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RIPK1 downregulation in keratinocyte enhances TRAIL signaling in psoriasis
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RIPK1 downregulation in keratinocyte enhances TRAIL signaling in psoriasis$ Nao Saito* , Masaru Honma, Takashi Shibuya, Shin Iinuma, Satomi Igawa, Mari Kishibe, Akemi Ishida-Yamamoto Department of Dermatology, Asahikawa Medical University, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 September 2017 Received in revised form 20 February 2018 Accepted 5 April 2018
Background: Psoriasis, a common inflammatory skin disorder characterized by scaly erythema and plaques, is induced by dysregulation of dendritic cell- and T cell-mediated immune reaction. Receptorinteracting protein kinase 1 (RIPK1) regulates inflammatory signaling in response to stimuli such as TNFa, TRAIL, and TLRs, resulting in apoptosis, necroptosis and NF-kB activation. However, the physiological relevance in human epidermis remains elusive. Objective: In this study, we examined whether RIPK1 is involved in the pathogenesis of psoriasis vulgaris. Methods: Skin samples of eight patients with psoriasis vulgaris were investigated by western blotting and immunohistochemistry. The functions of RIPK1 in keratinocytes were examined by RT-PCR and ELISA in vitro. TRAIL-neutralization-experiment was employed in an imiquimod-induced murine psoriasis model. Results: In lesional psoriatic epidermis, RIPK1-expression was decreased compared with that in normal epidermis. Cytokines involved in the pathomechanism of psoriasis, such as IL-1b, IL-17A, IL-22 and TRAIL, reduced RIPK1-expression in normal human epidermal keratinocytes (HEK) in vitro. In addition, RIPK1-knockdown enhanced TRAIL-mediated expression of psoriasis-relating cytokines, such as IL-1b, IL-6, IL-8, TNF-a, in HEK. Numerous TRAIL-positive cells were detected in the dermis of lesional psoriatic skin, and TRAIL receptors were expressed in psoriatic epidermis and HEK in conventional cultures. Moreover, TRAIL-neutralization in an imiquimod-induced murine psoriasis model remarkably improved skin phenotypes, such as ear thickness, and TNF-a expression in lesional skin. Conclusions: These results lead us to conclude that RIPK1-downregulation in keratinocytes increases their susceptibility to TRAIL stimulation, and plays a role in the pathogenesis of psoriasis vulgaris. © 2018 Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology.
Keywords: RIPK1 TRAIL Psoriasis vulgaris IMQ-model mice
1. Introduction Psoriasis, a common inflammatory skin disorder characterized by scaly erythemas and plaques, affects around 2% of the population [1]. Dysregulated interactions of innate and adaptive immunities are deeply involved in the pathomechanism. Complexes of epidermis-derived antimicrobial peptide, LL-37, and
Abbreviations: HEK, human epidermal keratinocytes; RIPK1, receptor-interacting protein kinase 1; RIPK1-KD-NHK, RIPK1 knockdown keratinocyte; Th17, T helper cell 17; TRAIL, TNF-related apoptosis-inducing ligand; TNF, tumor necrosis factor. $ Work was done in Asahikawa, Japan. * Corresponding author at: Department of Dermatology, Asahikawa Medical University, 2 1 1 1 Midorigaoka, Higashi, Asahikawa, Hokkaido, 078 8510, Japan. E-mail address: [email protected] (N. Saito).
host DNA are thought to act as an initiating factor via stimulating IFN-a-production from dermal plasmacytoid dendritic cells [2]. Consequently, activated myeloid dendritic cell (DC) populations release TNF-a and IL-23, and stimulate Th1 cells and T helper cell 17 (Th17) to produce IFN-g, IL-17 and IL-22 [3,4]. These cytokines induce proliferation of epidermal keratinocytes and mobilize immune cells into lesional skin [5]. In the exacerbating inflammatory chain, DC/Th17 axis is a major research focus as well as being the therapeutic target in the pathomechanism of psoriasis [6,7]. Receptor-interacting protein kinase 1 (RIPK1), one of the receptor-interacting serine/threonine-protein kinases, was originally identified as a death domain-containing kinase. It is a key regulator of inflammation, apoptosis and necroptosis downstream of tumor necrosis factor (TNF)-a, TRAIL, and Toll-like receptors (TLRs). Expression or function of RIPK1 is regulated in complex and intricate
https://doi.org/10.1016/j.jdermsci.2018.04.007 0923-1811/ © 2018 Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology.
Please cite this article in press as: N. Saito, et al., RIPK1 downregulation in keratinocyte enhances TRAIL signaling in psoriasis, J Dermatol Sci (2018), https://doi.org/10.1016/j.jdermsci.2018.04.007
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mechanisms including ubiquitylation, deubiquitylation, and phosphorylation [8]. Recent studies have shown that RIPK1 is also involved in oncogenicity of melanoma, axonal degeneration of amyotrophic lateral sclerosis, and apoptosis in breast cancer [9–11]. Moreover, epidermis-specific RIPK1-knockout mice developed significant inflammatory skin reaction characterized by thickened epidermis with various types of cell death [12]. Given this background, we examined whether RIPK1 is involved in the pathogenesis of psoriasis vulgaris and found that keratinocytes become highly sensitive to TRAIL signaling by RIPK1-downregulation.
2.6. ELISA Human IL-1b and human TNF-a ELISA on culture supernatants of keratinocytes was performed using ELISA kits from R&D systems in accordance with the manufacturer’s instructions. 2.7. Mice C57BL/6J female mice (six weeks old) were purchased from Oriental Yeast (Tokyo, Japan). All the animal experiments were performed with the approval of the ethical committee for animal studies of Asahikawa Medical University.
2. Materials and methods 2.8. IMQ-induced psoriasis model and Ab treatment 2.1. Patient samples A total of eight patients with psoriasis vulgaris at Asahikawa Medical University Hospital participated in this study. The collection of samples was performed with approval by the local ethical committee and the institutional review board of Asahikawa Medical University, and each patient gave written informed consent.
The skin of the back and the right ear of each mouse was treated daily for seven days with 62.5 mg and 31.25 mg of 5% IMQ cream (Beselna cream; Mochida Pharmaceutical, Tokyo, Japan). Vaseline petroleum jelly was used as control. In some experiments, mice received ip injections with 250 mg of anti-TRAIL (clone N2B2) or control rat IgG2a (Biolegend, San Diego, CA) per mouse before application of IMQ cream on days 0, 2, 4, and 6.
2.2. Keratinocyte culture and stimulation Primary keratinocytes were cultured in CnT-PR medium (CELLnTEC, Bern, Switzerland) at 37 C with 5% CO2, and used for the assay with no more than fourth passages. Recombinant human TRAIL (100 ng/ml, Peprotech, Rocky Hill, NJ), recombinant human IL-1b (R&D systems, Minneapolis, MN), recombinant human IL-17A (Peprotech), recombinant human IL-22 (Peprotech), recombinant human IFN-g (Peprotech), recombinant human TNFa (100 ng/ml, Peprotech) and recombinant LL37 (2.56 mM, 7.68 mM, Eurogentec, Seraing, Belgium) were added into the culture medium. 2.3. siRNA transfection Normal human keratinocytes (NHK) were transfected with siRNA of RIPK1 (40 pM) or si-control (40 pM) (Silencer1 Select Negative Control No.1 siRNA, Ambion, Austin, TX) using the electroporation system NucleofectionTM (Lonza, Basel Schweiz). At 72 h following transfection, cells were used in experiments.
