IL-22/STAT3-Induced Increases in SLURP1 Expression within Psoriatic Lesions Exerts Antimicrobial Effects against Staphylococcus aureus

Background SLURP1 is the causal gene for Mal de Meleda (MDM), an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma and transgressive keratosis. Moreover, although SLURP1 likely serves as an important proliferation/differentiation factor in keratinocytes, the possible relation between SLURP1 and other skin diseases, such as psoriasis and atopic dermatitis, has not been studied, and the pathophysiological control of SLURP1 expression in keratinocytes is largely unknown. Objectives Our aim was to examine the involvement of SLURP1 in the pathophysiology of psoriasis using an imiquimod (IMQ)-induced psoriasis model mice and normal human epidermal keratinocytes (NHEKs). Results SLURP1 expression was up-regulated in the skin of IMQ-induced psoriasis model mice. In NHEKs stimulated with the inflammatory cytokines IL-17, IL-22 and TNF-α, which are reportedly expressed in psoriatic lesions, SLURP1 mRNA expression was significantly up-regulated by IL-22 but not the other two cytokines. The stimulatory effect of IL-22 was completely suppressed in NHEKs treated with a STAT3 inhibitor or transfected with siRNA targeting STAT3. Because IL-22 induces production of antimicrobial proteins in epithelial cells, the antibacterial activity of SLURP1 was assessed against Staphylococcus aureus (S. aureus), which is known to be associated with disease severity in psoriasis. SLURP1 significantly suppressed the growth of S. aureus. Conclusions These results indicate SLURP1 participates in pathophysiology of psoriasis by regulating keratinocyte proliferation and differentiation, and by suppressing the growth of S. aureus.


Introduction
Psoriasis is a commonly occurring inflammatory skin disorder, affecting approximately 3% of the population worldwide. Psoriasis is characterized by over-proliferation and/or disturbed differentiation of keratinocytes [1], as well as by enhanced expression of tumor necrosis factor-α (TNF-α), IL-17 and IL-22 within the psoriatic skin [2][3][4]. Activated CD4 + and CD8 + T cells populate the psoriatic plaques [5], and symptoms of the disease are ameliorated by drugs targeting T cells [6]. These findings suggest that the abnormal turnover of keratinocytes in psoriasis is caused by cytokines secreted by activated T cells within psoriatic plaques.
Mal de Meleda (MDM; OMIM2483000) is a rare autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. As with psoriasis, it is suspected that MDM is related to defective T-cell function [7]. Homozygous mutations of the SLURP1 gene (previously known as ARS component B), encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor related protein-1 (SLURP1), have been identified as a cause of MDM [8]. SLURP1 is a secreted protein that assumes the three-finger folded structure found in a family of neurotoxins from snakes or frogs [9], and reportedly accelerates differentiation and apoptosis in keratinocytes [10]. Furthermore, SLURP1-deficient mice show severe palmoplantar keratoderma characterized by increased keratinocyte proliferation and water barrier defects [11]. SLURP1 also acts as an allosteric agonist that enhances acetylcholine (ACh)-evoked macroscopic currents in Xenopus oocytes expressing recombinant human α7-nicotinic acetylcholine receptors (α7-nAChRs) [12]. α7-nAChRs reportedly control skin homeostasis and the terminal differentiation of epidermal keratinocytes required for formation of the skin barrier [13]. These effects of SLURP1 on the skin suggest SLURP1 may be involved in regulating the proliferation and differentiation of keratinocytes, not only in MDM but also in other skin disorders, including psoriasis.
Staphylococcus aureus (S. aureus) is detected in about 50% of skin lesions in patients with psoriasis [14,15] and is associated with a higher Psoriasis Area Severity Index (PASI) score [16]. In addition, Niebuhr et al. [17] reported that staphylococcal enterotoxin B (SEB) and αtoxin from S. aureus induce secretion of IL-22 from peripheral blood mononuclear cells and CD4 + T cells, and several reports have shown that IL-22 production is associated with psoriasis [3,18,19]. IL-22 is an IL-10 family cytokine that acts mainly on epithelial cells [20]. Within the skin, IL-22 mediates keratinocyte proliferation and epidermal hyperplasia, inhibits terminal differentiation of keratinocytes, and induces production of antimicrobial proteins (AMPs) [19,21,22]. IL-22-induced AMPs such as β-defensin2, S100A7 and CAP18/LL37 all have the ability to suppress growth of S. aureus [23,24].
