Cholecystokinin Downregulates Psoriatic Inflammation by Its Possible Self-Regulatory Effect on Epidermal Keratinocytes

This information is current as Atsuko Funakoshi, Kazuki Tatsuno, Takatoshi Shimauchi, of September 23, 2021. Toshiharu Fujiyama, Taisuke Ito and Yoshiki Tokura J Immunol published online 22 March 2019 http://www.jimmunol.org/content/early/2019/03/21/jimmun ol.1801426 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 22, 2019, doi:10.4049/jimmunol.1801426 The Journal of Immunology

Cholecystokinin Downregulates Psoriatic Inﬂammation by Its Possible Self-Regulatory Effect on Epidermal Keratinocytes

Atsuko Funakoshi, Kazuki Tatsuno, Takatoshi Shimauchi, Toshiharu Fujiyama, Taisuke Ito, and Yoshiki Tokura

Cholecystokinin (CCK) is a peptide hormone that functions in digestive organs and the CNS. We previously showed that CCK downregulates peripheral pruritus by suppressing degranulation of mast cells. In this study, we demonstrated that CCK octapeptide (CCK8) was constitutively expressed in the epidermis of normal skin, whereas its expression was lost in acanthotic lesions of psoriasis. In contrast, CCKA receptor (CCKAR), a high-afﬁnity receptor for CCK, was constitutively expressed in the epidermis of psoriatic skin lesions. Expression of CCK was also reduced in skin lesions of an imiquimod (IMQ)-induced psoriatic mouse model. Notably, the expression level of CCK inversely correlated with the severity of epidermal inﬂammation, raising the possibility that

CCK from epidermal keratinocytes suppresses the psoriatic inflammation. To verify this hypothesis, we investigated the effects of Downloaded from sulfated CCK octapeptide (CCK8S) on the development of IMQ-induced psoriatic inflammation. i.p. injection of CCK8S suppressed the IMQ-induced psoriatic inflammation accompanied by reduced mRNA expression of IL-17, IL-22, and IL-6 but not of IL-23. The suppressive effect of CCK8S was completely restored by administration of CCKAR antagonist. In vitro studies showed that exogenous CCK8S suppressed IL-6 production in CCKAR-expressing cultured human keratinocytes, and blocking the endogenous CCK signaling with CCKAR antagonist markedly enhanced IL-6 production. When keratinocytes were stimulated with IL-17, the

expression of endogenous CCK was significantly decreased. These findings suggest that CCK physiologically functions as a negative http://www.jimmunol.org/ regulator of keratinocyte-based inflammation in an autocrine or paracrine manner, although decreased CCK may pathologically contribute to continuous and aggravated skin lesions such as psoriasis. The Journal of Immunology, 2019, 202: 000–000.