2.9. Evaluation of skin inflammation severity and measurement of ear thickness To evaluate severity of mouse skin inflammation, an objective scoring system based on the clinical Psoriasis Area and Severity Index (PASI) was employed. Severity of erythema and scaling was rated independently as score 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, severe. The ear thickness was measured using a caliper (Peacock Ozaki MFG. CO., LTD., Tokyo, Japan). 2.10. Statistics In all figures, data are presented as mean SD from pooled data of at least 3 independent experiments. P values were calculated with one-way analysis of variance (post hoc Tukey or Dunnett) and two-tailed independent Student’s t-tests, and P < 0.05 was considered significant. 3. Results
2.4. Quantitative real-time PCR
3.1. Decreased RIPK1 expression in lesional epidermis of psoriasis
Total RNA extracted using RNeasy Kit (Qiagen, Hilden, Germany) was applied for quantitative real-time PCR (RT-PCR) performed by a LightCycler 480 Instrument (Roche, Basel Schweiz) or TaqMan gene expression assays (Applied Biosystems, Carlsbad, CA). Primer and probe sets used were TNFRSF10A (Hs00269492_m1), TNFRSF10 B (Hs00366278_m1), TNFRSF10D (Hs00174664_m1), GAPDH (Hs02758991_g1), mIL17A (Mm00439618_ m1), mIL17F (Mm00521423_ m1), mIL-22 (Mm01226722_ g1) and mGAPDH (Mm99999915_g1). For each data point, similar results were obtained at least in two independent experiments.
To clarify the function of RIPK1 in the epidermis, we first performed immunohistochemistry for RIPK1. In healthy control epidermis, atopic dermatitis and erythema multiforme, abundant RIPK1-expression was detected throughout whole layers of the epidermis. In contrast, it was minimal in psoriatic epidermis (Fig. 1A). Moreover, protein expression levels of RIPK1 was significantly decreased in lesional and non-lesional epidermis of psoriasis as compared with that in healthy control (Figs. 1B, C and S1). In lesional skin of IMQ-treated mice, an animal model recapitulating key features of psoriasis [13], qRT-PCR and western blotting revealed 80 to 97% decrease of RIPK1-expression in psoriasiform skin (Fig. 1D). To further clarify the regulatory mechanism, RIPK1-expession was analyzed in HEK treated with psoriasis-relating cytokines. IL-1b, IL-17A, IL-22, TRAIL, and IL17A + IL-22 significantly reduced RIPK1-expression in HEK (Fig. 1E). These cytokines are produced by immune cells relating to both innate and adaptive immunities, and they are deeply involved in the pathogenesis of psoriasis. In addition to IL-1b and Th17 cytokines, TRAIL is expressed in inflammatory DC in psoriatic lesions, and can directly act on keratinocytes [14]. RIPK1 mainly regulates inflammatory signaling relating to NF-kB, apoptosis, and
2.5. Immunohistochemistry Immunostaining on human and mouse skin was performed with antibodies against RIPK1 (Thermo Fisher Scientific, Waltham, MA), human CD4, CD8, CD11c (BD Biosciences, San Jose, CA), DR4, DR5, DcR2 (abcam, Cambridge, UK), and TRAIL (Cell signaling, Danvers, MA). Fluorescence staining was detected using a confocal laser scanning fluorescence microscope (Fluoview FV1000, Olympus, Tokyo, Japan).
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Fig. 1. Decreased RIPK1 expression in lesional skin of psoriasis vulgaris. (A) Immunohistochemistry analysis of RIPK1 in healthy control, atopic dermatitis, erythema multiforme and psoriasis vulgaris. Scale bar = 50 mm. (B) RIPK1 expression was evaluated using western blotting. The RIPK1 protein levels ware quantified using densitometry and expressed as the ratio of RIPK1 to b actin. N = 5 **p < 0.01 vs the control. (C) Immunohistochemistry analysis of RIPK1 in lesional and non-lesional skin for psoriasis vulgaris. Scale bar = 50 mm. (D) RIPK1 expression levels in imiquimod-treated mice were determined by RT-PCR and western blotting. N = 4 **p < 0.01 vs the control. (E) HEK were stimulated with psoriasis-related cytokines. RIPK1 expression levels were determined by RT-PCR. Similar results were obtained in two independent experiments. **p < 0.01 vs the control.
necroptosis under the stimuli of TNF, TRAIL, and TLRs. We hypothesized that the RIPK1-downregulation in keratinocyte enhances TRAIL signaling and contributes to exacerbation of psoriasis.
These results indicate that RIPK1-KD-keratinocyte is more sensitive to TRAIL signaling, which induces IL-1b, IL-6, IL-8, IL-20, IL-33 and TNF-a via NF-kB pathway. TRAIL did not induce any type of cell death in each group (Fig. S4).
3.2. RIPK1 downregulation in keratinocytes increased their susceptibility to TRAIL stimulation
3.3. TRAIL positive cells and TRAIL receptors were detected in psoriasis vulgaris
To verify our hypothesis, TRAIL was added to RIPK1-knockdown keratinocyte (RIPK1-KD-keratinocyte) in conventional culture conditions. RIPK1-expression was suppressed to 7 to 14% of control (Sic-NHK) in RIPK1-KD-keratinocytes (Fig. S2). TRAIL induced three to eight times higher mRNA levels of IL-1b, IL-6, IL-8, IL-20, IL-33 and TNF-a in RIPK1-KD keratinocytes (Fig. 2A). While mRNA-expression-levels of IL-20, IL-24 and IL-33 presented differences at the baseline, TRAIL-treatment did not significantly alter the expression levels. Protein levels of TNF-a and IL-1b were also increased in the culture supernatant of RIPK1-KD-keratinocytes without any difference in gene-expression at baseline (Figs. 2B and S3). An NF-kB inhibitor inhibited TRAIL-induced IL-6- and TNF-a-expression in RIPK1-KD-keratinocyte (Fig. 2C).
TRAIL, one of the TNF-superfamily members presenting potent cytotoxicity to tumor cells and transformed cells, is a transmembranous protein which can be shed by ADAM proteases. Four types of human TRAIL receptors, DR4, DR5, DcR2 and DcR1, have been reported. Among them, DcR1 works as an intrinsic antagonist against other TRAIL receptors [15]. Immunohistochemistry showed numerous TRAIL positive cells and abundant expression of TRAIL receptors, DR4 and DR5, in psoriasis lesional dermis and epidermis, respectively (Fig. 3A). DcR2-expression was not detected in either psoriasis lesions or in healthy controls. To clarify the regulatory mechanism of TRAIL receptors in the epidermis, HEK were cultured with psoriasis-relating cytokines and LL-37. LL-37 highly upregulated TRAIL receptor-expression, but
Please cite this article in press as: N. Saito, et al., RIPK1 downregulation in keratinocyte enhances TRAIL signaling in psoriasis, J Dermatol Sci (2018), https://doi.org/10.1016/j.jdermsci.2018.04.007
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Fig. 2. Expression of psoriasis-related cytokines in RIPK1-decreased keratinocytes. (A) Sic-NHK and RIPK1-KD-NHK were stimulated with TRAIL for 1, 3, and 6 h. After stimulation, the cytokine expression was determined by RT-PCR. Similar results were obtained in two independent experiments. *p < 0.05, **p < 0.01 vs the control. (B) Sic-NHK and RIPK1-KD-NHK were stimulated with TRAIL for 48 h. IL-1b and TNF-a concentrations in culture supernatant were determined by ELISA. Similar results were obtained in two independent experiments. *p < 0.05, **p < 0.01 vs the control. (C) Sic-NHK and RIPK1-KD-NHK were stimulated with TRAIL for 3 h in the presence or absence of NF-kB inhibitor BAY11-7082 (5 mM). The expression of IL-6 and TNF-a was determined by RT-PCR. Similar results were obtained in two independent experiments. **p < 0.01 vs the control.
