A common feature of antimicrobial
peptides is their cationic and amphiphatic structure (15). The major
antimicrobial peptides in human skin are the defensins comprising
multiple cysteine-rich b-sheet peptides and the cathelicidins, a
family having only one member with a helical structure, called
LL37 (16). The knowledge on biological functions of antimicrobial peptides has expanded in recent years beyond direct microbial
killing to also include immune modulation. For instance, LL37 is
responsible for directing DNA to the endosomal compartment in
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Keratinocytes are involved in protecting the body from infections and environmental challenges, but also in inflammatory conditions
like psoriasis. DNA has emerged as a potent stimulator of innate immune responses, but there is largely no information of how
keratinocytes respond to cytosolic DNA. In this study, we report that human keratinocytes are tolerant to cytoplasmic DNA. However, if treated with inflammatory cytokines, keratinocytes gained the capacity to respond to DNA through a mechanism antagonized by the antimicrobial peptide LL37, proposed to be involved in activation and regulation of skin inflammation. The DNA
sensor IFN-inducible protein 16 (IFI16) colocalized with DNA and the signaling molecule stimulator of IFN genes (STING) in the
cytoplasm only in cytokine-stimulated cells, correlating with recruitment of the essential kinase TANK-binding kinase 1. Moreover,
IFI16 was essential for DNA-driven innate immune responses in keratinocytes. Finally, IFI16 was upregulated in psoriasis skin
lesions and localized to the cytoplasm in a subpopulation of cells. Collectively, this work suggests that inflammatory environments
in the skin can lead to breakdown of tolerance for DNA in keratinocytes, which could contribute to the development of inflammatory
diseases. The Journal of Immunology, 2014, 192: 2395–2404.
2396
Materials and Methods
Cells
Normal adult human keratinocytes were obtained by trypsinization of skin
samples from patients undergoing plastic surgery as previously described
(20). Second-passage keratinocytes were grown in serum-free keratinocyte
medium supplemented with human recombinant epidermal growth factor,
bovine pituitary extract, and gentamicin (all from Life Technologies/
Invitrogen Carlsbad, CA). Cells were grown at 37˚C and 5% CO2 in
a humidified incubator. We have previously reported that this protocol
routinely results in very pure keratinocyte cultures with .95% keratin 14–
positive cells (21). Fresh medium was added every second day. By the time
the cells achieved 60–80% confluency, the medium was changed to growth
factor–free keratinocyte medium, and the cells were incubated for various
stimulations. To generate monocyte-derived macrophages (MDMs), buffy
coats from Aarhus University Hospital blood bank were used to collect
PBMCs by Ficoll-Paque (GE Healthcare) gradient centrifugation. Monocytes were purified by plastic adherence using 1 3 108 PBMCs seeded in
6-cm UpCell plates (Nunc, Thermo-Scientific) precoated with poly-L-lysine (0.01% w/v; Cultrex) and allowed to stabilize overnight in IMDM
supplemented with 10% FCS, 600 mg/ml glutamine, 200 IU/ml penicillin,
and 100 mg/ml streptomycin. The following day, unattached cells were
washed away, and adherent cells were differentiated into MDMs by culturing for an additional 5 d in culture media: IMDM supplemented with
10% (v/v) pooled AB+ human sera (Invitrogen), 600 mg/ml glutamine,
200 IU/ml penicillin, 100 mg/ml streptomycin (Life Technologies), and
20 ng/ml M-CSF (Sigma-Aldrich).
Reagents
Cells were transfected with oligo dsDNA and FITC-dsDNA (2 mg/ml)
synthesized chemically (DNA Technology, Risskov, Denmark) and stimulated with either TNF-a (50 ng/ml) or IL-1b (50 ng/ml), both from R&D
Systems (Abingdon, U.K.). The transfection was done by Lipofectamine
2000 (Invitrogen) and LL37 (Innovagen, Lund, Sweden). ODN2216 and
ODN4084F were obtained from InvivoGen. In some experiments, keratinocytes were pretreated with an endosomal acidification inhibitor Bafilomycin A1 (100 nM) and TBK1/IkB kinase (IKK) ε Inhibitor BX-795
(1 mM) (both from Invivogen, San Diego, CA), IΚΚb inhibitor Manumycin A (10 mM), and a p38 MAPK inhibitor SB202190 (20 mM) (both from
Sigma-Aldrich) were also employed.
Genomic DNA isolation and sonication
Keratinocytes were washed and trypsinized. Genomic DNA was purified
using DNA Isolation kit for cells and tissues (Roche Applied Science). Cells
were counted and resuspended in PBS to wash the cells and centrifuged at
1500 rpm for 10 min. Cellular lysis buffer was added and homogenized to
a fine suspension. Proteinase K was added and incubated at 65˚C for 2 h.
Further RNase solution was added and incubated at 37˚C for 15 min.
Proteins were precipitated, and the supernatant was separated. The genomic DNA was precipitated by using isopropanol. The isolated genomic
DNA was diluted and sonicated into small fragments using Sonifier B-12
(Branson Sonic Power, Dietzenbach, Germany). Sonication produced a
concoction of random small fragments of DNA of varied lengths.