soriasis is a chronic inflammatory skin disease mediated (7, 8). Inflammatory DCs play an indispensable role in the path- by immunocompetent cells and nonhematopoietic cells ogenesis of psoriasis with their released IL-23, thereby main- P like keratinocytes. Th17 cells play a central role in the taining Th17 cells, whereas plasmacytoid DCs are stimulated pathogenesis of psoriasis, and Th17 cell–derived IL-17A and via TLRs and trigger psoriasis lesions by releasing IFN-a (1, 9). A IL-22 induce proliferation and activation of epidermal keratino- mouse model of psoriasis-like epidermal inflammation induced by by guest on September 23, 2021 cytes, leading to epidermal hyperplasia (acanthosis) and para- imiquimod (IMQ) has been widely used to investigate the path- keratosis (1, 2). In mice, gd T cells replace Th17 cells as IL-17A ogenesis of psoriasis. Topical application of IMQ, a synthetic producers (3, 4). Activated keratinocytes produce various cyto- ligand for TLR7, onto mouse skin activates plasmacytoid DCs and kines and chemokines, such as IL-1, IL-6, IL-8, CXCL10, CCL20, induces epidermal inflammation with histological changes con- and vascular endothelial growth factor, which promote cytokine siderably similar to human psoriasis (10–13). production and skin recruitment of T cells, neutrophils, and Cholecystokinin (CCK) is largely expressed in the CNS and dendritic cells (DCs) (5). Compelling evidence has been accu- small intestine, where it serves as a peptide hormone with specific mulated that IL-6 is associated with pathogenesis of psoriasis functions (14). CCK undergoes tissue-specific posttranscriptional (6–8). IL-6 promotes Th17 cell function by downregulating de- processing: brain makes mainly CCK8, whereas CCK12, 22, 33, velopment of regulatory T cells and induces proliferation of and 58 are found more often in the gastrointestinal tract. Another keratinocytes, resulting in continuous psoriatic inflammation important posttranscriptional modification is sulfation of the ty- rosine residue (14, 15). Recent studies have highlighted that CCK plays an immunomodulatory role by affecting immunocompe- Department of Dermatology, Hamamatsu University School of Medicine, Hama- matsu 431-3192, Japan tent cells such as T cells, B cells, macrophages, and DCs (16–19). ORCIDs: 0000-0002-0369-869X (T.S.); 0000-0002-9274-7050 (T.I.). In vitro studies have shown that sulfated CCK octapeptide Received for publication October 23, 2018. Accepted for publication February 27, (CCK8S), biologically active CCK, inhibits IgG1 production in 2019. LPS-stimulated B cells and differentiation of Th1 and Th17 cells, This work was supported by Grants-in-Aid for Scientific Research (18K16025 and whereas it enhances differentiation of regulatory T cells (16, 17). 25293243) from the Ministry of Education, Culture, Sports, Science and Technology It is noteworthy that CCK suppresses development of collagen- and a grant from Otsuka Pharmaceutical. induced arthritis by inhibiting differentiation of Th17 cells (18, Address correspondence and reprint requests to Dr. Atsuko Funakoshi, Department of 20). These findings indicate that CCK plays downregulatory roles in Dermatology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan. E-mail address: [email protected] immune-mediated inflammatory responses. To assess the function The online version of this article contains supplemental material. of CCK in the skin, we investigated the effect of CCK on peripheral Abbreviations used in this article: AEC, 3-amino-9-ethylcarbazole; CCK, cholecys- pruritus in mice (21). We found that CCK8S exerts an antipruritic tokinin; CCKAR, CCKA receptor; CCKBR, CCKB receptor; CCK8S, sulfated CCK activity by suppressing degranulation of mast cells. However, a role octapeptide; DC, dendritic cell; IMQ, imiquimod; NHEK, normal human epidermal of CCK in T cell–mediated skin diseases has not been elucidated. keratinocyte. Two types of CCK receptors, CCKA receptor (CCKAR) and Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 CCKB receptor (CCKBR), have been characterized (15). CCKAR