not other cytokines (Fig. 3B, C). While TRAIL is expressed in a variety of immune cells in a stimulation dependent manner, TRAILpositive cells in psoriasis lesions expressed CD4+ or CD11c+ (Fig. 3D), suggesting a crucial role of CD4+ and CD11c+ cells in the pathogenesis of psoriasis. 3.4. Neutralization of TRAIL inhibits development of psoriasis-like dermatitis in IMQ-model mice Next, we examined the correlation between RIPK1 and TRAILpositive cells in an IMQ-induced psoriasis model. In IMQ-model mice, numerous TRAIL-positive cells were detected in the dermis, and the TRAIL receptor, DR5, was highly expressed in the epidermis as compared with that in the control (Fig. 4A, B). Inversely, RIPK1expression in the epidermis was decreased similarly to that in psoriatic epidermis (Figs. 1D and S5). To confirm involvement of TRAILsignaling in development of psoriatic lesions, a TRAIL-neutralizationexperiment was employed. Intraperitoneal injection of TRAIL neutralization antibody every other day substantially suppressed the skin phenotype of IMQ-treated mice, such as erythema, infiltration and scales, with histopathological improvement of acanthosis,
hyperkeratosis, and parakeratosis (Figs. 5 A, C and S6). Moreover, TRAIL neutralization antibody canceled inhibition of RIPK1-expression in epidermis of IMQ-treated mouse (Figs. 5A and S7, S8), and ear swelling and thickness of the back skin were also suppressed significantly (Figs. 5B and S9). None of cell death type was detected in epidermal keratinocytes of each mouse group (Figs. S10, 11, 12). These favorable changes resulting from TRAIL neutralization were accompanied by a decrease of TNF-a mRNA (Fig. 5D). Collectively, these results indicate that TRAIL signaling is likely to be involved in the exacerbation of psoriasis lesions through RIPK1- downregulation. 4. Discussion In this study, we found that RIPK1 downregulation in keratinocytes increased their susceptibility to TRAIL stimulation. TRAIL, the best known apoptosis-inducer [16], is a member of the TNF superfamily that can induce tumor-selective cell death. In allergic conditions, it shows bidirectional functions, such as inflammatory role on epithelia in asthma [17] and anti-inflammatory role in atopic dermatitis [18]. In the pathogenesis of psoriasis, it has been reported as a factor presenting an inflammatory role
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Fig. 3. Lesional skin of psoriasis vulgaris expressed TRAIL receptors and TRAIL positive cells. (A) Immunohistochemistry analysis of TRAIL receptors and TRAIL positive cells in psoriasis vulgaris and healthy control. Scale bar = 50 mm. (B) DR4 and DR5 expression was evaluated using western blotting in keratinocytes stimulated with LL37 (2.56 mM). The DR4 and DR5 protein levels were quantified using densitometry and expressed as the ratio of DR4 and DR5 to b actin. N = 4. (C) Cultured human keratinocytes were stimulated with psoriasis-related cytokines and LL37. DR4, DR5 and DcR2 expression levels were determined by RT-PCR. Similar results were obtained in two independent experiments. **p < 0.01 vs the control. (D) Skin sections obtained from psoriasis vulgaris were stained with antibodies against CD4, CD8, CD11c (green) and TRAIL (red). Scale bar = 50 mm.
Fig. 4. TRAIL positive cells and TRAIL receptors in IMQ-model mice. (A) Phenotypical presentation of mouse back skin after 7 days of treatment. (B) H&E staining of the mice back skin and immunohistochemistry analysis of TRAIL positive cells and TRAIL receptor in mice. Scale bar = 50 mm.
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Fig. 5. TRAIL neutralization antibody inhibits psoriasis vulgaris in IMQ-model mice. (A) Mice were treated with IMQ and control antibody or TRAIL neutralization antibody. For each group, n = 4 (left panel). H&E staining (middle panel) and immunohistochemistry analysis of RIPK1 in mice (right panel). Scale bar = 50 mm. (B) Ear swelling of the right ear was measured: IMQ with a control antibody (circles); IMQ and TRAIL neutralization antibody (squares) (n = 4). *p < 0.05, **p < 0.01 vs the control. (C) Erythema and scale of the back skin were scored: IMQ with a control antibody (circles); IMQ and TRAIL neutralization antibody (squares) (n = 4). (D) Cytokine expression of mice skin was determined by RT-PCR. Similar results were obtained in two independent experiments. *p < 0.05, **p < 0.01 vs the control.
[14,19]. TRAIL expressed on CD11c+CD1c inflammatory DCs in psoriatic lesions can induce epidermal keratinocytes to express CCL20, which recruits CCR6-positive Th17 cells into the lesion. TRAIL can also stimulate NF-kB and MAP kinase signaling pathways depending on RIPK1 [20]. A recent report presented that epidermis-specific inhibition of NF-kB in mice induces psoriasis-like skin inflammation involving increased expression of IL-24 and activated STAT3 signaling via TNF receptor 1-induced and MAP kinase-dependent mechanism [21]. In this study, RIPK1-downregulation in epidermal keratinocytes enhanced TRAIL-mediated expression of psoriasis-relating cytokines, such as IL-1b, IL-6, IL-8, IL-20, IL-33 and TNF-a, suggesting a crucial role of NF-kB pathway on enhanced expression of these cytokines. Moreover, expression of TRAIL receptors can be detected more strongly in both lesional and non-lesional epidermis of psoriasis vulgaris compared with healthy epidermis [14]. Therefore, TRAILsignaling through TRAIL receptors can act as an important regulator of the immune responses. TRAIL can also induce apoptosis in tumor cells but not normal cells depending on contrastive expression and activation levels of TRAIL receptors [22]. In idiopathic pulmonary fibrosis, upregulated expression of TRAIL receptors contributes to susceptibility to TRAIL signaling
[23]. In this study, we have newly identified LL-37 as an inducer of TRAIL receptor expression. This finding strongly supports involvement of TRAIL signaling in psoriasis. A complex of LL-37 and host DNA can stimulate dermal plasmacytoid DCs to produce IFN-a at the disease onset and during the exacerbation stage [2]. This upregulation system of TRAIL receptors via LL-37 can contribute the pathomechanism at an initiating stage of psoriasis. RIPK1 was originally identified as a death domain-containing kinase bound to intracellular death domains of activated FAS and TNF receptor 1 via ligand stimulation. RIPK1 also modulates the inflammatory response, apoptosis and necroptosis mediated by TRAIL, Fas, LPS and polyIC. RIPK1 is consisted of three independent domains, an amino-terminal kinase domain, an intermediate domain and a carboxy-terminal death domain. The intermediate domain engaging RIPK1 to mediate cell survival via NF-kB pathway is subjected to Lys63-linked ubiquitylation at Lys377. The N-terminal kinase domain relates to necrosis-specific RIPK1 phosphorylation which stabilizes binding of RIPK1 and RIPK3 in pro-necrotic complex, resulting in upregulation of pro-necrotic kinase activity and inducing downstream reactive oxygen species production [8]. MTT assay did not show any type of cell death in Sic-NHK and RIPK1-KD-NHK by TRAIL-treatment (Fig. S4). In