Small interfering RNA transfections
Primary keratinocytes were seeded 24 h prior to transfection. Cell culture
medium was changed to keratinocyte basal medium before the transfection.
Small interfering RNAs (siRNAs; Life Technologies; silencing select: IFI16
siRNA catalog number s7138; control siRNA catalog number 4390846) and
Lipofectamine 2000 (Invitrogen, Carlsbad, CA) were incubated with keratinocyte basal medium for 20 min to allow complex formation. The
siRNA–Lipofectamine complexes were added to the cells and incubated
for 6 h before the medium was changed to keratinocyte serum-free medium. The cells were allowed to silence IFI16 for 48 h before verifying
knockdown efficiency by quantitative RT-PCR (RT-qPCR) and further
analysis.
RNA isolation
Keratinocytes were washed with ice-cold PBS. RNA was purified with
a High Pure RNA Isolation kit (Roche Applied Science). Cells were
resuspended in PBS, and Roche RNA lysis buffer was added to the cells.
The lysates were transferred to high-pure filter tube assembly and centrifuged at 8000 3 g for 15 s. The subsequent filtrate was subjected to DNase
treatment for 15 min. The DNase-treated samples were washed by a series
of buffers and centrifuged at 8000 3 g for 15 s in each step. An extra
centrifugation at 13,000 3 g for a 2-min step was performed to remove the
residual buffer. Finally, 50–100 ml elution buffer was added to the filter
tube to elute high-pure RNA and stored at 280˚C until further use.
RNA analysis
RNA for human CCL20, CXCL10, IFN-b, IFI16, and b-actin was analyzed
using SYBR Green-based expression analysis with Brilliant II QRT-PCR 1step kit (Agilent Technologies) and the following primers: CCL20 forward,
59-TACTCCACCTCTGCGGCGAATCAGAA-39 and reverse 59-GTGAAACCTCCAACCCCAGCAAGGTT-39; IFI16: forward, 59-TAGGCCCAGCTGAGAGCCATCC-39 and reverse, 59-TGAGGTCACTCTGGGCACTGTCTT-39 (both from DNA Technology); and CXCL10 Qiagen
QuantiTect primer assay Hs_CXCL10_1_SG, and b-actin Qiagen Quanti Tect
primer assay Hs_ACTB_2_SG (Qiagen). RNA expression was normalized to
b-actin expression and to expression in the relevant untreated control sample.
IFIT1 and GAPDH quantification analysis of the RNA was performed by
a two-step process involving cDNA synthesis by using M-MLV RT (Invitrogen) followed by QuantiFast SYBR Green Kit (Qiagen) with IFIT1 forward primer 59-CCTCCTTGGGTTCGTCTACA-39 and reverse primer 59GGCTGATATCTGGGTGCCTA-39 and GAPDH forward primer 59-TCTTTTGCGTCGCCAGCCGAG-39 and reverse primer 59-ACCAGGCGCCCAATACGACCA-39 (DNA Technology). For detection of IFI16 mRNA in the
psoriasis biopsies, TaqMan primer-probe sets from Life Technologies were
used (catalog number Hs00194261_m1 for IFI16 and catalog number
Hs99999902_m1 for RPLP0 used for normalization).
ELISA
Human CXCL10 protein was measured on culture supernatants using the
Duoset ELISA Development System (R&D systems) and following the
instructions of the manufacturer.
Western blotting
Cytosolic and nuclear fractions of keratinocytes were separated and isolated
using the protocol previously described (22). For SDS-PAGE, 20 mg protein
was loaded per sample and run on Bio-Rad premade Criterion gels (BioRad) and then transferred to polyvinylidene difluoride membranes. For
Western blotting, the following specific Abs were used: IFI16 (1G7; Santa
Cruz Biotechnology, Santa Cruz, CA), cGAS (C6orf150; Sigma-Aldrich),
RCC1 (sc-1162; Santa Cruz Biotechnology), and b-actin (AC-15 HRP;
Abcam).
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pDCs, where TLR9-mediated recognition takes place (5). LL37
production is elevated in psoriasis skin compared with normal skin
and has been proposed to be involved in disease progression.
However, this does not fit with the finding that treatments ameliorating psoriasis symptoms, like vitamin D analogs, narrow-band
UVB light, and fumaric acid esters, induce expression of LL37
(17–19). This indicates that LL37 has properties to dampen inflammation in the skin, and one report (6) has demonstrated that
this antimicrobial peptide decreases activation of the IL-1b–activating inflammasome in keratinocytes. However, there is no
knowledge on whether LL37 is able to control the responses
stimulating antimicrobial and inflammatory gene expression in
keratinocytes.