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801426 2 DOWNREGULATORY ROLE OF CHOLECYSTOKININ IN PSORIASIS serves as a high-affinity receptor for CCK8S, whereas nonsulfated into mice according to the experimental protocol described in Figs. 5A CCK8 binds to CCKAR with much lower affinity. CCK33, 39, and and 8A. Ear thickness was sequentially measured using a micrometer 58 bind to CCKAR with similar affinity to CCK8 (15). Gastrin is (Mitutoyo, Kanagawa, Japan). The net increase in ear thickness was calculated by subtracting the ear thickness measured before IMQ treatment. Ears considered to be a major physiological ligand of CCKBR and were collected at the indicated time points and stored in RNAlater Stabi- is mainly expressed in the gastrointestinal tract. The expression lization Solution (Thermo Fisher Scientific, Waltham, MA) until RNA and functional role of CCK receptors in immunocompetent cells purification for real-time PCR analysis. Experiments were performed with have been investigated. CD4+ T cells express both CCKAR and at least five mice per group. CCKBR, whereas CCK8S suppresses IgG1 production of B cells Immunohistochemistry through CCKBR (16, 17). We found that fetal skin-derived mast Human skin and mouse ear samples were fixed in 10% buffered formalin cells expressed both CCKAR and CCKBR (21). Because the ex- and embedded in paraffin. Sections were deparaffinized and stained with pression of CCKBR on mast cells is upregulated by substance P, it H&E. For immunostaining of CCK and CCKAR, deparaffinized sections is more likely that CCK8S suppresses degranulation of mast cells were stained with anti-CCK8 Ab (ab27441; Abcam) (22, 23) overnight at via CCKBR, resulting in reduction of peripheral pruritus. 4˚C or with anti-CCKAR Ab (Bioss, Woburn, MA) for 1 h at room tem- Our preliminary study of the expression of CCK in the epidermis perature. Immunoreactivity was detected with Histofine Simple Stain Max PO (Nichirei Biosciences, Tokyo, Japan). Color was developed by using of various skin diseases showed that CCK was decreased in pso- 3-amino-9-ethylcarbazole (AEC) as a substrate. Percentages of CCK8+ epi- riasis, a representative Th17-mediated skin disorder. In this study, dermal keratinocytes were determined by Patholoscope image analysis soft- we investigated the expression and role of CCK in psoriasis by ware (MITANI Corporation, Tokyo, Japan). Concerning the specificity of the using an IMQ-induced psoriatic epidermal inflammation model. anti-CCK8 Ab, two images of normal human skin stained with rabbit anti- CCK8 Ab or rabbit IgG isotype control Ab are shown in Supplemental Fig. 1. Results suggest that CCK downregulates the development of IMQ- Downloaded from induced epidermal inflammation. CCK inhibits keratinocyte IL-6 Cell culture and cytokine measurement production and Th17 cytokine production. We propose a novel Normal human epidermal keratinocytes (NHEKs) obtained from Kurabo role of CCK in the pathogenesis of T cell–mediated skin disease. (Osaka, Japan) were maintained in EpiLife medium (Thermo Fisher Sci- entific) with a culture additive (Thermo Fisher Scientific). A subconfluent monolayer of NHEKs was washed with PBS and treated with CCK8S Materials and Methods (10 nM) for 2 h in EpiLife medium without any other growth factors, followed Skin samples by stimulation with IMQ (10 mM) or IL-17 (20 ng/ml). In some experi- http://www.jimmunol.org/ ments, NHEKs were pretreated with lorglumide (10 mM) for 30 min before Skin biopsy samples obtained from patients with psoriasis vulgaris or the treatment with CCK8S. To investigate the effect of CCKAR or CCKBR atopic dermatitis and normal subjects were used for immunohistochemical blockade on IL-6 production from unstimulated NHEKs, NHEKs were stainings. All the patients and normal subjects were seen at the Department cultured with indicated concentrations of lorglumide or YM022 (Sigma, of Dermatology, Hamamatsu University School of Medicine. The experi- St. Louis, MO). After 24 h, the culture supernatant was collected, and the mental protocol for human skin sample collections was conducted in ac- amount of IL-6 was measured using Human IL-6 ELISA Max Deluxe cordance with the Declaration of Helsinki principles and was approved by (BioLegend, San Diego, CA) according to the manufacturer’s instructions. the medical ethics committees of Hamamatsu University School of Med- For real-time PCR analysis of CCK mRNA, NHEKs were stimulated with icine (No. 17-188). Written informed consent was obtained from the pa- IL-17 (20 ng/ml) for 24 h. Total RNA was purified by Isogen II (Nippon tients, and if not, the opt-outs for the protocol were also opened to the