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addition, expression level of phospho-RIPK3 was minimal in IMQtreated mouse epidermis with or without TRAIL-neutralization (Fig. S12). Collectively, we conclude that total amount of RIPK1 is more important than its phosphorylation level. Downregulation of RIPK1 can increase sensitivity to TRAIL-induced apoptosis in cancer cells which were originally resistant to TRAIL-signals [24]. Thus, TRAIL is considered to control multiple cellular signaling pathways relating to inflammation and cell death process. Regarding regulatory effects on various inflammation, RIPK1 can work in diverse ways. Reduced RIPK1 levels or function can contribute to suppression of inflammation. SHARPIN-deficient mice, in which no NF-kB activation is detectable, develop a TNFdependent multi-organ inflammation, mainly relating to apoptosis and necroptosis, and the TNF-driven inflammation can be prevented by crossing with RIPK1 kinase-dead mice [25]. Moreover, loss of single RIPK1 allele weakens cutaneous inflammation in both SHARPIN-deficient mice and inhibitor of apoptosis protein (IAP)-deleted mice, and it extends lifespan of the IAP-deficient mice significantly. In wild-type mice, toxic epidermal necrolysis-like inflammation is induced by subcutaneous injection of an IAP antagonist compound (SM), but the inflammation can be canceled in TNF receptor 1 (TNFR1)-deficient mice. The cancellation effect does not work in TRAIL- and TWEAK receptor-deficient mice, suggesting that SMs-induced skin inflammation depends on a TNFR1 but not on TRAIL or TWEAK [26]. On the other hand, RIPK1-deficient mice are lethal at birth because of systemic inflammation. In the neonates, expression of IL-33, which can be released as a processed and inflammatory form, was elevated in the skin and intestine [27]. These evidences suggest that reduced expression level of RIPK1 possibly induces both anti-inflammatory and pro-inflammatory effects depending on the situation. In this study, IL-1b, IL-17A, IL-22 and TRAIL reduced RIPK1expression in keratinocytes. These psoriasis-relating cytokines are possibly involved in a vicious circle. In the RIPK1 gene promoter, p53, NF-kappa-B1, Evi-1, N-Myc, Pax-6, Arnt, GR-alpha, GR and CUTL1 binding sites have been detected by Gene Cards1 (QIAGEN). Among these transcription factors, NF-kappa-B1 has been reported to be in the signaling pathways in downstream of IL-1b, IL-17A, IL22 and TRAIL, suggesting its contribution on regulating RIPK1expression under the stimuli of IL-1b, IL-17A, IL-22 and TRAIL. Psoriasis vulgaris is induced by dysregulation of immune reaction with complex feedback loops originating from antigen presenting cells, neutrophils and keratinocytes. Cytokines derived from Th17 cells activated in the downstream of DC-mediated TNFa and IL-23 have been considered as the main responsible factors and therapeutic targets for psoriasis treatment. RIPK1-expression was decreased in lesional and non-lesional epidermis of psoriasis (Fig. 1B, C). These data suggest that reduced RIPK1-expression in keratinocytes can be involved in initiation, exacerbation and persistence mechanism of psoriasis. In psoriasis, even normal skin contains abundant stores of T lymphocytes as well as resident populations of DCs. Xenografting experiment of non-lesional psoriatic skin to immunodeficient mice has verified that resident populations of T cells and DCs are sufficient to induce psoriasis [28]. We speculate that latently activated immune response associated with several cytokines, such as IL-1b, IL-17A, IL-22 and TRAIL, might decrease RIPK1 expression in non-lesional psoriatic epidermis as well as in lesional psoriatic epidermis. In addition, RIPK1-KD keratinocytes showed much higher sensitivity to TRAIL signaling. These results are consistent with the findings in psoriatic lesional skin: TRAIL released from dermal DCs enhances IL-1b, IL-6, IL-8, IL-20, IL-33 and TNF-a-expression in epidermal keratinocytes via downregulation of RIPK1-expression. Lesional skin of IMQ-induced psoriasis model mice also showed decreased expression of RIPK1. The RIPK1-expression of IMQ-model mice was
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gradually decreased by day 4, and the clinical symptoms such as erythema and scale emerged after three days of IMQ-treatment. These results indicate that RIPK1-downregulation in IMQ-model mice may amplify IMQ-induced psoriasis-like inflammation. Also, TRAIL neutralization suppressed psoriatic inflammation possibly by reduced production of TNF-a, which is a pro-inflammatory cytokine produced by macrophages, lymphocytes, keratinocytes and endothelial cells. TNF-a induces secondary mediators and adhesion molecules, all of which have been implicated in pathogenesis of psoriasis vulgaris [1]. Interestingly, TRAIL-neutralization resulted in upregulation of RIPK1-expression in the epidermis. Thus, TRAIL-neutralization antibody may modulate inflammation and suppress the production of cytokines that induce RIPK1 downregulation. In conclusion, we presented a novel modification mechanism of TRAIL-signaling by RIPK1 in epidermal keratinocytes. This mechanism can be involved in the pathogenesis of psoriasis, and may be a novel therapeutic target of psoriasis. Conflict of interest The authors state no conflict of interest. Acknowledgements The authors wish to thank Ms. K Nishikura, Ms. Y Nishinome and Ms. R Shinokawa for their technical expertise. This work was supported by JSPS KAKENHI Grant Number 15K19669 and 24591620, and the Lydia O‘leary Memorial Pias Dermatological Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jdermsci.2018.04.007. References [1] W.H. Boehncke, M.P. Schon, Psoriasis, Lancet 386 (2015) 983–994. [2] R. Lande, J. Gregorio, V. Facchinetti, B. Chatterjee, Y.H. Wang, et al., Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide, Nature 449 (2007) 564–569. [3] I. Kryczek, A.T. Bruce, J.E. Gudjonsson, A. Johnston, A. Aphale, et al., Induction of IL-17+ T cell trafficking and development by IFN-gamma: mechanism and pathological relevance in psoriasis, J. Immunol. 181 (2008) 4733–4741. [4] L.C. Zaba, J. Fuentes-Duculan, N.J. Eungdamrong, M.V. Abello, I. Novitskaya, et al., Psoriasis is characterized by accumulation of immunostimulatory and Th1/Th17 cell-polarizing myeloid dendritic cells, J. Invest. Dermatol. 129 (2009) 79–88. [5] K.E. Nograles, B. Davidovici, J.G. Krueger, New insights in the immunologic basis of psoriasis, Semin. Cutan. Med. Surg. 29 (2010) 3–9. [6] J.T. Elder, A.T. Bruce, J.E. Gudjonsson, A. Johnston, P.E. Stuart, et al., Molecular dissection of psoriasis: integrating genetics and biology, J. Invest. Dermatol. 130 (2010) 1213–1226. [7] A. Di Cesare, P. Di Meglio, F.O. Nestle, The IL-23/Th17 axis in the immunopathogenesis of psoriasis, J. Invest. Dermatol. 129 (2009) 1339–1350. [8] D. Ofengeim, J. Yuan, Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death, Nat. Rev. Mol. Cell Biol. 14 (2013) 727–736. [9] X.Y. Liu, F. Lai, X.G. Yan, C.C. Jiang, S.T. Guo, et al., RIP1 kinase is an oncogenic driver in melanoma, Cancer Res. 75 (2015) 1736–1748. [10] Y. Ito, D. Ofengeim, A. Najafov, S. Das, S. Saberi, et al., RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS, Science 353 (2016) 603–608. [11] C. Palacios, A.I. Lopez-Perez, A. Lopez-Rivas, Down-regulation of RIP expression by 17-dimethylaminoethylamino-17-demethoxygeldanamycin promotes TRAIL-induced apoptosis in breast tumor cells, Cancer Lett. 287 (2010) 207–215. [12] M. Dannappel, K. Vlantis, S. Kumari, A. Polykratis, C. Kim, et al., RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis, Nature 513 (2014) 90–94. [13] L. van der Fits, S. Mourits, J.S. Voerman, M. Kant, L. Boon, et al., Imiquimodinduced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL17 axis, J. Immunol. 182 (2009) 5836–5845. [14] L.C. Zaba, J. Fuentes-Duculan, N.J. Eungdamrong, L.M. Johnson-Huang, K.E. Nograles, et al., Identification of TNF-related apoptosis-inducing ligand and
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[22] S.R. Wiley, K. Schooley, P.J. Smolak, W.S. Din, C.P. Huang, et al., Identification and characterization of a new member of the TNF family that induces apoptosis, Immunity 3 (1995) 673–682. [23] K.M. Akram, N.J. Lomas, N.R. Forsyth, M.A. Spiteri, Alveolar epithelial cells in idiopathic pulmonary fibrosis display upregulation of TRAIL, DR4 and DR5 expression with simultaneous preferential over-expression of pro-apoptotic marker p53, Int. J. Clin. Exp. Pathol. 7 (2014) 552–564. [24] Y.Y. Huang, H. Liu, Y. Li, L.J. Pu, C.C. Jiang, et al., Down-regulation of RIP1 by 2deoxy-D-glucose sensitizes breast cancer cells to TRAIL-induced apoptosis, Eur. J. Pharmacol. 705 (2013) 26–34. [25] S.B. Berger, V. Kasparcova, S. Hoffman, B. Swift, L. Dare, et al., Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice, J. Immunol. 192 (2014) 5476–5480. [26] H. Anderton, J.A. Rickard, G.A. Varigos, N. Lalaoui, J. Silke, Inhibitor of apoptosis proteins (IAPs) limit RIPK1-Mediated skin inflammation, J. Invest. Dermatol. 137 (2017) 2371–2379. [27] J.A. Rickard, J.A. O'Donnell, J.M. Evans, N. Lalaoui, A.R. Poh, et al., RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis, Cell 157 (2014) 1175–1188. [28] M.A. Lowes, A.M. Bowcock, J.G. Krueger, Pathogenesis and therapy of psoriasis, Nature 445 (2007) 866–873.