In this work, we report that keratinocytes in contrast to macrophages are largely tolerant to cytoplasmic DNA. However, when
treated in combination with either of the inflammatory cytokines
TNF-a or IL-1b, the keratinocytes displayed a potent response to
synthetic and purified human DNA. Interestingly, we found that
LL37 modulated the DNA-driven innate responses in cytokinetreated keratinocytes. This correlated with mobilization of IFI16
to the areas containing DNA in the cytoplasm and recruitment of
STING and TANK binding kinase 1 (TBK1). Finally, we demonstrate that IFI16 is upregulated in psoriasis skin and can be
found in the cytoplasm in a subpopulation of keratinocytes in
psoriatic lesions. In summary, we suggest that an inflammatory
environment in the skin can render keratinocytes sensitive to cytoplasmic DNA, which could initiate an uncontrolled inflammatory response eventually promoting the pathological Th17 immune
response.
DNA-MEDIATED IMMUNE ACTIVATION IN KERATINOCYTES
The Journal of Immunology
Confocal microscopy
Cells were fixed at the indicated time point posttreatment, permeabilized
with methanol for 5 min at 220˚C, labeled with Abs against IFI16 (sc-8023;
Santa Cruz Biotechnology), STING (Imgenex IMG-6485A), or TBK1 (sc9910; Santa Cruz Biotechnology). Images were acquired on a Zeiss LSM
710 confocal microscope using a 633 1.4 oil-immersion objective (Zeiss).
Image processing was performed using Zen 2010 (Zeiss) and ImageJ
(National Institutes of Health). All images are representative of at least two
or three independent experiments.
Immunofluorescence analysis
Biopsies
Biopsies were obtained from nonlesional and lesional psoriatic skin.
Keratome and 4-mm punch biopsies from untreated lesional plaque-type
psoriatic skin were taken from the center of a chronic plaque from either the upper or lower extremities. For each patient, biopsies were taken
from only one anatomical site, and the biopsies from nonlesional skin were
taken at a distance of at least 5 cm from a lesional plaque. For quantitative
PCR, 4-mm punch biopsies were immediately snap-frozen in liquid nitrogen and stored until further use. For immunofluorescence and immunohistochemical analysis, biopsies were embedded in paraffin. The study
was conducted according to the Declaration of Helsinki Principles. The
regional ethical committee, Region Midtjylland, approved all described
studies, and signed informed consent was obtained from each patient
(permission numbers M-20090102 and M-20110027).
Donor variations
All in vitro work with primary human keratinocytes was done with cells
from two to three different donors. Data included in the article are all from
series of experiments in which the conclusions drawn were independent
of the donor used.
Statistical analysis
The data are shown as mean 6 SD. The statistical significance was determined by two-tailed Student t test or Wilcoxon rank sum test. The p
values ,0.05 were considered to be statistically significant.
Results
Intracellular DNA alone is a weak inducer of innate immune
responses in keratinocytes
To test whether synthetic DNA localizes to the cytoplasm in primary human epidermal keratinocytes, we transfected such cells
with a dsDNA oligonucleotide labeled with FITC. As shown in
Fig. 1A, synthetic dsDNA localized to the cytoplasm when transfected by either Lipofectamine or LL37, and for both DNA delivery methods, .95% of the DNA-positive spots localized to the
cytoplasm. We also observed that the DNA localized to specific
foci in the cytoplasm, consistent with what has previously been
found in macrophages (10, 23).
To evaluate the cellular response to cytosolic DNA, we measured
expression of the chemokines CCL20 and CXCL10. CCL20 is
reported to play a key role in recruitment of Th17 CD4+ T cells
(24), and CXCL10 is a signature of IFN-stimulated genes, also
reported to be involved in skin inflammation (25). For comparison,
we examined the expression of chemokines in response to TNF-a
and LL37. Surprisingly, DNA delivered into the cytoplasm of
keratinocytes did not evoke significant expression of CCL20 or
CXCL10 (Fig. 1B, 1C), whereas TNF-a stimulation did lead to
expression of the two chemokines. Expression of IFN-b was also
not induced by DNA, and the highly induced IFN-stimulated gene
IFIT1 was induced only marginally (Supplemental Fig. 1A, 1B).
Treatment with LL37 alone did modestly stimulate expression of
CCL20 and CXCL10. To examine whether the observed lack of
response to DNA in keratinocytes was due to the use of a too low
concentration of DNA or harvest of RNA at the wrong time point,
we performed dose-response and kinetics experiments and also
collected supernatants after 24 h of treatment to allow accumulation of chemokine in the supernatant to occur. DNA stimulation
did not evoke a chemokine response at any of the DNA concentrations tested (Fig. 1D), and up to 24 h of treatment did not lead
to production of CXCL10 mRNA or protein (Fig. 1E, 1F). By
contrast, primary human macrophages responded to DNA stimulation with strong induction of CXCL10 at both the RNA and
protein level (Fig. 1D, 1E). In contrast to the modest response of
the keratinocytes to DNA stimulation, we found that RNA delivery into the cytoplasm of keratinocytes, by use of a virus that
stimulates though the RNA-sensing RIG-I pathway (26), led to
potent induction of CXCL10 (Fig. 1F, 1G).
Despite the lack of response to cytoplasmic DNA in keratinocytes, these cells did express the DNA sensors IFI16 and cGAS
(Fig. 1H), with cGAS localizing exclusively to the cytoplasm and
IFI16 localizing mainly to the nucleus but with a distinct and
easily detectable pool of IFI16 localizing to the cytoplasm. For
AIM2, we were able to detect the mRNA in the keratinocytes, but
did not detect AIM2 protein (data not shown), similar to what has
been reported previously (6, 27). Thus, DNA alone is a weak inducer of innate immune responses in keratinocytes.
