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Cathepsin K Is Involved in Development of
Psoriasis-like Skin Lesions through
TLR-Dependent Th17 Activation
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of June 17, 2017.
Toshitake Hirai, Takashi Kanda, Kenji Sato, Mikiro
Takaishi, Kimiko Nakajima, Mayuko Yamamoto, Reiko
Kamijima, John DiGiovanni and Shigetoshi Sano
J Immunol published online 29 March 2013
http://www.jimmunol.org/content/early/2013/03/28/jimmun
ol.1200901
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Published March 29, 2013, doi:10.4049/jimmunol.1200901
The Journal of Immunology
Cathepsin K Is Involved in Development of Psoriasis-like Skin
Lesions through TLR-Dependent Th17 Activation
Toshitake Hirai,* Takashi Kanda,* Kenji Sato,* Mikiro Takaishi,* Kimiko Nakajima,*
Mayuko Yamamoto,* Reiko Kamijima,* John DiGiovanni,†,‡ and Shigetoshi Sano*
P
soriasis is a common chronic inflammatory skin disease
characterized by increased proliferation, altered differentiation of the epidermis, and inflammatory cell infiltrates
(1). Those cell infiltrates are composed of T cells, neutrophils, and
dendritic cells (DCs). A number of recent studies have demonstrated that IL-23, which is essential for the development of Th17
cells, is functionally involved in the pathogenesis of psoriasis (2).
A human mAb against the p40 subunit shared by IL-12 and IL-23,
termed ustekinumab, shows a therapeutic efficacy in psoriasis (3).
Therefore, amelioration of psoriasis is closely associated with
reduced Th17 responses in patients with psoriasis (4, 5). In addition to adaptive immunity, innate immunity also plays an important role in the pathogenesis of psoriasis. It has been
demonstrated that stimulation of TLR9 by complexes of the antimicrobial peptide LL37 and self-DNA results in the activation of
plasmacytoid DCs (pDCs), which initiate the onset of psoriasis
(6). Also, a recent study has shown that self-RNA-LL37 complexes trigger TLR8 of myeloid DCs (mDCs) as well as TLR7 of
pDCs in human, and drive the autoinflammatory responses in
psoriasis (7). Furthermore, psoriasis is susceptible to an increase
*Department of Dermatology, Kochi Medical School, Kochi University, Kochi 7838505, Japan; †Division of Pharmacology and Toxicology, University of Texas, Austin, TX; and ‡Department of Nutritional Sciences, University of Texas, Austin, TX
Received for publication March 22, 2012. Accepted for publication February 27,
2013.
This work was supported in part by a grant from the Ministry of Education, Science,
Sports, and Culture in Japan.
Address correspondence and reprint requests to Prof. Shigetoshi Sano, Department of
Dermatology, Kochi Medical School, Kochi University, Kohasu, Okocho, Nankoku
783-8505, Japan. E-mail address: [email protected]
Abbreviations used in this article: AEP, asparagine endopeptidase; CTS, cathepsin;
CTSK, cathepsin K; DC, dendritic cell; mDC, myeloid DC; pDC, plasmacytoid DC;
TPA, 12-O-tetradecanoylphorbol-13-acetate.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200901
of the copy number variation in the b-defensin gene locus (8).
These findings suggest that an altered innate defense mechanism
and the resulting enhanced IL-23 signaling might initiate psoriasis.
Lysosomal cysteine proteases, generally known as the cathepsins (CTSs), exert enzymatic activity under low pH conditions (9,
10). CTSS have been implicated in the degradation of the invariant
chain in B cells and in DCs, thereby generating CLIP (11). Cathepsin K (CTSK) is highly expressed in osteoclasts, and is involved in the degradation of bone matrices, such as type I collagen
(12). CTSK does not show Ag-processing capabilities, but facilitates the signaling of innate immune responses to pathogen DNA
through TLR9 (13). CTSK-deficient mice were resistant to experimental autoimmune encephalomyelitis in which Th17 cells
play an important role (13). Therefore, inhibition of CTSK activity
might be therapeutically effective for the treatment of Th17mediated autoimmune diseases, including rheumatoid arthritis
and psoriasis.