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RIPK1 downregulation in keratinocyte enhances TRAIL signaling in psoriasis$ Nao Saito* , Masaru Honma, Takashi Shibuya, Shin Iinuma, Satomi Igawa, Mari Kishibe, Akemi Ishida-Yamamoto Department of Dermatology, Asahikawa Medical University, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 September 2017 Received in revised form 20 February 2018 Accepted 5 April 2018
Background: Psoriasis, a common inflammatory skin disorder characterized by scaly erythema and plaques, is induced by dysregulation of dendritic cell- and T cell-mediated immune reaction. Receptorinteracting protein kinase 1 (RIPK1) regulates inflammatory signaling in response to stimuli such as TNFa, TRAIL, and TLRs, resulting in apoptosis, necroptosis and NF-kB activation. However, the physiological relevance in human epidermis remains elusive. Objective: In this study, we examined whether RIPK1 is involved in the pathogenesis of psoriasis vulgaris. Methods: Skin samples of eight patients with psoriasis vulgaris were investigated by western blotting and immunohistochemistry. The functions of RIPK1 in keratinocytes were examined by RT-PCR and ELISA in vitro. TRAIL-neutralization-experiment was employed in an imiquimod-induced murine psoriasis model. Results: In lesional psoriatic epidermis, RIPK1-expression was decreased compared with that in normal epidermis. Cytokines involved in the pathomechanism of psoriasis, such as IL-1b, IL-17A, IL-22 and TRAIL, reduced RIPK1-expression in normal human epidermal keratinocytes (HEK) in vitro. In addition, RIPK1-knockdown enhanced TRAIL-mediated expression of psoriasis-relating cytokines, such as IL-1b, IL-6, IL-8, TNF-a, in HEK. Numerous TRAIL-positive cells were detected in the dermis of lesional psoriatic skin, and TRAIL receptors were expressed in psoriatic epidermis and HEK in conventional cultures. Moreover, TRAIL-neutralization in an imiquimod-induced murine psoriasis model remarkably improved skin phenotypes, such as ear thickness, and TNF-a expression in lesional skin. Conclusions: These results lead us to conclude that RIPK1-downregulation in keratinocytes increases their susceptibility to TRAIL stimulation, and plays a role in the pathogenesis of psoriasis vulgaris. © 2018 Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology.
Keywords: RIPK1 TRAIL Psoriasis vulgaris IMQ-model mice
1. Introduction Psoriasis, a common inflammatory skin disorder characterized by scaly erythemas and plaques, affects around 2% of the population [1]. Dysregulated interactions of innate and adaptive immunities are deeply involved in the pathomechanism. Complexes of epidermis-derived antimicrobial peptide, LL-37, and
Abbreviations: HEK, human epidermal keratinocytes; RIPK1, receptor-interacting protein kinase 1; RIPK1-KD-NHK, RIPK1 knockdown keratinocyte; Th17, T helper cell 17; TRAIL, TNF-related apoptosis-inducing ligand; TNF, tumor necrosis factor. $ Work was done in Asahikawa, Japan. * Corresponding author at: Department of Dermatology, Asahikawa Medical University, 2 1 1 1 Midorigaoka, Higashi, Asahikawa, Hokkaido, 078 8510, Japan. E-mail address: [email protected] (N. Saito).
host DNA are thought to act as an initiating factor via stimulating IFN-a-production from dermal plasmacytoid dendritic cells [2]. Consequently, activated myeloid dendritic cell (DC) populations release TNF-a and IL-23, and stimulate Th1 cells and T helper cell 17 (Th17) to produce IFN-g, IL-17 and IL-22 [3,4]. These cytokines induce proliferation of epidermal keratinocytes and mobilize immune cells into lesional skin [5]. In the exacerbating inflammatory chain, DC/Th17 axis is a major research focus as well as being the therapeutic target in the pathomechanism of psoriasis [6,7]. Receptor-interacting protein kinase 1 (RIPK1), one of the receptor-interacting serine/threonine-protein kinases, was originally identified as a death domain-containing kinase. It is a key regulator of inflammation, apoptosis and necroptosis downstream of tumor necrosis factor (TNF)-a, TRAIL, and Toll-like receptors (TLRs). Expression or function of RIPK1 is regulated in complex and intricate
https://doi.org/10.1016/j.jdermsci.2018.04.007 0923-1811/ © 2018 Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology.
Please cite this article in press as: N. Saito, et al., RIPK1 downregulation in keratinocyte enhances TRAIL signaling in psoriasis, J Dermatol Sci (2018), https://doi.org/10.1016/j.jdermsci.2018.04.007
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mechanisms including ubiquitylation, deubiquitylation, and phosphorylation [8]. Recent studies have shown that RIPK1 is also involved in oncogenicity of melanoma, axonal degeneration of amyotrophic lateral sclerosis, and apoptosis in breast cancer [9–11]. Moreover, epidermis-specific RIPK1-knockout mice developed significant inflammatory skin reaction characterized by thickened epidermis with various types of cell death [12]. Given this background, we examined whether RIPK1 is involved in the pathogenesis of psoriasis vulgaris and found that keratinocytes become highly sensitive to TRAIL signaling by RIPK1-downregulation.
2.6. ELISA Human IL-1b and human TNF-a ELISA on culture supernatants of keratinocytes was performed using ELISA kits from R&D systems in accordance with the manufacturer’s instructions. 2.7. Mice C57BL/6J female mice (six weeks old) were purchased from Oriental Yeast (Tokyo, Japan). All the animal experiments were performed with the approval of the ethical committee for animal studies of Asahikawa Medical University.
2. Materials and methods 2.8. IMQ-induced psoriasis model and Ab treatment 2.1. Patient samples A total of eight patients with psoriasis vulgaris at Asahikawa Medical University Hospital participated in this study. The collection of samples was performed with approval by the local ethical committee and the institutional review board of Asahikawa Medical University, and each patient gave written informed consent.
The skin of the back and the right ear of each mouse was treated daily for seven days with 62.5 mg and 31.25 mg of 5% IMQ cream (Beselna cream; Mochida Pharmaceutical, Tokyo, Japan). Vaseline petroleum jelly was used as control. In some experiments, mice received ip injections with 250 mg of anti-TRAIL (clone N2B2) or control rat IgG2a (Biolegend, San Diego, CA) per mouse before application of IMQ cream on days 0, 2, 4, and 6.
2.2. Keratinocyte culture and stimulation Primary keratinocytes were cultured in CnT-PR medium (CELLnTEC, Bern, Switzerland) at 37 C with 5% CO2, and used for the assay with no more than fourth passages. Recombinant human TRAIL (100 ng/ml, Peprotech, Rocky Hill, NJ), recombinant human IL-1b (R&D systems, Minneapolis, MN), recombinant human IL-17A (Peprotech), recombinant human IL-22 (Peprotech), recombinant human IFN-g (Peprotech), recombinant human TNFa (100 ng/ml, Peprotech) and recombinant LL37 (2.56 mM, 7.68 mM, Eurogentec, Seraing, Belgium) were added into the culture medium. 2.3. siRNA transfection Normal human keratinocytes (NHK) were transfected with siRNA of RIPK1 (40 pM) or si-control (40 pM) (Silencer1 Select Negative Control No.1 siRNA, Ambion, Austin, TX) using the electroporation system NucleofectionTM (Lonza, Basel Schweiz). At 72 h following transfection, cells were used in experiments.