DNA synergizes with TNF-a and IL-1b to induce gene
expression in keratinocytes
Cytosolic DNA has been reported to be an important dangerassociated molecular pattern (DAMP) in keratinocytes (6). In a
recent study, it was reported that skin inflammation induced by
synergistic action of IL-17A, IL-22, IL-1a, and TNF-a recapitulates some features of psoriasis (28). To address the potential role
of cytosolic DNA and its synergy with proinflammatory cytokines
in inflamed keratinocytes, we stimulated keratinocytes with
transfected synthetic dsDNA together with TNF-a or IL-1b. Interestingly, this dual treatment led to very potent induction of the
chemokines CCL20 and CXCL10 (Fig. 2A–D) as well as IFN-b
and IFIT1 (Supplemental Fig. 1A, 1B). Interestingly, the strong
synergistic induction of CXCL10 expression after stimulation
with DNA and inflammatory cytokines was observed irrespective
of whether the cytokine stimulation was given before or after
DNA (Supplemental Fig. 1C, 1D), although we observed the
synergy to be strongest when cytokine stimulation was given after
DNA transfection. The observed chemokine response was independent of endosomal acidification (Supplemental Fig. 1E, 1F), as
known for the TLR9 pathway (29). Furthermore, a TLR9 agonist
induced only limited gene expression, and the response evoked by
dsDNA in cytokine-treated cells was not affected by cotreatment
with the TLR9 antagonist ODN4084F (Supplemental Fig. 1G,
1H). By the use of a series of well-described small-molecule
inhibitors of the signaling pathways through IKK–NF-kB, TBK1–
IFN regulatory factor 3 (IRF3), and MAPK, we found that cytokine/
DNA-driven expression of CCL20 was dependent on the IKK–
NF-kB pathway but not the TBK1, IRF3, and MAPK pathways,
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Four-micrometer paraffin-embedded sections of lesional and nonlesional
psoriatic skin were deparaffinized and rehydrated. Ag retrieval was performed by boiling in TEG buffer (pH 9) in a microwave. Blockage of the
nonspecific Ab binding sites was achieved by incubating the samples for
30 min with Image-iT FX Signal Enhancer (Invitrogen). Sections were then
incubated overnight at 4˚C with mouse monoclonal anti-IFI16 (sc-8023;
Santa Cruz Biotechnology), diluted in TBS buffer with 5% goat serum.
Secondary staining was obtained incubating with Alexa Fluor 488 Dye
(Invitrogen; 1:300) diluted in TBS buffer with 5% goat serum for 1 h.
Finally, the samples were washed, and nuclear staining was performed by
embedding samples in Prolong Gold antifade reagent with DAPI (Invitrogen). Samples were evaluated by confocal microscopy. As a negative
control, sections were incubated with blocking buffer without primary Ab
and as an isotype control with normal mouse IgG (Santa Cruz Biotechnology) instead of primary Ab.
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DNA-MEDIATED IMMUNE ACTIVATION IN KERATINOCYTES
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FIGURE 1. Intracellular DNA alone is a weak inducer of innate immune responses in keratinocytes. (A) Human primary keratinocytes were treated with
FITC-dsDNA (delivered with Lipofectamine [Lipo] or LL37, 2 mg/ml). Two hours posttreatment, the cells were fixed, stained with DAPI, and analyzed by
confocal microscopy. Scale bar, 10 mm. (B–D) Human primary keratinocytes were treated with dsDNA (Lipo 2 mg/ml, or the indicated concentrations in C),
TNF-a (50 ng/ml), or LL37 (5 mg/ml) as indicated for 6 h. Total RNA was isolated, and mRNA levels of CCL20 and CXCL10 were quantified by RT-qPCR.
In (D), RNA was also harvested from MDMs treated with Lipofectamine-delivered DNA for 6 h and analyzed for levels of CXCL10 mRNA. (E) Human
primary keratinocytes and MDMs were treated as indicated for 24 h using the same concentrations as in (A). Culture supernatants were harvested, and
CXCL10 protein levels were determined by ELISA. (F) Human primary keratinocytes were treated with DNA (2 mg/ml, delivered with Lipofectamine or
LL37) or infected with Sendai virus (multiplicity of infection of 1). Total RNA was harvested at the indicated time points posttreatment and analyzed for
levels of CXCL10 mRNA. All PCR data in this figure were normalized to b-actin and are presented as mean fold induction of triplicate cultures 6 SD. (G)
Schematic illustration of the pathways activated by cytosolic RNA and DNA. (H) Western blotting of cytosolic (Cyt) and nuclear (Nuc) fractions of
keratinocytes. Conc, Concentration; NR, normalized ratio; UT, untreated.
whereas induction of CXCL10 was dependent on all pathways
(Supplemental Fig. 2).
To investigate whether human genomic DNA could induce analogous synergistic responses, we isolated genomic DNA from primary
keratinocytes and transfected keratinocytes cultures with this DNA.