We previously reported that Stat3 is activated in keratinocytes
in the majority of human psoriatic lesions (14). K5.Stat3C transgenic mice, in which Stat3 is constitutively active in keratinocytes,
develop psoriasis-like lesions following wounding stimuli or topical treatment with the tumor promoter 12-O-tetradecanoylphorbol13-acetate (TPA), which strongly suggests that Stat3 activation is
required for the development of psoriasis. Furthermore, topical
treatment with a Stat3 inhibitor attenuates human psoriatic
plaques as well as the development of psoriasis-like lesions in
TPA-treated K5.Stat3C mice (15). Human psoriatic lesions and
TPA-induced psoriasis-like lesions in K5.Stat3C mice both show
increased transcript levels for IL-23, IL-17A, IL-17F, and IL-22,
which all belong to the IL-23/Th17 pathway. Furthermore, psoriatic lesions in human and TPA-induced psoriasis-like lesions in
K5.Stat3C mice are sensitive to administration of anti–IL-12/23p40
or anti–IL-23p19 Abs (16), suggesting an essential role for the
IL-23/Th17 axis.
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Cathepsins (CTSs) are lysosomal cysteine proteases that play an important role in the turnover of intracellular proteins and extracellular proteins, such as the degradation of extracellular matrices and the processing of antigenic proteins. A CTS inhibitor,
NC-2300, not only suppresses bone erosion by inhibition of cathepsin K (CTSK), but also ameliorates paw swelling at inflamed
joints in adjuvant-induced arthritis in rats. It has been demonstrated that the amelioration of joint inflammation by NC-2300 is
mediated by the downregulation of cytokine expression in dendritic cells, which are essential for Th17 activation. In this work, we
studied the role for CTSs in the pathogenesis of psoriasis-like lesion in K5.Stat3C mice, a mouse model of psoriasis, in which Th17
contributes to lesion development similar to psoriasis. Psoriatic lesions expressed increased levels of Ctsk and Ctss mRNA
compared with uninvolved skin and normal control skin. Similarly, the epidermis and dermis in K5.Stat3C mice demonstrated
increased CTSK activities, which were sensitive to NC-2300. Topical treatment with NC-2300 significantly ameliorated 12-Otetradecanoylphorbol-13-acetate–induced psoriasis-like lesions in K5.Stat3C mice, and downregulated the expression of IL-12, IL23, and Th17 cytokines. In vitro experiments revealed that TLR7 activation of bone marrow–derived myeloid dendritic cells led to
increase in IL-23 at mRNA and protein levels, which were downregulated by NC-2300. These results suggest that CTSK plays
a role in development of psoriatic lesions through TLR7-dependent Th17 polarization. The Journal of Immunology, 2013, 190:
000–000.
2
In this study, we investigated whether CTSK is involved in the
development of psoriasis-like lesions in TPA-treated K5.Stat3C
mice. The results show that CTSK activity is higher in the epidermis and the dermis of K5.Stat3C mice than in wild-type mice.
Furthermore, TPA-induced psoriasis-like lesions are ameliorated
by topical treatment with a CTSK inhibitor. Our data suggest that
CTSK plays a role in development of psoriatic lesions through the
TLR7-mediated IL-23/Th17 pathway.
Materials and Methods
Patients and skin samples
The study protocol was conducted in accordance with the guidelines of the
World Medical Association’s Declaration of Helsinki and was approved
by the Institute Ethical Review Board of the Kochi Medical School,
Kochi University. Skin biopsy specimens were taken from lesions and from
contiguous uninvolved skins from 10 patients with plaque psoriasis (9
males, 1 female). Before taking skin biopsy, the patients had not been
treated with topical steroids, vitamin D3, UV irradiation, or systemic
therapy for at least 1 mo. Normal control skins were obtained from four
donors who had plastic surgery.
All experimental procedures performed on mice were approved by the
Institutional Animal Care and Use Committee of Kochi Medical School.
K5.Stat3C mice were generated as previously reported (17). Briefly, Stat3C
cDNA (a gift from Dr. J. Bromberg, Memorial Sloan Kettering Cancer
Center, New York, NY) was ligated into the pBK5 construct, followed by
digestion with EcoRI. The construct was then used to generate transgenic
founder mice on an FVB background. Transgenic mice were identified
by PCR of their genomic DNAs with primers specific for the gene encoding rabbit b-globin, as follows: 59-TTCAGGATGTTGTTTAGAATGG39 and 59-CAATAAGAATATTTCCACGCCA-39. In some experiments, the
transgenic mice were backcrossed at least 10 generations with C57BL/6
mice to obtain spontaneous psoriasis-like lesions. The CTSK inhibitor,
NC-2300, was provided by Nippon Chemiphar (Saitama, Japan) and has
been previously described (13, 18). To examine the in vivo effect of NC2300 on the CTSK activity in skin, we topically treated K5.Stat3C mouse
onto the ear skin with either 20 ml NC-2300 in acetone at the indicated
concentrations at every 12 h or acetone alone. Skin samples were taken at
2 h after the last treatment with NC-2300. The ear skins were placed dermal
side down on PBS containing 20 mM EDTA for 80 min at 37˚C. The
epidermis was separated from the underlying dermis using forceps and
washed several times in PBS. The CTSK activity was determined using
a Cathepsin K Activity Assay Kit (BioVision), according to the manufacturer’s instruction.