Gene, Tokyo, Japan) according to the manufacturer’s instructions. by guest on September 23, 2021 general public online at Hamamatsu University School of Medicine. Flow cytometric analysis Mice For analysis of CCK receptor expression, NHEKs were stained with anti- Female C57BL/6 mice were obtained from Japan SLC (Shizuoka, Japan). CCKAR Ab (Bioss) or anti-CCKBR Ab (Alamone Labs, Jerusalem, Israel) Mice were rested for at least 1 wk before use in the Laboratory Animal for 1 h on ice, followed by incubation with Alexa Fluor 647–conjugated Facilities & Services of Hamamatsu University School of Medicine and anti-rabbit IgG (BioLegend) for 30 min on ice. After incubation with were used at the age of 8–10 wk. Experiments were performed according 7AAD (BD Biosciences, San Diego, CA) for 10 min, expression of CCK to the guidelines of Hamamatsu University School of Medicine. receptors on 7AAD2 live cells was determined using a FACSCanto II (BD Biosciences). Data were analyzed by FlowJo software (BD Biosciences). Induction of IMQ-induced psoriasis-like epidermal inflammation Real-time PCR analysis Mice received a topical application of 5% IMQ cream (4.17 mg IMQ/ Total RNA was extracted by homogenizing ears in TRIzol reagent (Thermo mouse; Mochida Pharmaceuticals, Tokyo, Japan) onto both sides of the Fisher Scientific) and purified using the PureLink RNA Mini Kit (Thermo ears daily for 6 consecutive d. Two hundred microliters of 150 mM CCK8S Fisher Scientific) according to the manufacturer’s protocol. Total RNA (1.4 mg/kg; Peptide Institute, Osaka, Japan) and 100 mg/ml lorglumide purified from ears and NHEKs was reverse-transcribed using TaqMan (0.8 mg/kg; Abcam, Cambridge, U.K.) or vehicle (saline) was i.p. injected Reverse Transcription Reagents (Thermo Fisher Scientific) and PrimeScript

FIGURE 1. Expression of CCK8 is impaired in skin lesions of psoriasis. Immunostaining for CCK8 in normal skin and lesional skin obtained from psoriasis and atopic dermatitis patients (upper panels, low-power ﬁeld; lower panels, high-power ﬁeld). Color was developed by using AEC as a substrate. Arrows indicate perinuclear positive (brown) staining for CCK8 in keratinocytes. Images are representative of four to six individuals. The Journal of Immunology 3

might be decreased in certain inﬂammatory skin diseases. Our study of the expression of CCK by epidermal keratinocytes in various disorders revealed that psoriasis represents the CCK8- reduced disease. By immunohistochemistry, we evaluated CCK8 and CCKAR expressions in skin biopsy specimens of psoriasis (n = 6) and atopic dermatitis (n = 4) along with normal skin of healthy subjects (n = 4). All specimens in each group showed virtually the same staining pattern. As represented by Fig. 1, CCK8 was expressed in epidermal keratinocytes of normal individuals, as perinuclear staining was observed in keratinocytes of the prickle layer. No positive staining was observed when normal skin was stained with rabbit IgG isotype control Ab (Supplemental Fig. 1). In contrast, CCK8 expression was completely lost in acanthotic lesions of psoriasis. In atopic dermatitis, chronic acanthotic lesions showed moderately positive intracytoplasmic staining for CCK8. Quantitative analysis of the images conﬁrmed that CCK8+ keratinocytes were abundant in normal and atopic dermatitis skin, whereas they were completely lost in psoriatic

skin lesions (one-way ANOVA, F =34.3,p , 0.001; Fig. 2). As Downloaded from represented by two cases of psoriasis (Fig. 3), CCK8 was expressed in the nonlesional skin of psoriasis at a comparable + FIGURE 2. Quantitative analysis of skin CCK8. Percentages of CCK8 level to that in normal skin, whereas it was absent in the acan- keratinocytes in normal skin and lesional skin obtained from psoriasis thotic lesional skin. or atopic dermatitis patients were determined using Patholoscope image We also examined CCKAR expression and observed that analysis software. At least 300 keratinocytes were counted to determine percentages of CCK8+ keratinocytes. **p , 0.01. CCKAR was constitutively expressed in the epidermis of both http://www.jimmunol.org/ nonlesional and lesional skin of psoriasis, as its positive staining was accentuated by the basal and upper prickle layer cells (Fig. 3). RT Reagent Kit with Genomic DNA Eraser (TaKaRa, Shiga, Japan), respectively. Real-time PCR analysis was carried out to compare the levels of Thus, although CCK8 expression was decreased, its receptor’s gene expression using Thermal Cycler Dice Real Time System II (TaKaRa). expression was retained in the lesional skin of psoriasis. TaqMan probes obtained from Thermo Fisher Scientific were as follows: human GAPDH, Hs99999905_m1; human CCK, Hs00174937_m1; mouse Increased keratinocyte CCK expression is associated with GAPDH, Mm99999915_g1; mouse IL-6, Mm00446190_m1; mouse regression of IMQ-induced psoriatic mouse model IL-17A, Mm00439618_m1; mouse IL-22, Mm01226722_g1; mouse IL-23a, The above immunohistochemical finding raised the possibility that Mm00518984_m1; and mouse CCK, Mm00446170_m1. CCK from epidermal keratinocytes suppresses the psoriatic in- by guest on September 23, 2021 Statistical analysis flammation and CCK reduction allows the psoriatic lesion to All statistical analysis was carried out using SPSS Statistics version 25 occur. To verify this hypothesis, we employed an IMQ-induced (IBM, Armonk, NY). To determine statistical significance, ANOVA was psoriatic epidermal inflammation model. IMQ has been shown performed, followed by the Bonferroni test for multiple comparisons. Two- to induce skin inflammation with various characteristics of tailed Student t tests were used to compare means between two groups. The psoriasis through TLR7 on skin-resident cells such as keratinocytes level of significance was set at p , 0.05. and plasmacytoid DCs (10–13). IMQ was applied onto mouse ears daily for 6 consecutive d to induce psoriatic epidermal in- Results flammation, and CCK mRNA expression in the lesional skin was Expression of CCK is decreased in psoriasis skin lesions measured by real-time PCR analysis. We found a statistically sig- Because topical CCK has an antipruritic, anti-inflammatory action nificant effect of IMQ in the expression of CCK mRNA in ears in a mouse model (21), it was hypothesized that CCK expression (one-way ANOVA, F = 23.94, p , 0.001). The expression level of