Keratinocytes transfected with genomic human DNA and stimulated
with TNF-a induced significant chemokine responses with a very
strong synergy between the stimuli (Fig. 2E, 2F). Altogether, these
results suggest that inflammatory cytokines enable keratinocytes to
respond to cytoplasmic DNA with a potent innate immune response.
LL37 selectively inhibits DNA-driven gene expression
It has been previously reported that LL37 transports extracellular
self-DNA fragments into TLR9-containing endosomal compart-
The Journal of Immunology
2399
ments of pDCs, in turn inducing type I IFN production (5). To test
the effect of LL37-mediated DNA delivery into the cytosol on
chemokine responses, we used the primary epidermal keratinocytes and compared with the response evoked by DNA delivered
with Lipofectamine. In contrast to what was observed after
DNA delivery with Lipofectamine, cytokine-treated keratinocytes transfected with synthetic DNA using LL37 as delivery
agent induced only marginal expression of CCL20 and CXCL10
(Fig. 3A, 3B).
LL37 couples with DNA and converts it into a potent DAMP,
a trigger of type I IFN in pDCs (5). A recent study also reported
LL37-mediated DNA delivery to be able to activate type I IFN
responses in monocytes (30). On the contrary, Dombrowski et al.
(6) suggested an anti-inflammatory function of LL37 by interfering with DNA-triggered inflammasome activation. Based on these
data, we were interested in evaluating whether the ability of LL37
to inhibit DNA-driven gene expression in keratinocytes was dependent on the antimicrobial peptide being the DNA transfection
agent. To address this, we transfected DNA into keratinocytes
using Lipofectamine and stimulated in parallel with LL37, which
had not been preincubated with DNA. Interestingly, chemokine
responses induced by the combined treatment of Lipofectaminedelivered DNA and TNF-a were found to be significantly inhibited
by LL37 (Fig. 3C, 3D). To assess the LL37 activity on self-DNA,
genomic DNA was used as stimulus. Similar to what was found
when stimulating with synthetic DNA, LL37 inhibited chemokine
expression induced by combined stimulation with Lipofectaminedelivered genomic DNA and TNF-a (Fig. 3E, 3F). The ability of
LL37 to inhibit DNA-driven gene expression was not dependent on
the antimicrobial peptide being given at the same time as the DNA,
because LL37 treatment 4 h post–DNA stimulation still led to
strong inhibition of the cellular response (Supplemental Fig. 3). We
also investigated the effect of LL37 on the modest gene induction
stimulated by TNF-a alone to exclude the cytokine being the target of
LL37. As expected, LL37 did not modulate TNF-a–induced gene
expression, hence demonstrating DNA or DNA-stimulated activities to
be the target of LL37 (Fig. 3G, 3H).
Assembly of the DNA-activated signalsome in keratinocytes is
supported by cytokine treatment
The chemokine expression data presented above suggested that
stimulation with inflammatory cytokines endowed keratinocytes
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FIGURE 2. DNA synergizes with TNF-a and IL-1b to induce gene
expression in keratinocytes. Human primary keratinocytes were treated with synthetic dsDNA (Lipofectamine [Lipo], 2 mg/ml) (A–D),
human keratinocyte genomic DNA (gDNA; Lipo, 2 mg/ml) (E, F),
TNF-a (50 ng/ml) (A, B, E, F), or IL-1b (10 ng/ml) (C, D) as indicated
for 6 h. Total RNA was isolated, and mRNA levels of CCL20 and
CXCL10 were quantified by RT-qPCR. The data were normalized to
b-actin and are presented as mean fold induction of triplicate cultures 6
SD relative to untreated (UT). Similar results were obtained in at least
three independent experiments. *p , 0.05. NR, Normalized ratio.
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DNA-MEDIATED IMMUNE ACTIVATION IN KERATINOCYTES
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FIGURE 3. LL37 selectively inhibits DNA-driven gene expression.
Human primary keratinocytes were treated with synthetic dsDNA
(delivered with Lipofectamine [Lipo] or LL37, 2 mg/ml) (A–D), human keratinocyte genomic DNA (gDNA; Lipo, 2 mg/ml), LL37 (5 mg/
ml), or TNF-a (50 ng/ml) (E, F), as indicated for 6 h. Total RNA was
isolated, and mRNA levels of CCL20 and CXCL10 were quantified by
RT-qPCR. The data were normalized to b-actin and are presented as
mean fold induction of triplicate cultures 6 SD relative to untreated
(UT). Similar results were obtained in at least three independent
experiments. *p , 0.05. NR, Normalized ratio.
with the ability to respond to intracellular DNA and that LL37
inhibited this response. To get molecular information on what may
be underlying these observations, we fixed keratinocytes treated
with FITC-dsDNA (Lipofectamine transfection) and TNF-a for 2
or 6 h and stained with Abs specific for IFI16, STING, and TBK1.