2.9. Evaluation of skin inflammation severity and measurement of ear thickness To evaluate severity of mouse skin inflammation, an objective scoring system based on the clinical Psoriasis Area and Severity Index (PASI) was employed. Severity of erythema and scaling was rated independently as score 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, severe. The ear thickness was measured using a caliper (Peacock Ozaki MFG. CO., LTD., Tokyo, Japan). 2.10. Statistics In all figures, data are presented as mean SD from pooled data of at least 3 independent experiments. P values were calculated with one-way analysis of variance (post hoc Tukey or Dunnett) and two-tailed independent Student’s t-tests, and P < 0.05 was considered significant. 3. Results
2.4. Quantitative real-time PCR
3.1. Decreased RIPK1 expression in lesional epidermis of psoriasis
Total RNA extracted using RNeasy Kit (Qiagen, Hilden, Germany) was applied for quantitative real-time PCR (RT-PCR) performed by a LightCycler 480 Instrument (Roche, Basel Schweiz) or TaqMan gene expression assays (Applied Biosystems, Carlsbad, CA). Primer and probe sets used were TNFRSF10A (Hs00269492_m1), TNFRSF10 B (Hs00366278_m1), TNFRSF10D (Hs00174664_m1), GAPDH (Hs02758991_g1), mIL17A (Mm00439618_ m1), mIL17F (Mm00521423_ m1), mIL-22 (Mm01226722_ g1) and mGAPDH (Mm99999915_g1). For each data point, similar results were obtained at least in two independent experiments.
To clarify the function of RIPK1 in the epidermis, we first performed immunohistochemistry for RIPK1. In healthy control epidermis, atopic dermatitis and erythema multiforme, abundant RIPK1-expression was detected throughout whole layers of the epidermis. In contrast, it was minimal in psoriatic epidermis (Fig. 1A). Moreover, protein expression levels of RIPK1 was significantly decreased in lesional and non-lesional epidermis of psoriasis as compared with that in healthy control (Figs. 1B, C and S1). In lesional skin of IMQ-treated mice, an animal model recapitulating key features of psoriasis [13], qRT-PCR and western blotting revealed 80 to 97% decrease of RIPK1-expression in psoriasiform skin (Fig. 1D). To further clarify the regulatory mechanism, RIPK1-expession was analyzed in HEK treated with psoriasis-relating cytokines. IL-1b, IL-17A, IL-22, TRAIL, and IL17A + IL-22 significantly reduced RIPK1-expression in HEK (Fig. 1E). These cytokines are produced by immune cells relating to both innate and adaptive immunities, and they are deeply involved in the pathogenesis of psoriasis. In addition to IL-1b and Th17 cytokines, TRAIL is expressed in inflammatory DC in psoriatic lesions, and can directly act on keratinocytes [14]. RIPK1 mainly regulates inflammatory signaling relating to NF-kB, apoptosis, and
2.5. Immunohistochemistry Immunostaining on human and mouse skin was performed with antibodies against RIPK1 (Thermo Fisher Scientific, Waltham, MA), human CD4, CD8, CD11c (BD Biosciences, San Jose, CA), DR4, DR5, DcR2 (abcam, Cambridge, UK), and TRAIL (Cell signaling, Danvers, MA). Fluorescence staining was detected using a confocal laser scanning fluorescence microscope (Fluoview FV1000, Olympus, Tokyo, Japan).
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Fig. 1. Decreased RIPK1 expression in lesional skin of psoriasis vulgaris. (A) Immunohistochemistry analysis of RIPK1 in healthy control, atopic dermatitis, erythema multiforme and psoriasis vulgaris. Scale bar = 50 mm. (B) RIPK1 expression was evaluated using western blotting. The RIPK1 protein levels ware quantified using densitometry and expressed as the ratio of RIPK1 to b actin. N = 5 **p < 0.01 vs the control. (C) Immunohistochemistry analysis of RIPK1 in lesional and non-lesional skin for psoriasis vulgaris. Scale bar = 50 mm. (D) RIPK1 expression levels in imiquimod-treated mice were determined by RT-PCR and western blotting. N = 4 **p < 0.01 vs the control. (E) HEK were stimulated with psoriasis-related cytokines. RIPK1 expression levels were determined by RT-PCR. Similar results were obtained in two independent experiments. **p < 0.01 vs the control.
necroptosis under the stimuli of TNF, TRAIL, and TLRs. We hypothesized that the RIPK1-downregulation in keratinocyte enhances TRAIL signaling and contributes to exacerbation of psoriasis.
These results indicate that RIPK1-KD-keratinocyte is more sensitive to TRAIL signaling, which induces IL-1b, IL-6, IL-8, IL-20, IL-33 and TNF-a via NF-kB pathway. TRAIL did not induce any type of cell death in each group (Fig. S4).
3.2. RIPK1 downregulation in keratinocytes increased their susceptibility to TRAIL stimulation
3.3. TRAIL positive cells and TRAIL receptors were detected in psoriasis vulgaris
To verify our hypothesis, TRAIL was added to RIPK1-knockdown keratinocyte (RIPK1-KD-keratinocyte) in conventional culture conditions. RIPK1-expression was suppressed to 7 to 14% of control (Sic-NHK) in RIPK1-KD-keratinocytes (Fig. S2). TRAIL induced three to eight times higher mRNA levels of IL-1b, IL-6, IL-8, IL-20, IL-33 and TNF-a in RIPK1-KD keratinocytes (Fig. 2A). While mRNA-expression-levels of IL-20, IL-24 and IL-33 presented differences at the baseline, TRAIL-treatment did not significantly alter the expression levels. Protein levels of TNF-a and IL-1b were also increased in the culture supernatant of RIPK1-KD-keratinocytes without any difference in gene-expression at baseline (Figs. 2B and S3). An NF-kB inhibitor inhibited TRAIL-induced IL-6- and TNF-a-expression in RIPK1-KD-keratinocyte (Fig. 2C).
TRAIL, one of the TNF-superfamily members presenting potent cytotoxicity to tumor cells and transformed cells, is a transmembranous protein which can be shed by ADAM proteases. Four types of human TRAIL receptors, DR4, DR5, DcR2 and DcR1, have been reported. Among them, DcR1 works as an intrinsic antagonist against other TRAIL receptors [15]. Immunohistochemistry showed numerous TRAIL positive cells and abundant expression of TRAIL receptors, DR4 and DR5, in psoriasis lesional dermis and epidermis, respectively (Fig. 3A). DcR2-expression was not detected in either psoriasis lesions or in healthy controls. To clarify the regulatory mechanism of TRAIL receptors in the epidermis, HEK were cultured with psoriasis-relating cytokines and LL-37. LL-37 highly upregulated TRAIL receptor-expression, but
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Fig. 2. Expression of psoriasis-related cytokines in RIPK1-decreased keratinocytes. (A) Sic-NHK and RIPK1-KD-NHK were stimulated with TRAIL for 1, 3, and 6 h. After stimulation, the cytokine expression was determined by RT-PCR. Similar results were obtained in two independent experiments. *p < 0.05, **p < 0.01 vs the control. (B) Sic-NHK and RIPK1-KD-NHK were stimulated with TRAIL for 48 h. IL-1b and TNF-a concentrations in culture supernatant were determined by ELISA. Similar results were obtained in two independent experiments. *p < 0.05, **p < 0.01 vs the control. (C) Sic-NHK and RIPK1-KD-NHK were stimulated with TRAIL for 3 h in the presence or absence of NF-kB inhibitor BAY11-7082 (5 mM). The expression of IL-6 and TNF-a was determined by RT-PCR. Similar results were obtained in two independent experiments. **p < 0.01 vs the control.