FIGURE 3. Expression of CCK8 but not CCKAR is impaired in skin lesions of psoriasis. Immunostaining of nonlesional skin and lesions of psoriasis for CCK8 (A) and CCKAR (B). Color was developed by using AEC as a substrate. Arrows in high-power ﬁeld images of Case 1 (lower panels) indicate positive staining of CCK8 (A) and CCKAR (B) in keratinocytes. Images are representative of four to six individuals. 4 DOWNREGULATORY ROLE OF CHOLECYSTOKININ IN PSORIASIS

immune cells (Fig. 6). We found that CCK8S significantly suppressed epidermal inflammation as assessed by ear thickness (two-way ANOVA, F = 100, p , 0.001; Fig. 5B) and histopa- thology (Fig. 6, upper panel). Immunohistochemical analysis of skin lesions revealed that epidermal keratinocytes continuously expressed CCKAR, a high-affinity receptor to CCK8S, suggesting that keratinocytes are a possible target of exogenous CCK8S (Fig. 6, middle and lower panels). Various types of cells, such as T cells, DCs, and keratinocytes, contribute to the development of IMQ-induced psoriatic changes by releasing cytokines at the local tissue (11–13). We therefore conducted real-time PCR analysis to evaluate the effect of CCK8S on cytokine production in the IMQ-induced psoriatic skin lesion. FIGURE 4. Expression of local CCK mRNA is downregulated by IMQ At 4 d after the initiation of IMQ application, the mRNA A treatment. Mice were treated with IMQ daily for 6 d. ( ) mRNA levels of expression levels of IL-17 (Fig. 7A) and IL-22 (Fig. 7B) were CCK in ears harvested from mice at indicated time points. Data are increased 11 and 29 times, respectively, compared with the un- expressed as mean 6 SE of three independent experiments. **p , 0.01. (B) The net increase in ear thickness measured at indicated time points. treated ears. i.p. injection of CCK8S significantly suppressed the Data are expressed as mean 6 SE of three independent experiments. IMQ-induced elevation of IL-17 and IL-22. IL-6, a keratinocyte- *p , 0.05, **p , 0.01 versus day 1 after the last IMQ treatment. and DC-derived cytokine, was increased earlier at day 2 and de- Downloaded from creased at day 4 (Fig. 7C). Administration of CCK8S strongly CCK was remarkably decreased 1 d after the last application of suppressed the IMQ-induced IL-6 elevation to a level comparable IMQ (12% of the untreated control; Fig. 4A), indicating marked to that in the untreated control at both day 2 and day 4. In contrast, reduction of CCK during the 6-d IMQ treatment. The CCK ex- CCK8S did not effect a transient increase in IL-23 expression pression was gradually increased thereafter and reached more than (Fig. 7D). Results of ANOVA were summarized in Supplemental