treated with TPA, snap-frozen sections were treated with 5% BSA for
blocking at room temperature for 1 h. They were then treated overnight at
4˚C with armenian hamster anti-mouse CD11 mAb (clone N418; BioLegend), rabbit anti-CTSK polyclonal Ab, or rabbit anti-mouse IL-23p19
polyclonal Ab (Abcam). This was followed by treatment with goat antiarmenian hamster IgG-FITC (Santa Cruz Biotechnology) and goat antirabbit IgG-Alexa 594 (Invitrogen), respectively, and counterstaining with
DAPI.
Generation of mDCs from bone marrow cells and in vitro
culture
Immature mDCs were generated from mouse bone marrow cells, as previously described (13). In brief, bone marrow cells were cultured with 10
ng ml21 murine GM-CSF (PeproTech) and 0.3 ng ml21 murine IL-4
(PeproTech) in RPMI 1640 medium supplemented with 10% FBS for 6
d. Loosely adherent cells were harvested by gentle pipetting and were used
as mDCs. To stimulate mDCs, cells were cultured in the presence of either
10 mM imiquimod (Enzo Life Sciences) or 10 nM R848 (resiquimod; Enzo
Life Sciences) for 24 h. NC-2300 was added to cells 3 h before stimulation
with the TLR ligands. Concentration of IL-12/23p40 and IL-23p19 in the
culture supernatant was determined by ELISA kits (eBioscience), according
to the manufacturer’s instructions.
RNA isolation and quantitative real-time PCR
Skin tissues were minced with scissors into small pieces on ice, and were
disrupted by sonication. Total RNAs were extracted using a RNA isolation
kit (Promega), according to the manufacturer’s protocol, and were reverse
transcribed using Moloney murine leukemia virus reverse transcriptase
(Invitrogen) with random oligonucleotide hexamers (Invitrogen). PCRs
TPA-induced psoriasis-like lesions in the ear skin of K5.Stat3C
mice
The generation of psoriasis-like lesions in the ears of K5.Stat3C mice was
as described previously (15, 16). In brief, the early TPA response was
conducted by topical treatment with 0.68 nmol TPA (Sigma-Aldrich) in
20 ml acetone on the right ear of each mouse, and with 20 ml acetone on
the left ear as a control once per day at days 1 and 3. NC-2300 (1, 5, 10,
and 50 mg ml21, twice per day) or vehicle alone (acetone) was topically
applied on the ears for consecutive 3 d, and ear skin was biopsied at day 4.
All mouse experiments were performed with strict adherence to institutional guidelines for minimizing distress.
Histology and immunostaining
Ear skin tissues were fixed in 20% buffered formalin and embedded in
paraffin. Sections were obtained from the paraffin blocks and stained by
H&E using standard methods. For staining of CTSK in the skin, sections
were deparaffinized in xylene and rehydrated through a 100% ethanol,
immersed in pH 9.0 Target Retrieval Solution (Dako) in a microwave for
10 min, and then placed in 0.3% H2O2 in PBS for 30 min at room temperature to block endogenous peroxidase activity. After the blocking of
nonspecific protein binding by incubation for 30 min with PBS containing
1% BSA (Sigma-Aldrich) and 5% normal goat serum (ImmunoBioScience), sections were incubated overnight at 4˚C with rabbit anti-CTSK
polyclonal Ab (BioVision). Subsequently, sections were stained using the
EnVision+ System (Dako). For detection of IL-23–producing CD11c+ cells
and CTSK-expressing CD11c+ cells in the ear skin that had been topically
FIGURE 1. CTS expression in psoriatic skin and CTSK activity in
K5.Stat3C mouse. (A) Quantitative mRNA expression of Ctsk, Ctsb, Ctsl,
and Ctss of control skin from normal donors, lesion, and uninvolved skin
from psoriatic patients (pso). The data are shown as mean 6 SD, *p ,
0.05, **p , 0.01, ***p , 0.001, n = 5–10 per group. Tukey-Kramer test.
(B) Enzymatic activity of CTSK in the epidermis and dermis from the ear
skin of K5.Stat3C mice (black bars) and wild-type mice (white bars) before
and after treatment with NC-2300. The ears of the mice were treated with
NC-2300 at concentrations of 1, 5, 10, and 50 mg ml21 (stepwise increments indicated by triangles). The data represent mean 6 SD, ##p , 0.01,
###
p , 0.001 versus vehicle-treated FVB/N mice; *p , 0.05, **p , 0.01,
***p , 0.001 versus vehicle-treated K5.Stat3C mice, n = 5–8 per group.
Tukey-Kramer test.