In the present study, we explored the involvement of SLURP1 in the pathophysiology of psoriasis using imiquimod (IMQ)-induced psoriatic model mice and normal human epidermal keratinocytes (NHEKs). We found that SLURP1 expression is markedly increased via STAT3 signaling in psoriatic skin, and that recombinant SLURP1 suppresses the growth of S. aureus. These results suggest SLURP1 expression is controlled by IL-22 and is involved in the maintenance of skin homeostasis as well as the pathogenesis of psoriasis.

Mice and treatments
All experiments were reviewed and approved by the Keio University Animal Care and Use Committee, and care was taken to minimize suffering and limit the number of animals used.
Female BALB/c mice were purchased from Sankyo Labo Service (Tokyo, Japan). IMQinduced skin inflammation was induced as described previously [25]. In brief, 8-to 11-weekold mice received a daily topical dose of 62.5 mg of commercially available IMQ cream (5%: a daily dose of 3.125 mg of the active compound, Beselna Cream; Mochida Pharmaceutical Co., Ltd., Shizuoka, Japan) on their shaved back for 2 or 4 consecutive days. Control mice were treated similarly with a control vehicle cream.
NHEKs were seeded into 6-well plates at a density of 2 × 10 4 cells/well and cultured for 5 days. Using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions, the cells were then transfected with 100 nM siRNA targeting STAT3 (Cell Signaling Technology, Inc., Danvers, MA, USA) or STAT5 (Cell Signaling Technology, Inc.), or with a scrambled control siRNA. Forty-eight hours after siRNA transfection, NHEKs were transferred to growth medium with or without 50 ng/mL IL-22, and were cultured for another 24 h.
Immunohistochemistry BALB/c mice were deeply anesthetized with sodium pentobarbital and then perfused through the aortic cone with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) at pH 7.4. Thereafter, skin from the shaved back of each mouse was removed and post-fixed in the same fixative overnight at 4°C, after which they were immersed in 20% sucrose in PB overnight at 4°C. The tissues were then frozen in OCT, cut into 15-μm sections on a cryostat, and thaw-mounted on MAS-coated slides (Matsunami Glass, Osaka, Japan). Immunofluorescent labeling was performed using our rabbit polyclonal anti-SLURP1 antibody (0.7 μg/ml; 26). Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200 dilution; Life Technologies) was used as the secondary antibody. Sections were then wetmounted in Fluoremount-G (SouthernBiotech, Birmingham, AL, USA) and examined using an Olympus FV-1000 confocal microscope system (Tokyo, Japan) equipped with a 60x objective lens (N.A. = 1.35). Confocal images of skin sections were acquired using a single sectioning.
In vitro antibacterial activity assay RK13 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (ICN Biomedical, Inc., Cleveland, OH, USA), 50 U/ml penicillin and 50 U/ml streptomycin at 37°C under an atmosphere of 95% air/5% CO2. Plasmids encoding human SLURP1 cDNAs were transfected into cells using Lipofectamine 2000 according to the manufacturer's instructions. After incubating the transfectant cells for 72 h, the culture supernatants were collected and recombinant human (rh)SLURP1 was partially purified using an anti-human SLURP1 antibody-conjugated column. α-defensin2 and magainin1 were purchased from Peptide Institute, Inc. (Osaka, Japan) and used as a positive control.
The bacterial strains used for antibacterial activity assays were S. aureus 209P and Escherichia coli (E. coli). Bacterial cultures were grown to mid-exponential growth phase, and the cell concentrations were photometrically estimated based on the OD 620 . After dilution to a concentration of 1 x 10 4 CFU/ml, 50-μl aliquots of the dilutions were incubated at 37°C for 18 h with 50 μl of the respective protein samples in a total volume of 100 μl in a sterile 96-well microtiter plate (Corning Inc., Corning, NY, USA). Inhibition of bacterial growth was evaluated by comparing the change in the OD 620 using a microplate reader (TECAN-200, Tecan Trading AG, Männedorf, Switzerland). The results are presented as the percentage of growth relative to the untreated control. This assay was repeated in six independent experiments performed in triplicate.