not other cytokines (Fig. 3B, C). While TRAIL is expressed in a variety of immune cells in a stimulation dependent manner, TRAILpositive cells in psoriasis lesions expressed CD4+ or CD11c+ (Fig. 3D), suggesting a crucial role of CD4+ and CD11c+ cells in the pathogenesis of psoriasis. 3.4. Neutralization of TRAIL inhibits development of psoriasis-like dermatitis in IMQ-model mice Next, we examined the correlation between RIPK1 and TRAILpositive cells in an IMQ-induced psoriasis model. In IMQ-model mice, numerous TRAIL-positive cells were detected in the dermis, and the TRAIL receptor, DR5, was highly expressed in the epidermis as compared with that in the control (Fig. 4A, B). Inversely, RIPK1expression in the epidermis was decreased similarly to that in psoriatic epidermis (Figs. 1D and S5). To confirm involvement of TRAILsignaling in development of psoriatic lesions, a TRAIL-neutralizationexperiment was employed. Intraperitoneal injection of TRAIL neutralization antibody every other day substantially suppressed the skin phenotype of IMQ-treated mice, such as erythema, infiltration and scales, with histopathological improvement of acanthosis,
hyperkeratosis, and parakeratosis (Figs. 5 A, C and S6). Moreover, TRAIL neutralization antibody canceled inhibition of RIPK1-expression in epidermis of IMQ-treated mouse (Figs. 5A and S7, S8), and ear swelling and thickness of the back skin were also suppressed significantly (Figs. 5B and S9). None of cell death type was detected in epidermal keratinocytes of each mouse group (Figs. S10, 11, 12). These favorable changes resulting from TRAIL neutralization were accompanied by a decrease of TNF-a mRNA (Fig. 5D). Collectively, these results indicate that TRAIL signaling is likely to be involved in the exacerbation of psoriasis lesions through RIPK1- downregulation. 4. Discussion In this study, we found that RIPK1 downregulation in keratinocytes increased their susceptibility to TRAIL stimulation. TRAIL, the best known apoptosis-inducer [16], is a member of the TNF superfamily that can induce tumor-selective cell death. In allergic conditions, it shows bidirectional functions, such as inflammatory role on epithelia in asthma [17] and anti-inflammatory role in atopic dermatitis [18]. In the pathogenesis of psoriasis, it has been reported as a factor presenting an inflammatory role
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Fig. 3. Lesional skin of psoriasis vulgaris expressed TRAIL receptors and TRAIL positive cells. (A) Immunohistochemistry analysis of TRAIL receptors and TRAIL positive cells in psoriasis vulgaris and healthy control. Scale bar = 50 mm. (B) DR4 and DR5 expression was evaluated using western blotting in keratinocytes stimulated with LL37 (2.56 mM). The DR4 and DR5 protein levels were quantified using densitometry and expressed as the ratio of DR4 and DR5 to b actin. N = 4. (C) Cultured human keratinocytes were stimulated with psoriasis-related cytokines and LL37. DR4, DR5 and DcR2 expression levels were determined by RT-PCR. Similar results were obtained in two independent experiments. **p < 0.01 vs the control. (D) Skin sections obtained from psoriasis vulgaris were stained with antibodies against CD4, CD8, CD11c (green) and TRAIL (red). Scale bar = 50 mm.
Fig. 4. TRAIL positive cells and TRAIL receptors in IMQ-model mice. (A) Phenotypical presentation of mouse back skin after 7 days of treatment. (B) H&E staining of the mice back skin and immunohistochemistry analysis of TRAIL positive cells and TRAIL receptor in mice. Scale bar = 50 mm.
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Fig. 5. TRAIL neutralization antibody inhibits psoriasis vulgaris in IMQ-model mice. (A) Mice were treated with IMQ and control antibody or TRAIL neutralization antibody. For each group, n = 4 (left panel). H&E staining (middle panel) and immunohistochemistry analysis of RIPK1 in mice (right panel). Scale bar = 50 mm. (B) Ear swelling of the right ear was measured: IMQ with a control antibody (circles); IMQ and TRAIL neutralization antibody (squares) (n = 4). *p < 0.05, **p < 0.01 vs the control. (C) Erythema and scale of the back skin were scored: IMQ with a control antibody (circles); IMQ and TRAIL neutralization antibody (squares) (n = 4). (D) Cytokine expression of mice skin was determined by RT-PCR. Similar results were obtained in two independent experiments. *p < 0.05, **p < 0.01 vs the control.
[14,19]. TRAIL expressed on CD11c+CD1c inflammatory DCs in psoriatic lesions can induce epidermal keratinocytes to express CCL20, which recruits CCR6-positive Th17 cells into the lesion. TRAIL can also stimulate NF-kB and MAP kinase signaling pathways depending on RIPK1 [20]. A recent report presented that epidermis-specific inhibition of NF-kB in mice induces psoriasis-like skin inflammation involving increased expression of IL-24 and activated STAT3 signaling via TNF receptor 1-induced and MAP kinase-dependent mechanism [21]. In this study, RIPK1-downregulation in epidermal keratinocytes enhanced TRAIL-mediated expression of psoriasis-relating cytokines, such as IL-1b, IL-6, IL-8, IL-20, IL-33 and TNF-a, suggesting a crucial role of NF-kB pathway on enhanced expression of these cytokines. Moreover, expression of TRAIL receptors can be detected more strongly in both lesional and non-lesional epidermis of psoriasis vulgaris compared with healthy epidermis [14]. Therefore, TRAILsignaling through TRAIL receptors can act as an important regulator of the immune responses. TRAIL can also induce apoptosis in tumor cells but not normal cells depending on contrastive expression and activation levels of TRAIL receptors [22]. In idiopathic pulmonary fibrosis, upregulated expression of TRAIL receptors contributes to susceptibility to TRAIL signaling
[23]. In this study, we have newly identified LL-37 as an inducer of TRAIL receptor expression. This finding strongly supports involvement of TRAIL signaling in psoriasis. A complex of LL-37 and host DNA can stimulate dermal plasmacytoid DCs to produce IFN-a at the disease onset and during the exacerbation stage [2]. This upregulation system of TRAIL receptors via LL-37 can contribute the pathomechanism at an initiating stage of psoriasis. RIPK1 was originally identified as a death domain-containing kinase bound to intracellular death domains of activated FAS and TNF receptor 1 via ligand stimulation. RIPK1 also modulates the inflammatory response, apoptosis and necroptosis mediated by TRAIL, Fas, LPS and polyIC. RIPK1 is consisted of three independent domains, an amino-terminal kinase domain, an intermediate domain and a carboxy-terminal death domain. The intermediate domain engaging RIPK1 to mediate cell survival via NF-kB pathway is subjected to Lys63-linked ubiquitylation at Lys377. The N-terminal kinase domain relates to necrosis-specific RIPK1 phosphorylation which stabilizes binding of RIPK1 and RIPK3 in pro-necrotic complex, resulting in upregulation of pro-necrotic kinase activity and inducing downstream reactive oxygen species production [8]. MTT assay did not show any type of cell death in Sic-NHK and RIPK1-KD-NHK by TRAIL-treatment (Fig. S4). In