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Animals used, topical application of NC-2300,
and determination of CTSK activity
CATHEPSINS PLAY A ROLE IN PSORIASIS-LIKE SKIN
The Journal of Immunology
were performed using Power SYBER Green PCR Master Mix (Applied
Biosystems), and amplification conditions were as follows: 50˚C for 2 min,
90˚C for 10 min for 1 cycle, followed by 40 cycles of 95˚C for 15 s and
60˚C for 1 min. The primers for mouse genes are as previously listed (16),
except for human Ctsk, sense, 59-GCCAGACAACAGATTTCCATC-39
and antisense, 59-CAGAGCAAAGCTCACCACAG-39; human Ctsb, sense,
59-TGGAGGGAGCTTTCTCTGTG-39 and antisense, 59-TGACGTGTTGGTACACTCCTG-39; human Ctss, sense, 59-TACGATCTGGGCATGAACC-39 and antisense, 59-GGGAACTCTCAGGGAACTCA-39; and human
Ctsl, sense, 59-GGGAGGGCAGTTGAGGAC-39 and antisense, 59-GCAAGGATGAGTGTAGGATTCA-39. The quantity of each transcript was
analyzed using the 7300 Fast System Software (Applied Biosystems) and
was normalized to Hprt according to the ΔΔCt method.
Flow cytometry and Abs
Statistical analysis
Statistical analysis was conducted using GraphPad Prism (version 4.03;
GraphPad Software). Statistical differences were evaluated by either unpaired t test or ANOVA, wherever applicable, followed by the Tukey or
Tukey-Kramer.
Results
Increased cathepsin K expression in psoriatic lesion and the
skin of K5.Stat3C mice
Previous studies demonstrated increased levels of CTSs B, D, L,
and S in psoriasis (19, 20). We analyzed the expressions of Ctsk,
Ctsb, Ctsl, and Ctss in human psoriasis (Fig. 1A). These four genes
were all upregulated significantly in psoriatic lesions compared
with normal skin. In particular, transcript levels of Ctsk and Ctss
were significantly increased in the psoriatic lesions compared
with uninvolved skin. We next examined the enzymatic activity of
CTSK in the skin from K5.Stat3C, in which the psoriasis-like lesion
develops spontaneously or is induced by wounding stimuli or
topical treatment with TPA (14). Interestingly, the CTSK activity
from K5.Stat3C mice was higher than that from wild-type mice
both in the epidermis and dermis (Fig. 1B). Because the epidermis
FIGURE 2. Effect of topical treatment with NC-2300 on the development of the psoriasis-like lesions in K5.Stat3C mice. (A) Immunostaining of
K5.Stat3C mouse skin treated for 3 d with acetone (top) and TPA (middle and bottom). Note that TPA-treated epidermis (ep) is positive for CTSK compared
with control epidermis. Arrows indicate CTSK-expressing dermal inflammatory cells. Asterisks, unspecific staining in the cornified layer. Immunofluorescence double staining of psoriasis-like lesion with anti-CD11c and anti-CTSK Abs (bottom panel). Arrows indicate CTSK-expressing CD11c+ cells,
which were both positive for CTSK (red) and CD11c (green). Scale bars, 50 mm. (B) The dose-dependent effect of NC-2300 on the psoriasis-like lesion.
The ear skin of K5 Stat3C mice was treated with acetone (open circles, n = 4) or TPA (filled circles, n = 4) and then acetone instead of NC-2300, respectively. NC-2300 was topically applied at 1 mg mL21 (open squares, n = 5), 5 mg mL21 (filled squares, n = 5), 10 mg mL21 (open triangles, n = 6), and
50 mg mL21 filled triangles, n = 4) on TPA-treated K5.Stat3C mice. Results are shown as mean increased ear thickness (mm) 6 SD, ###p , 0.001 versus
vehicle-treated mice; ***p , 0.001 versus TPA-treated mice. Tukey-Kramer test. (C) Representative histological features of TPA-treated ear skin following
treatment with 10 mg mL21 NC-2300. H&E staining. Scale bar, 100 mm. (D) Assessment of the epidermal thickness. White bar, vehicle control (n = 6);
black bar, TPA control (n = 6); gray bar, TPA plus 10 mg mL21 NC-2300 treatment (n = 6). The data are reported as mean thickness (mm) 6 SD, ***p ,
0.001. Tukey test.
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Cervical lymph nodes were collected as the skin-draining lymph nodes
from K5.Stat3C mice following TPA treatment. The single-cell suspensions were stimulated for 4 h with 0.2 mg ml21 PMA (WAKO), 2.7 mM
ionomycin (Sigma-Aldrich), and 40 mg ml21 brefeldin A (Sigma-Aldrich). Cells were then stained for 20 min with FITC-conjugated antiCD3 and Cy5-conjugated anti-CD4 mouse Abs (BD Pharmingen),
followed by permeabilization with Cytofix/Cytoperm buffer (BD
Pharmingen) and intracellular staining with PE-conjugated anti–IL-17A
mAb (BD Pharmingen). Samples were separated using a FACSCalibur
flow cytometer, and data analysis was conducted using Cell-Quest Pro
software (BD Biosciences).