Results
Increased SLURP1 expression in the skin of psoriatic model mice IMQ-treated mice develop a psoriasis-like skin disorder [25]. We assessed SLURP1 expression in skin from the backs of mice treated with IMQ for 2 or 4 consecutive days. As shown in Fig  1A, we found that IMQ-treated skin developed signs of erythema, scaling and thickening over the course of treatment. Acanthosis is caused by keratinocyte hyperproliferation and scaling is caused by altered epidermal differentiation; both are seen in psoriatic skin lesions and were observed in the IMQ-treated mice (data not shown). Within the psoriasis-like skin lesions, SLURP1 protein expression was significantly increased in hyperproliferative keratinocytes ( Fig  1B), and quantitative real-time PCR revealed corresponding elevation of SLURP1 mRNA expression (Fig 1C). In addition, the induction of IL-22, which appears to contribute to keratinocyte proliferation and epidermal hyperplasia, was also detected in the same skin samples (Fig 1C). The result in Fig 1B was   Expression of SLURP1 mRNA was enhanced by IL-22, but not IL-1β, IL-17A, IFN-γ or TNF-α. Moreover, the stimulatory effect of IL-22 was dose-dependent ( Fig 2B) and was inhibited by simultaneous addition of actinomycin D, but not by cycloheximide (Fig 2C). Expression of SLURP1 was enhanced by 50 ng/mL IL-22, and the protein was detected as a secreted form in the culture supernatant (Fig 2D). Taken together, these results suggest that SLURP1 is a direct target of IL-22 and that the increase in SLURP1 protein expression was transcriptionally regulated.
IL-17A and TNF-α have been shown to act cooperatively with IL-22 to enhance AMP expression (i.e., β Defensin2, S100A7/psoriacin and other S100 family peptides), chemokine expression (e.g., CXCL10, CCL5) and proinflammatory Th22 responses in T cells [23,27]. On the other hand, IFN-γ reportedly suppresses expression of SLURP1 [28]. To examine these effects qPCR analysis was performed for SLURP1, CCL5 and S100A7. As shown in Fig 2E, IL-22-induced SLURP1 mRNA expression was not cooperatively enhanced by IL-17A or TNF-α, but it was suppressed with IFN-γ. This suppression was also confirmed at the protein level, as IL-22 did not increased SLURP1 levels in the supernatants of cultures treated with IFN-γ (Fig 2D).
TNF-α and IFN-γ stimulation significantly increased CCL5 expression (Fig 3A) and IL-22 in combination with IL-17A or TNF-α augmented the effect of IL-22 on S100A7 expression (Fig 3B). These results clearly demonstrated both IL-17A and TNF-α are active. However, similar to SLURP1 expression, IFN-γ combined with IL-22 abolished the effect of IL-22 on S100A7 expression (Fig 3B). These results indicate that IFN-γ somehow affects IL-22-induced gene expression.

SLURP1 expression is under control of the IL-22-STAT3 axis
To investigate the signal transduction pathway leading from IL-22 stimulation to enhanced SLURP1 transcription, IL-22-stimulated NHEKs were treated with a panel of signal transduction inhibitors. The enhancement of SLURP1 mRNA expression was completely blocked by  the STAT3 inhibitor S3I-201, but was unaffected by the STAT5 inhibitor 573108 or the MAP kinase kinase inhibitor PD980589 (Fig 4A). To further confirm this result, NHEKs were transfected with siRNA targeting STAT3 or STAT5. Expression of the respective proteins was greatly suppressed in cells expressing STAT3 or STAT5 siRNA (Fig 4B). Moreover, IL-22-induced expression of SLURP1 mRNA was also suppressed in the STAT3 knockdown cells (Fig 4C). On the other hand, SLURP1 induction was unaffected by STAT5 knockdown. These results suggest that SLURP1 expression is under the control of the IL-22-STAT3 axis.
Antibacterial activities of SLURP1 against S. aureus IL-22 induces production of various AMPs, including β-defensin2, S100A7 and LL37, in regions of skin inflammation [23,24]. Because it has been reported that prostate and testis expressed protein (PATE), which is a secreted protein containing a Ly6 domain, has antibacterial activity [29], we investigated the antibacterial activity of partially purified rhSLURP1 secreted from SLURP1-transfected RK13 cells. As shown in Fig 5, SLURP1 dose dependently suppressed proliferation of the gram-positive bacterium S. aureus but not gram-negative bacterium E. coli. Although antibacterial activity of α-defensin2 to gram-negative bacterium E. coli was quite high, the suppressive effect of α-defensin2 on S. aureus proliferation was almost similar to that of SLURP1 (Fig 5). Magainin1 which does not kill gram-positive bacteria had no effect on proliferation of S. aureus [30], however magainin1 significantly suppressed proliferation of gram-negative bacterium E. coli at the same dose used for gram-positive bacteria ( Fig  5). These results clearly demonstrate that SLURP1 is effective in suppression of proliferation in the gram-positive bacterium S. aureus but not gram-negative bacterium E. coli. More concentration of SLURP1 protein may completely suppress the proliferation of S. aureus, however the physiological concentration of SLURP1 has been reported around 10 ng/ml [28].