In the untreated cells, STING was broadly distributed in the cytoplasm, and IFI16 was primarily found in the nucleus (Fig. 4A),
consistent with the data presented in Fig. 1H. At 2 h posttreatment,
STING was found to be mobilized to specific foci colocalizing
with DNA in the cytoplasm (Fig. 4A). This was strongly stimulated by TNF-a treatment. However, only in keratinocytes treated
with cytokine did we observe mobilization of IFI16 to the DNA
and colocalization among DNA, IFI16, STING, and TBK1, indicative of assembly of a functional signalsome (Fig. 4A, Supplemental Fig. 4). At 6 h posttreatment, the DNA foci in the
cytoplasm in the cytokine-treated cells remained positive for IFI16
and STING (Fig. 4B). Moreover, abundant colocalization between
STING and TBK1 was observed, often in foci not containing
DNA or IFI16. If the Lipofectamine-delivered DNA was given to
cells also receiving LL37, colocalization between DNA and IFI16
in the cytoplasm was still observed in TNF-a–treated cells (Fig.
4B). However, we found significantly less colocalization between
The Journal of Immunology
2401
DNA and STING and between STING and TBK1 (Fig 4B). Given
the correlation between IFI16–DNA association and DNA-driven
gene induction (Figs. 2, 4A, Supplemental Fig. 4), we were also
interested in evaluating how cytokine treatment may allow the
association between IFI16 and DNA. To address this, we treated
keratinocytes with TNF-a and IL-1b for 2 h and isolated cytoplasmic extracts. Interestingly, cytosolic extracts from cells stimulated with IL-1b, and to a lesser extent also TNF-a, contained
more IFI16 that extracts from untreated keratinocytes (Fig. 4C).
Collectively, these data suggest that treatment of keratinocytes
with inflammatory cytokines induces translocation of a small pool
of IFI16 into the cytoplasm, which enables the cells to assemble
signaling complexes including IFI16, STING, and TBK1 and
suggests that LL37 inhibits assembly of a functional STING signalsome.
IFI16 is essential for DNA-driven innate immune responses in
cytokine-treated keratinocytes. Given the correlation between asso-
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FIGURE 4. Assembly of the DNA-activated signalsome in keratinocytes is supported by cytokine treatment. (A) Human primary keratinocytes were treated with FITC-dsDNA (delivered with Lipofectamine
[Lipo], 2 mg/ml), LL37 (5 mg/ml), or TNF-a (50 ng/ml), as indicated. The
cells were fixed after 2 h, stained for IFI16, STING, and TBK1, and
analyzed by confocal microscopy. White box indicates area displayed in
zoom column. Scale bar, 10 mm. (B) The cells were treated as in (A), but
fixed after 6 h of treatment for subsequent staining and analysis. Similar
results were obtained in at least three independent experiments. (C) Keratinocytes were left untreated (UT) or stimulated with 50 ng/ml of
TNF-a or IL-1b for 2 h. Cytoplasmic extracts were analyzed for
IFI16 and b-actin by Western blotting.
ciation of DNA with IFI16 and formation of STING foci, we were
interested in evaluating whether IFI16 was essential for DNAstimulated gene expression in cytokine-treated cells. For this
purpose, we performed siRNA-mediated knockdown of IFI16
expression in primary human keratinocytes and were able to reduce
levels of IFI16 mRNA by ∼70% (Fig. 5A). Importantly, the strong
DNA-mediated induction of CCL20 and CXCL10 in TNF-a–
treated cells was significantly reduced in cells with knockdown of
IFI16 (Fig. 5B, 5C). These data demonstrate an essential role for
IFI16 in DNA-driven innate immune responses in human primary
keratinocytes.
IFI16 exhibits subcellular localization in psoriasis lesions
similar to what is observed after DNA transfection of
cytokine-treated keratinocytes
With the aim to examine the potential relevance of the findings
described above in human skin diseases, we isolated skin biopsies
2402
DNA-MEDIATED IMMUNE ACTIVATION IN KERATINOCYTES
FIGURE 5. DNA-driven gene expression in cytokine-treated keratinocytes is dependent on IFI16. (A)
Human primary keratinocytes were transfected with
control (Ctrl) and IFI16-specific siRNA. Total RNA
was harvested 48 h later, and levels of IFI16 mRNA
were measured by RT-qPCR. (B and C) Keratinocytes
treated with siRNA for 48 h were stimulated with
synthetic dsDNA (Lipofectamine [Lipo], 2 mg/ml) or
TNF-a (50 ng/ml) as indicated for 6 h. Total RNA was
isolated, and mRNA levels of CCL20 and CXCL10
were quantified by RT-qPCR. The data were normalized to b-actin and are presented as mean fold induction of triplicate cultures 6 SD. Similar results
were obtained in at least three independent experiments. *p , 0.05. NR, Normalized ratio.
due to the protocol used, including deparaffinization and rehydrated of paraffin-embedded sections. In the lesional psoriasis
skin, IFI16 was upregulated at both the mRNA and protein level
(Fig. 6A, 6B). More importantly, in between 5 and 8% of the cells,
IFI16 could be found in the cytoplasm, where it was observed
to either distribute throughout the cytoplasm or to localize to
specific perinuclear foci (Fig. 6A, zoom panels). The latter pattern
exhibited remarkable similarity to what was observed after DNA
transfection of cytokine-treated keratinocytes in vitro (Fig 4).