3
4
CATHEPSINS PLAY A ROLE IN PSORIASIS-LIKE SKIN
least 2 mo (data not shown). This result allows us to speculate that
CTSK is required for initiation of psoriasis-like pathology at the
early phase, when dermal inflammation is more prominent.
Effect of NC-2300 on Th17 cell activation in TPA-treated skin
of K5.Stat3C mice
NC-2300 downregulates IL-12/IL-23 transcripts and decreases
IL-23–producing DCs in K5.Stat3C mice
FIGURE 3. Effect of NC-2300 (10mg ml-1) on Th17 cytokines in the
psoriasis-like lesions. (A) The mRNA expression levels of Th17 cytokines
in the lesions. The values are shown by mean mRNA levels relative to Hprt
transcripts 6 SD, *p , 0.05, **p , 0.01, n = 4–10 per group. TukeyKramer test. (B) Intracellular expression of Th17 cells in skin-draining
cervical lymph nodes. Representative data are shown from three independent experiments. Number represents percentages of Th17 cells. (C)
Bar graphs represent the frequency of Th17 cells in the cervical lymph
nodes. The data are shown as mean 6 SD, *p , 0.05, n = 3. Tukey test.
of K5.Stat3C mice has constitutively activated Stat3, this result
suggests that Stat3 signaling contributes to the upregulation of
CTSK enzymatic activity, although it is unknown whether this
effect is direct or indirect. Furthermore, topical treatment with
NC-2300 inhibited the CTSK activity in the epidermis and the
dermis of K5.Stat3C mice in a dose-dependent manner. The same
trend of inhibitory effect of NC-2300 on the enzyme activity was
obtained in those from wild-type mice (Fig. 1B).
Topical treatment with NC-2300 inhibits the development of
the psoriasis-like lesions in K5.Stat3C mice
We previously established the psoriasis-like skin lesion in the ear
of K5.Stat3C mouse with short-term TPA treatment (16). This
lesion demonstrated cytokine and antimicrobial profiles that closely
resembled human psoriatic lesions, and sensitive to administration
of anti-IL17A, anti-IL12/23p40, or anti-IL23p19 Abs. We found
that the expression of CTSK was increased in the epidermis and
dermis in K5.Stat3C mice when topically treated with TPA (Fig.
2A). In particular, the dermal-infiltrating cells, including CD11c+
cells, were strongly positive for CTSK (arrows in Fig. 2A middle
and arrowheads in Fig. 2A bottom). To examine the effect of
NC-2300 on lesion development, NC-2300 was topically applied
twice per day during the course of TPA induction. The ear skin
of K5.Stat3C mice showed psoriasis-like epidermal hyperplasia
following TPA treatment (Fig. 2B, 2C), whereas the ear skin of
wild-type mice did not (data not shown) (15, 16). Notably, topical
application of NC-2300 dose dependently reduced the TPA-induced
ear swelling over time (Fig. 2B) and attenuated the epidermal
hyperplasia (Fig. 2C, 2D). However, NC-2300 did not attenuate
spontaneously developed, chronic lesions, which persisted for at
Because IL-23 stimulation is essential for Th17 development, we
next studied the effect of NC-2300 on IL-23 expression in TPAinduced psoriasis-like lesions. We have previously demonstrated
that TPA-induced skin lesions in K5.Stat3C mice have increased
mRNA transcript levels of both subunits of IL-23, namely IL-12/
23p40 and IL-23p19 (Il12b and Il23a, respectively) (Fig. 4A) (16).
The expression of Il12a and Il23a mRNAs was significantly inhibited by topical treatment with NC-2300. This result suggests
a role for CTSK in facilitating IL-12/IL-23 production in the skin
lesions. Because the psoriasis-like lesions did not show increased
IFN-g gene expression, Th1 skewing did not occur in K5.Stat3C
mice (16). In psoriasis, inflammatory mDCs have been identified
as Tip-DCs (TNF-a and inducible NO synthase–producing DCs)
that produce IL-23, contributing to Th17 cell differentiation and
proliferation (5, 21). Similar to human psoriasis, there were increased numbers of CD11c+ cells that produce IL-23 in the dermis
of TPA-induced lesions in K5.Stat3C mice (Fig. 4B, 4C). Notably,
IL-23–producing CD11c+ cells were significantly decreased in the
dermis by topical treatment with NC-2300 (Fig. 4B, 4D). This
result suggests that the impairment in Th17 induction by CTSK
inactivation might be, at least in part, attributed to a decrease of
IL-23–producing mDCs that accumulate in the lesions.