Discussion
In this study, we demonstrated that 1) SLURP1 expression is enhanced in the skin of IMQinduced psoriatic model mice; 2) expression of SLURP1 mRNA is induced by IL-22 in keratinocytes; and 3) this up-regulation of SLURP1 transcription is completely suppressed by a STAT3 inhibitor or by siRNA targeting STAT3, which is a major transcriptional factor downstream of IL-22. Wolk and colleagues [19] previously showed that IL-22 is present at high levels in the skin of patients with psoriasis, but is undetectable in healthy skin. Correspondingly, psoriasis patients exhibit elevated plasma IL-22 levels that correlate with disease severity [3,19]. It has also been reported that STAT3 is strongly expressed in keratinocytes within psoriatic skin lesions and that levels are especially high in the nucleus, where it participates in the hyper-proliferation of keratinocytes and inflammatory infiltration [31]. Consistent with these observations, our results suggest that SLURP1 expression is increased within psoriatic lesions. The increased expression of SLURP1 in hyperproliferative keratinocytes within psoriatic lesions is consistent with the increased turnover of the epidermis in psoriasis because SLURP1 accelerates differentiation and apoptosis in keratinocytes [10]. Furthermore, SLURP-1 expressed in keratinocytes was recently reported to regulate wound healing [32]. These findings suggest the possibility that the increased SLURP1 expression is one of compensatory reactions to maintain epidermal homeostasis in psoriatic lesions.
We also found that even a low concentration of rhSLURP1 significantly suppressed the growth of S. aureus (Fig 5). Importantly, S. aureus is detected in about 50% of skin lesions in patients with psoriasis [14,15], and is associated with a higher PASI score [16]. These observations indicate that SLURP1 is not only a mediator that regulates keratinocyte differentiation and apoptosis, it may also be an important factor that reduces the disease activity of psoriasis by suppressing the growth of S. aureus.
When applied in combination with IL-22, neither IL-17A nor TNF-α acted cooperatively with IL-22 to enhance SLURP1 expression, while IFN-γ completely abolished IL-22-induced SLURP1 expression (Fig 2D). It has been reported that IL-17A and IL-17F do not alter expression of the IL-22 receptor [23]. On the other hand, TNF-α is known to enhance the effect of IL-22 on keratinocytes by increasing expression of both the IL-22 receptor and STAT3 [22]. In the present study, IL-22 increased SLURP1 expression in a dose-dependent manner via a STAT3-mediated pathway. In addition, we also found that TNF-α increased expression of IL-22RA1 (data not shown). However, the fact that the TNF-α-mediated enhancement of IL-22 receptor expression did not increase SLURP1 expression indicates SLURP1 levels are tightly regulated by means other than STAT3 activation. SLURP1-deficient mice exhibit severe PPK characterized by increased keratinocyte proliferation and water barrier defects that resemble MDM [11]. SLURP1 binds directly to α7-nAChRs, altering their function [10,12]. Although α7-nAChRs reportedly control the terminal differentiation of epidermal keratinocytes and skin homeostasis required for formation of the water barrier [13], α7-nAChR-deficient mice do not show symptoms of PPK. These observations suggest SLURP1 may regulate keratinocyte proliferation and differentiation through as yet unknown targets in addition to α7-nAChR.
We recently reported that SLURP1 increases ACh synthesis in T cells and attenuates T cell proliferation, and that these effects are abolished by methyllycaconitine, an α7-nAChR antagonist [33]. Those results indicate SLURP1 modulates the functional development of T cells via α7-nAChR-mediated pathways, which is consistent with the findings of Nizri et al. [34], who observed that nicotine suppresses antigen-induced T cell proliferation in wild-type mice but not in α7-nAChR deficient mice. They also reported that nicotine suppresses expression of Th1 and Th17 cytokines, such as TNF-α, IL-17 and IL22, and attenuates Th1 differentiation and expression of T-bet, a key transcriptional factor supporting Th1 development. Given that IL-22 is mainly produced by Th17, Th22 and Tc22 cells, SLURP1 may ameliorate psoriatic disease activity by modulating T cell function. Further investigation will be needed to determine whether SLURP1 itself suppresses Th1 or Th17 cytokine production by modulating T cell differentiation.
In sum, we found that SLURP1 expression is greatly increased within the skin lesions in a psoriatic mouse model, and that SLURP1 expression is increased via IL-22-STAT3 signaling in NHEKs. In addition, recombinant SLURP1 suppressed the growth of S. aureus, which is associated with a higher PASI score. These results suggest that interaction between SLURP1 and IL-22 may contribute to skin homeostasis as well as the pathogenesis of psoriasis.