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addition, expression level of phospho-RIPK3 was minimal in IMQtreated mouse epidermis with or without TRAIL-neutralization (Fig. S12). Collectively, we conclude that total amount of RIPK1 is more important than its phosphorylation level. Downregulation of RIPK1 can increase sensitivity to TRAIL-induced apoptosis in cancer cells which were originally resistant to TRAIL-signals [24]. Thus, TRAIL is considered to control multiple cellular signaling pathways relating to inflammation and cell death process. Regarding regulatory effects on various inflammation, RIPK1 can work in diverse ways. Reduced RIPK1 levels or function can contribute to suppression of inflammation. SHARPIN-deficient mice, in which no NF-kB activation is detectable, develop a TNFdependent multi-organ inflammation, mainly relating to apoptosis and necroptosis, and the TNF-driven inflammation can be prevented by crossing with RIPK1 kinase-dead mice [25]. Moreover, loss of single RIPK1 allele weakens cutaneous inflammation in both SHARPIN-deficient mice and inhibitor of apoptosis protein (IAP)-deleted mice, and it extends lifespan of the IAP-deficient mice significantly. In wild-type mice, toxic epidermal necrolysis-like inflammation is induced by subcutaneous injection of an IAP antagonist compound (SM), but the inflammation can be canceled in TNF receptor 1 (TNFR1)-deficient mice. The cancellation effect does not work in TRAIL- and TWEAK receptor-deficient mice, suggesting that SMs-induced skin inflammation depends on a TNFR1 but not on TRAIL or TWEAK [26]. On the other hand, RIPK1-deficient mice are lethal at birth because of systemic inflammation. In the neonates, expression of IL-33, which can be released as a processed and inflammatory form, was elevated in the skin and intestine [27]. These evidences suggest that reduced expression level of RIPK1 possibly induces both anti-inflammatory and pro-inflammatory effects depending on the situation. In this study, IL-1b, IL-17A, IL-22 and TRAIL reduced RIPK1expression in keratinocytes. These psoriasis-relating cytokines are possibly involved in a vicious circle. In the RIPK1 gene promoter, p53, NF-kappa-B1, Evi-1, N-Myc, Pax-6, Arnt, GR-alpha, GR and CUTL1 binding sites have been detected by Gene Cards1 (QIAGEN). Among these transcription factors, NF-kappa-B1 has been reported to be in the signaling pathways in downstream of IL-1b, IL-17A, IL22 and TRAIL, suggesting its contribution on regulating RIPK1expression under the stimuli of IL-1b, IL-17A, IL-22 and TRAIL. Psoriasis vulgaris is induced by dysregulation of immune reaction with complex feedback loops originating from antigen presenting cells, neutrophils and keratinocytes. Cytokines derived from Th17 cells activated in the downstream of DC-mediated TNFa and IL-23 have been considered as the main responsible factors and therapeutic targets for psoriasis treatment. RIPK1-expression was decreased in lesional and non-lesional epidermis of psoriasis (Fig. 1B, C). These data suggest that reduced RIPK1-expression in keratinocytes can be involved in initiation, exacerbation and persistence mechanism of psoriasis. In psoriasis, even normal skin contains abundant stores of T lymphocytes as well as resident populations of DCs. Xenografting experiment of non-lesional psoriatic skin to immunodeficient mice has verified that resident populations of T cells and DCs are sufficient to induce psoriasis [28]. We speculate that latently activated immune response associated with several cytokines, such as IL-1b, IL-17A, IL-22 and TRAIL, might decrease RIPK1 expression in non-lesional psoriatic epidermis as well as in lesional psoriatic epidermis. In addition, RIPK1-KD keratinocytes showed much higher sensitivity to TRAIL signaling. These results are consistent with the findings in psoriatic lesional skin: TRAIL released from dermal DCs enhances IL-1b, IL-6, IL-8, IL-20, IL-33 and TNF-a-expression in epidermal keratinocytes via downregulation of RIPK1-expression. Lesional skin of IMQ-induced psoriasis model mice also showed decreased expression of RIPK1. The RIPK1-expression of IMQ-model mice was
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gradually decreased by day 4, and the clinical symptoms such as erythema and scale emerged after three days of IMQ-treatment. These results indicate that RIPK1-downregulation in IMQ-model mice may amplify IMQ-induced psoriasis-like inflammation. Also, TRAIL neutralization suppressed psoriatic inflammation possibly by reduced production of TNF-a, which is a pro-inflammatory cytokine produced by macrophages, lymphocytes, keratinocytes and endothelial cells. TNF-a induces secondary mediators and adhesion molecules, all of which have been implicated in pathogenesis of psoriasis vulgaris [1]. Interestingly, TRAIL-neutralization resulted in upregulation of RIPK1-expression in the epidermis. Thus, TRAIL-neutralization antibody may modulate inflammation and suppress the production of cytokines that induce RIPK1 downregulation. In conclusion, we presented a novel modification mechanism of TRAIL-signaling by RIPK1 in epidermal keratinocytes. This mechanism can be involved in the pathogenesis of psoriasis, and may be a novel therapeutic target of psoriasis. Conflict of interest The authors state no conflict of interest. Acknowledgements The authors wish to thank Ms. K Nishikura, Ms. Y Nishinome and Ms. R Shinokawa for their technical expertise. This work was supported by JSPS KAKENHI Grant Number 15K19669 and 24591620, and the Lydia O‘leary Memorial Pias Dermatological Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jdermsci.2018.04.007. References [1] W.H. Boehncke, M.P. Schon, Psoriasis, Lancet 386 (2015) 983–994. [2] R. Lande, J. Gregorio, V. Facchinetti, B. Chatterjee, Y.H. Wang, et al., Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide, Nature 449 (2007) 564–569. [3] I. Kryczek, A.T. Bruce, J.E. Gudjonsson, A. Johnston, A. Aphale, et al., Induction of IL-17+ T cell trafficking and development by IFN-gamma: mechanism and pathological relevance in psoriasis, J. Immunol. 181 (2008) 4733–4741. [4] L.C. Zaba, J. Fuentes-Duculan, N.J. Eungdamrong, M.V. Abello, I. Novitskaya, et al., Psoriasis is characterized by accumulation of immunostimulatory and Th1/Th17 cell-polarizing myeloid dendritic cells, J. Invest. Dermatol. 129 (2009) 79–88. [5] K.E. Nograles, B. Davidovici, J.G. Krueger, New insights in the immunologic basis of psoriasis, Semin. Cutan. Med. Surg. 29 (2010) 3–9. [6] J.T. Elder, A.T. Bruce, J.E. Gudjonsson, A. Johnston, P.E. Stuart, et al., Molecular dissection of psoriasis: integrating genetics and biology, J. Invest. Dermatol. 130 (2010) 1213–1226. [7] A. Di Cesare, P. Di Meglio, F.O. Nestle, The IL-23/Th17 axis in the immunopathogenesis of psoriasis, J. Invest. Dermatol. 129 (2009) 1339–1350. [8] D. Ofengeim, J. Yuan, Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death, Nat. Rev. Mol. Cell Biol. 14 (2013) 727–736. [9] X.Y. Liu, F. Lai, X.G. Yan, C.C. Jiang, S.T. Guo, et al., RIP1 kinase is an oncogenic driver in melanoma, Cancer Res. 75 (2015) 1736–1748. [10] Y. Ito, D. Ofengeim, A. Najafov, S. Das, S. Saberi, et al., RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS, Science 353 (2016) 603–608. [11] C. Palacios, A.I. Lopez-Perez, A. Lopez-Rivas, Down-regulation of RIP expression by 17-dimethylaminoethylamino-17-demethoxygeldanamycin promotes TRAIL-induced apoptosis in breast tumor cells, Cancer Lett. 287 (2010) 207–215. [12] M. Dannappel, K. Vlantis, S. Kumari, A. Polykratis, C. Kim, et al., RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis, Nature 513 (2014) 90–94. [13] L. van der Fits, S. Mourits, J.S. Voerman, M. Kant, L. Boon, et al., Imiquimodinduced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL17 axis, J. Immunol. 182 (2009) 5836–5845. [14] L.C. Zaba, J. Fuentes-Duculan, N.J. Eungdamrong, L.M. Johnson-Huang, K.E. Nograles, et al., Identification of TNF-related apoptosis-inducing ligand and
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Please cite this article in press as: N. Saito, et al., RIPK1 downregulation in keratinocyte enhances TRAIL signaling in psoriasis, J Dermatol Sci (2018), https://doi.org/10.1016/j.jdermsci.2018.04.007