TLR7/8 ligand induces IL-12 and IL-23 expression in bone
marrow–derived DCs, and this effect is inhibited by NC-2300
It has been shown that the cathelicidin antimicrobial peptide
LL37, which is produced by keratinocytes, is a key factor that
mediates pDC activation in psoriasis (6). LL37 binds to self-DNA
fragments that are released by stressed or dying cells in the skin,
which then trigger pDC activation through TLR9, resulting in
production of IFN-a and activation of the adaptive immune response toward psoriasis. Self-RNA-LL37 complexes not only induce IFN-a production by pDCs through TLR7, but also trigger
mDC maturation and activation through TLR8, leading to cytokine production and upregulation of CD80 and CD86 expression
(7). Furthermore, self-RNA-LL37 complexes are associated with
maturation of mDCs through activation of TLR8 in psoriatic skin
(7). We next examined whether NC-2300 affected TLR activation
within mDCs. TLR7/8 ligands, imiquimod, and R848 induced
transcriptional upregulation of all subunits of IL-12 and IL-23 in
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Th17 cells have been shown to play a crucial role in psoriasis (5).
Similarly, the skin lesions of K5.Stat3C mice depend on activation
of the IL-23/Th17 axis, and the inflamed skin has Th17 cell infiltrates (16). We examined whether NC-2300 affects Th17 cytokine mRNA levels in the psoriasis-like lesions in TPA-treated
K5.Stat3C mice. TPA treatment induced the transcript levels of
Il17a, Il17f, and Il22 in the skin of K5.Stat3C mice, indicating the
activation of Th17 cells (Fig. 3A). Topical application of NC-2300
tended to reduce expression levels of those Th17 cytokine genes,
suggesting that CTSK activity might be required for TPA-induced
Th17 activation. Upon TPA stimulation, Th17 cells were increased
in skin-draining lymph nodes, but this increase in Th17 cells was
reversed by NC-2300 treatment (Fig. 3B, 3C). These results imply
that CTSK might facilitate the Th17 polarization/activation induced by topical TPA treatment.
The Journal of Immunology
5
mDCs most likely through activation of TLR7, because TLR8 is
nonfunctional in mice (Fig. 5A, 5B). However, pretreatment with
NC-2300 inhibited the transcriptional activation of these genes
induced by imiquimod or R848. In addition, ELISA revealed that
imiquimod-induced production of IL-12/23p40 and IL-23p19 by
mDCs was significantly inhibited by NC-2300 (Fig. 5C). Taken
together, the impaired induction of Th17 cells and the resulting
attenuation of TPA-induced psoriasis-like lesions by CTSK inactivation are caused, at least in part, by downregulation of TLR7dependent production of IL-23 from mDCs.
Discussion
CTSs are members of the lysosomal cysteine protease family and
show strong collagenolytic and elastolytic activities. CTSK is expressed by osteoclasts, where it mediates bone resorption (22). In
this regard, CTSK has been considered to be a pharmacological
target for the treatment of osteoporosis (23). CTSK is also expressed by fibroblasts in keloids and in dermatofibromas (24).
Recent studies demonstrated that stromal CTSK is involved in
the invasive nature of cancers, such as squamous cell carcinoma,
Paget disease, and melanoma, presumably through its ability to
promote matrix degradation. Furthermore, it has been demonstrated that CTSK also participated in adjuvant-induced arthritis
in rat (13).
In the current study, we found that Ctsk was upregulated in
the psoriatic lesions compared with uninvolved skin and normal
control skin. This is the case with the skin of K5.Stat3C mice, a
mouse model of psoriasis. Similar to psoriatic epidermis, in
which Stat3 is activated, K5.Stat3C mice express constitutively
activated Stat3 in the epidermis (14). Previous studies demonstrated
that Stat3 directly suppresses cystatin C, which is an endogenous
inhibitor of cysteine proteases (25), and that expression of cystatin
C is detected in keratinocytes (26). The absence of cystatin C in
K14-HPV16 transgenic mice enhances epidermal dysplasia and
increases the expression and activities of CTSs in the skin (27). It
might be speculated that cystatin C levels would be reduced in
keratinocytes in psoriatic lesions as well as those in K5.Stat3C
mice, and thereby the CTSK activity might be enhanced. Notably,
CTSK activities were higher not only in the epidermis, but in the
dermis of K5.Stat3C mice than those in wild-type mice. This might
be attributed to an indirect influence from Stat3C-expressing keratinocytes. Topical application of NC-2300 indeed reduces CTSK
activity in the epidermis and the dermis. Immunohistochemical
study revealed CTSK expression was upregulated in the infiltrating
cells, including the CD11c+ cells, in the dermis as well as the
epidermal keratinocytes when topically treated with TPA, suggesting that an increased CTSK expression in the skin is associated
with psoriasis-like change.