Unfortunately, attempts to stain the tissue sections for STING
showed that the Ab was not compatible with the protocol used.
FIGURE 6. IFI16 is upregulated in psoriasis lesions
and exhibits a cellular localization pattern similar to
keratinocytes receiving dual stimulation with DNA and
inflammatory cytokines. (A) Tissue sections of punch
biopsies from nonlesional and lesional psoriatic skin
were stained for IFI16 and DNA (DAPI) and analyzed
by confocal microscopy. Arrows indicate cytosolic
IFI16 foci. Scale bar, 100 mm. (B) RNA was isolated
from nonlesional (NLS) and lesional psoriatic skin
(LS), and levels of IFI16 mRNA were quantified by RTqPCR. Data shown as means of normalized values
relative to nonlesional psoriasis skin 6 SD. n = 9. (C)
Tissue section of punch biopsies from lesional psoriatic
skin stained for DNA (DAPI) and analyzed by confocal
microscopy. Arrows indicate DAPI staining (DNA) in
the cytosol. Scale bar, 10 mm. *p , 0.05. DIC, Differential interference contrast; NR, Normalized ratio.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
from nonlesional and lesional psoriasis skin and stained tissue
sections for IFI16 and DNA. As expected, and in contrast to the
nonlesional skin, the lesional psoriasis skin contained a thickened
cell layer with nucleated cells in the epidermis (Fig. 6A). IFI16 was
expressed at low levels in the nonlesional skin and localized primarily to specific areas resembling nucleoli in the nuclei in the
keratinocytes (Fig. 6A). However, because we have previously
observed that extensive sample handling and harsh protocols (like
e.g., fluorescence in situ hybridization) affect the apparent localization of IFI16 as measured by immunofluorescence (31), it is not
clear at this stage whether this is a real biological phenomenon
The Journal of Immunology
Finally, we used the DAPI-stained tissue sections to look for
the potential presence of DNA in the cytosol. Interestingly, we
observed DAPI-positive foci outside the nucleus in between
3 and 5% of the skin cells, but with no significant difference
between cells from psoriasis lesions and nonlesional skin
(Fig. 6C).
Collectively, the presented data demonstrate that keratinocytes
do not evoke innate immune responses to cytoplasmic DNA
unless present in an inflammatory environment. The antimicrobial
peptide LL37, the expression of which is upregulated by several
treatments known to ameliorate psoriasis symptoms (17–19),
inhibits DNA-driven gene expression and interferes with assembly of functional signaling complexes downstream of DNA
sensing. In cells treated with TNF-a, the DNA sensor IFI16
colocalizes with DNA, which correlates with DNA-driven gene
expression. Finally, IFI16 is upregulated in lesional psoriasis
skin and can be found to localize to specific spots in the cytoplasm, similar to what is observed after stimulation with synthetic DNA in vitro.
Skin inflammation can be caused by autoimmune reactions, infections, or physical injury. DNA has recently emerged as an important
stimulator of innate immune responses (9) and is known to evoke
immune responses in keratinocytes (6). Given the abundant exposure of keratinocytes to DNA-associated danger signals, it is important that immune activation by DNA is tightly regulated in these
cells. In this study, we report that immunological reactions of human
primary keratinocytes after DNA exposure require parallel stimulation with inflammatory cytokines. This stimulation enables
keratinocytes to assemble functional signaling complexes acting
upstream of gene expression. The antimicrobial peptide LL37
interferes with this process, hence specifically inhibiting DNAdriven immune activation. Finally, we provide data on material
from psoriasis patients demonstrating that IFI16 is upregulated
in psoriasis lesions and that IFI16 and DNA under such conditions
can be found in the cytoplasm of keratinocytes in specific perinuclear foci, similar to what is seen after DNA stimulation of
keratinocytes in vitro.
A key finding of the present project is that human keratinocytes
normally do not respond to cytoplasmic DNA with an innate immune response and that stimulation with inflammatory cytokines
enables the cells to induce gene expression in response to this
DAMP. This indicates that cytokine treatment activates an essential
factor in the DNA sensing pathway acting upstream of STING,
hence breaking down the intrinsic immunological tolerance of
keratinocytes for cytosolic DNA. We found that the DNA sensor
IFI16 was primarily expressed in the nucleus in keratinocytes not
treated with cytokines, and this was not affected by delivery of
DNA into the cytoplasm. By contrast, in keratinocytes treated with
TNF-a, IFI16 was found to localize with DNA in the cytoplasm,
which correlated with DNA-induced gene expression. Interestingly, although DNA treatment stimulated some degree of STING
redistribution to the well-described punctuate foci (14), even in the
absence of cytokine treatment, recruitment of the IRF3 kinase
TBK1 was seen only in cytokine-treated cells in which IFI16 was
also recruited to the STING foci. We also observed that cytokine
treatment led to accumulation of IFI16 in the cytoplasm. This
could indicate that redistribution of a small pool of cellular IFI16
from the nucleus to the cytoplasm is a critical event in stimulation
of DNA sensitivity in keratinocytes. The authors do acknowledge
that this conclusion could have been strengthened with higher
quality data on the distribution of IFI16 and also if more data
addressing this key issue had been included. This is now an area of
focus in the laboratory. The concept of IFI16 redistribution in
response to inflammatory stimuli is in agreement with a previous
publication by Landolfo and associates (32), demonstrating that
exposure of human keratinocytes and human skin explants for
high doses of UVB irradiation induced IFI16 localization to the
cytoplasm. It has been reported that acetylation of two arginine
residues in the nuclear localization signal of IFI16 inhibits the
nuclear localization of the protein (33), and in this respect, it is
interesting that inflammatory cytokines induce acetyl transferase
activity (34), which could provide a mechanistic explanation for
the observed phenomenon.