NC-2300 ameliorates adjuvant-induced arthritis in rats, which
represents an animal model for rheumatoid arthritis (13). Furthermore, CTSK-deficient mice are resistant to experimental autoimmune encephalomyelitis, in which Th17 cells play an important
role (13, 28). Th17 cytokines also play a critical role in the immunopathogenesis of psoriasis and in TPA-induced psoriasis-like
lesions in K5.Stat3C mice (16). Treatment with NC-2300 attenuated psoriasis-like lesions, and simultaneous downregulation of
Il17a, Il17f, Il12a, and Il23a occurred. The decrease in IL-23
transcripts in the skin is in part due to the reduced recruitment
of IL-23–producing DCs. It should be noted that IFN-g is not
upregulated in psoriasis-like lesions, whereas Th17 cytokines are
highly upregulated (16). Therefore, Th17 cells have predominant
roles over Th1 cells in developing skin lesions, suggesting that
the IL-23/Th17 axis is one of the targets of NC-2300 in our experimental setting. We consider that NC-2300 inhibited the early
phase of lesion development, whereas the chronic, persistent
lesions were resistant to topical NC-2300 (data not shown). This
result may suggest the presence of an opportunistic window for
the treatment with NC-2300, because the dermal inflammation is
less prominent in the chronic lesions of this mouse model. In this
regard, the therapeutic effect of NC-2300 is required to validate
using more relevant models, such as the SCID-hu xenogeneic
transplantation model (1).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 4. Topical treatment of NC-2300 (10mg
ml-1) suppressed IL-23 production in the psoriasis-like
lesions. (A) Expression of Il12a, Il12b, and Il23a
mRNAs in the lesions. The data are shown as mean 6
SD, *p , 0.05, n = 4–10 per group. Tukey-Kramer test.
(B and C) Immunostaining of the ear skin of K5.Stat3C
mice treated with TPA demonstrated IL-23–producing
CD11c+ cells, which were stained with anti–IL-23p19
(red), anti-CD11c (green), and DAPI. Rectangular area
in (B) is shown at a higher magnification in (C).
Arrowheads indicate IL-23p19+CD11c+ cells. Scale
bars, 100 mm (B), 20 mm (C). (D) The number of IL23p19+CD11c+ was counted in the ears of K5.Stat3
mice treated with vehicle alone (white bar, n = 6), TPA
(black bar, n = 6), and TPA plus NC-2300 (gray bar,
n = 6). NC-2300 treatment significantly reduced the
number of IL-23p19+CD11c+ cells. The data represent
mean 6 SD, ***p , 0.001. Tukey test.
6
A
B
lesions compared with uninvolved skin or normal healthy skin (6).
Moreover, LL37 forms complexes with self-DNA and mediates
pDC activation through the TLR9 pathway that might drive the
autoimmunity in psoriasis. LL37 also forms complexes with selfRNA, by which TLR8-dependent mDC activation occurs in psoriatic skin lesions (7). We in this study demonstrate that triggering
with TLR7/8 ligands upregulates IL-12 and IL-23 in mDCs. Because TLR8 is nonfunctional in mice, this result suggests that
TLR7 activation in mDCs contributes to upregulation of IL-23.
Recombinant CTSs and asparagine endopeptidase (AEP) could
cleave TLR7 or TLR9, leading to TLR signaling (33, 34, 35). The
proteolysis of TLR7 and TLR9 occurs in a multistep fashion. The
first step includes a processing of the TLR ectodomain by AEP
and CTSs. However, NC-2300 did not affect the AEP activity
(data not shown), suggesting that NC-2300 prevents CTSK from
TLR cleavage. We do not exclude the possibility that species of
CTSs other than CTSK could also be pharmacological targets of
NC-2300, in particular, at higher concentrations (13).
Psoriasis is one of the most common skin diseases and is considered to develop through the crosstalk between the epidermal
keratinocytes and immunocytes. Recently developed biologics
are the most promising therapeutics for psoriasis. They include
TNF inhibitors and anti–IL-12/23p40 Abs (ustekinumab), both of
which target the IL-23/Th17 axis (36). The trend for anticytokine
therapies depends on the development of Abs to more specific
components of this axis, including IL-23p19, IL-17, IL-17R, and
IL-22 (37). However, there is a risk for systemic adverse events
with anticytokine therapies, including sepsis, opportunistic infections, and reactivation of latent tuberculosis. Whereas inhibitors
of CTSK have been used in studies of patients with osteopenia/
osteoporosis, they might also be applicable for the treatment
of rheumatoid arthritis because of its inhibitory effect on Th17
activation (13). The current study using a psoriasis mouse model
revealed that NC-2300 efficiently penetrated the skin and exerted
an antipsoriatic effect through downregulation of TLR7/IL-23/Th17
cascade. Thus, topical targeting CTSK by specific inhibitors might
be an alternative therapy, with less risk of systemic adverse reactions, for psoriasis.
Acknowledgments
FIGURE 5. Effect of NC-2300 on IL-12 and IL-23 in bone marrow–
derived mDCs after treatment of TLR7/8 ligands. (A) The mDCs were
pretreated with vehicle alone or 10 mM NC-2300 for 3 h prior to stimulation with 10 nM R848 for 24 h at 37˚C. Treatment of R848-induced
transcripts of Il12a, Il12b, and Il23a, all of which were inhibited by pretreatment with NC-2300. The data are shown as mean 6 SD, *p , 0.05,
**p , 0.01, ***p , 0.001, n = 6. Tukey test. (B) R848 was replaced with
imiquimod (IMQ, 10 mM) under the same experimental protocol. The
results were mostly reproduced when IMQ was used. The data are shown
as mean 6 SD, **p , 0.01, ***p , 0.001, n = 6. Tukey test. (C) The
mDCs were pretreated with vehicle alone or 1, 10, and 30 mM NC-2300
for 3 h prior to stimulation with 10 mM IMQ for 24 h at 37˚C. The data
represent mean 6 SD, ###p , 0.001 versus vehicle-treated mDCs; *p ,
0.05, **p , 0.01 versus IMQ-treated mDCs, n = 5. Tukey test.
The ability of cathelicidin to function as an antimicrobial peptide is required for optimal host defense against infection (29, 30).
A lack of cathelicidin antimicrobial peptides has been associated
with increased microbial growth both in mouse models and in
in vitro cell cultures (29, 30). Furthermore, recent in vitro studies
indicate that cathelicidin also modulates innate immunity. Deficiencies in cathelicidin and/or other antimicrobial peptides correlate with increased opportunities of infection in patients with
atopic dermatitis or Kostman syndrome (31, 32). LL37, an active
peptide of cathelicidin, is highly expressed in psoriatic-involved
We thank Drs. M. Tarutani and T. Yamakawa for discussion and technical
assistance. We are grateful to Nippon Chemiphar for generously providing
the NC-2300.
Disclosures
The authors have no financial conflicts of interest.
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Corrections
Hirai, T., T. Kanda, K. Sato, M. Takaishi, K. Nakajima, M. Yamamoto, R. Kamijima, J. DiGiovanni, and S. Sano. 2013. Cathepsin K is
involved in development of psoriasis-like skin lesions through TLR-dependent Th17 activation. J. Immunol. 190: 4805–4811
There was an error in the data of Fig. 5 as published. Data shown in Fig. 5A as Il12a gene expression was duplicated from the data for
Il23a. The correct data for Il12a gene expression is now shown in the revised Fig. 5 below. The figure legend was correct as published and
is shown below for reference. This error does not change the conclusion of the paper.
FIGURE 5. Effect of NC-2300 on IL-12 and IL-23 in bone marrow–derived mDCs after treatment of TLR7/8 ligands. (A) The mDCs were pretreated
with vehicle alone or 10 mM NC-2300 for 3 h prior to stimulation with 10 nM R848 for 24 h at 37˚C. Treatment of R848-induced transcripts of Il12a, Il12b,
and Il23a, all of which were inhibited by pretreatment with NC-2300. The data are shown as mean 6 SD, *p , 0.05, **p , 0.01, ***p , 0.001, n 5 6.
Tukey test. (B) R848 was replaced with imiquimod (IMQ, 10 mM) under the same experimental protocol. The results were mostly reproduced when IMQ
was used. The data are shown as mean 6 SD, **p , 0.01, ***p , 0.001, n 5 6. Tukey test. (C) The mDCs were pretreated with vehicle alone or 1, 10, and
30 mM NC-2300 for 3 h prior to stimulation with 10 mM IMQ for 24 h at 37˚C. The data represent mean 6 SD, ###p , 0.001 versus vehicle-treated mDCs;
*p , 0.05, **p , 0.01 versus IMQ-treated mDCs, n 5 5. Tukey test.
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