The antimicrobial peptide LL37 has intrinsic DNA-binding
function and has been reported to be able to deliver DNA into
endosomes and the cytoplasm (5, 6). Moreover, LL37 is upregulated in psoriasis skin (5), which could indicate a role for LL37 in
promotion of disease. Paradoxically, LL37 expression is further
upregulated in response to some treatments known to ameliorate
psoriasis (17–19). With focus on chemokines of relevance for type
I IFN function and Th17 cell recruitment, we found that DNA
delivered with LL37 did not stimulate expression of CCL20 and
CXCL10 in keratinocytes and that the antimicrobial peptide also
inhibited gene expression induced by Lipofectamine-delivered
DNA, even when given 4 h after DNA treatment. The peptide
did not affect chemokine expression induced by TNF-a. Regarding the mechanism of action of LL37, our data demonstrate that
treatment with LL37 has no impact on recruitment of IFI16 to
cytosolic DNA STING-positive foci, but does inhibit recruitment
of TBK1 to the signaling complex. This could indicate that LL37
inhibits DNA signaling by acting at a level downstream of IFI16
and STING but upstream of TBK1. The idea of LL37 acting
within the cells is consistent with previous data demonstrating
LL37 to inhibit inflammasome activation by DNA delivered with
lipofection (6). It should be mentioned that the mode of action of
LL37 may be cell-type specific, because pDCs and monocytes
have been reported to respond potently to LL37-delivered DNA,
through the TLR9–MyD88 and STING–TBK1 pathways, respectively (5, 30). Collectively, the data could indicate a regulatory
function for LL37 in keratinocytes by controlling DNA-driven
amplification of several inflammatory pathways involved in the
pathogenesis of psoriasis.
Psoriasis has for many years been considered a possible autoimmune-driven inflammatory skin disease, but an endogenous
trigger of the inflammatory responses has never been identified (2).
The disease is characterized by hyperproliferation of keratinocytes
and an excessive Th17 response (2). In addition, expression of
IFN-stimulated genes is elevated in early psoriasis plaques (4).
The data presented in this work point toward a role for the DNAsensing pathway in evoking some of these responses and could
also indicate that DNA may serve as an autoantigen in psoriasis.
We found that IFI16 was upregulated in psoriasis lesions and was
localized to specific foci in the cytoplasm in a fraction of cells,
reminiscent of what is observed after immunostimulatory DNA
recognition in vitro (10). Interestingly, it has been suggested that
aberrant DNA replication intermediates, as may be found in hyperproliferating keratinocytes, can trigger innate immune responses (35).
Physical injury, which is a classical activator of cutaneous inflammation in psoriasis and known as the Köbner phenomenon, has also
been reported to induce release of DNA to the cytoplasm in keratinocytes (6). Thus, it is possible that release of DNA into the cytoplasm, induced by physical damage, DNA replication byproducts, or
even infection, could amplify inflammatory reactions in keratinocytes
in which tolerance to DNA has been broken due to low-grade constitutive cytokine expression. Although the focus of this study has
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Discussion
2403
2404
been on cytosolic DNA sensing, it should be noted that the elevated
expression of IFI16 could also impact on exacerbation of psoriasis by
other mechanisms. Most notably, nuclear IFI16 has been proposed to
stimulate inflammasome activation (36) and modulate expression of
a range of inflammatory genes (37).
Altogether, we report that human keratinocytes have intrinsic
immunological tolerance to cytoplasmic DNA. However, treatment
with inflammatory cytokines leads to breakdown of this tolerance.
This correlates with cytosolic colocalization between DNA and the
DNA sensor IFI16 and assembly of a STING-TBK1–containing
signaling complex. The antimicrobial peptide LL37 inhibits DNAdriven innate immune responses and acts by preventing assembly
of a functional signaling complex. Finally, in psoriasis skin lesions,
IFI16 is upregulated and can be found in the cytoplasm in foci resembling DNA recognition/signaling complexes. Collectively, these
data have revealed the immunological DNA-sensing pathway as
a regulatory switch in keratinocytes and suggest a role for host or
microbial DNA as danger signals in skin inflammation.
We thank Kirsten Stadel Petersen for technical assistance.
Disclosures
The authors have no financial conflicts of interest.
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Acknowledgments
DNA-MEDIATED IMMUNE ACTIVATION IN KERATINOCYTES