50% of the untreated control at day 29. Inversely, the consecutive Table I. http://www.jimmunol.org/ 6-d treatment of IMQ induced a robust epidermal inflammation as We next clarified whether the suppressive effect of CCK8S on assessed by ear thickness at 1 d after the last IMQ treatment the IMQ-induced psoriatic inflammation is mediated through its (Fig. 4B). We found significant differences in ear thickness specific receptor. Based on the experimental protocol (Fig. 8A), between days after the last IMQ treatment (ANOVA, F = 64.55, mice received an i.p. injection of lorglumide, a highly selective p , 0.001). The ear thickness was significantly decreased at 4 d CCKAR antagonist (24), prior to CCK8S injection and IMQ after the last IMQ treatment and was reduced to 14% of maximum treatment. Again, CCK8S exerted a suppressive effect on the response on day 29. There are two possibilities in this temporal IMQ-induced ear swelling response (Fig. 8B). Injection of lor- inverse correlation between the expression level of CCK and the glumide blocked the CCK8S effect, suggesting that the effect of severity of epidermal inflammation. One is that the IMQ-induced CCK8S is mediated via CCKAR. Results of one-way ANOVA by guest on September 23, 2021 psoriatic change causes the reduction of CCK8 expression. The were as follows: F = 7.625 and p , 0.001. other possibility is that IMQ treatment downregulates CCK8 CCK8S suppresses IL-6 production by human expression, promoting the psoriatic inflammation. To address epidermal keratinocytes this issue, we therefore investigated the effect of CCK8S on the mouse model and cultured epidermal keratinocytes. Although DCs are one of the main sources of IL-6 and IL-23 (1, 7), the fact that the injection of CCK8S suppressed IL-6 but not IL-23 CCK8S suppresses IMQ-induced psoriatic epidermal expression in the psoriatic lesions provides an implication that inflammation through CCKAR CCK8S does not critically target DCs. Because keratinocytes also The aforementioned data suggest that the lack of CCK is associated produce a large amount of IL-6 and express CCKAR, it is more with the development of psoriasis and that CCK may play a likely that the impairment of local IL-6 expression by CCK8S regulatory role in its pathogenesis. To examine the in vivo function is due to its effect on keratinocytes. We therefore evaluated of CCK, we applied IMQ daily for 6 consecutive d onto mouse ears the direct effect of CCK8S on cultured human keratinocytes. By and i.p. injected CCK8S into the mice according to the experi- flow cytometric analysis, it was confirmed that NHEKs express mental protocol (Fig. 5A). Again, repeated application of IMQ CCKAR but not CCKBR (Fig. 9A). Pretreatment of NHEKs with induced an increase in ear thickness (Fig. 5B), which was associated CCK8S at 10 nM significantly suppressed IL-6 production by with histological changes typical of psoriatic epidermal inflamma- IMQ- or IL-17–stimulated NHEKs (two-way ANOVA, F = 36.9, tion, such as acanthosis, parakeratosis, and massive infiltration of p , 0.001; interaction, F = 2.43, p = 0.096; Fig. 9B). Similar

FIGURE 5. CCK8S suppresses IMQ-induced psoriatic epidermal inﬂammation. (A) Experimental protocol. (B) The net increase in ear thickness calculated by subtracting the ear thickness measured before the start of IMQ treatment. Data are expressed as mean 6 SE of three independent experiments. **p , 0.01 versus control group, ***p , 0.001. The Journal of Immunology 5

FIGURE 6. CCK8S attenuates IMQ-induced histological changes but not CCKAR expression. Psoriatic epidermal inflammation was induced according to the experimental protocol described in Fig. 5A. Repre- sentative images of ears harvested at day 6 and stained with H&E (upper panel) or anti-CCKAR Ab (middle panel, low-power field; lower panel, high-power field). For immunostaining of CCKAR, color was developed by using AEC as a substrate. Arrows indicate perinuclear staining of CCKAR in keratinocytes. Downloaded from

levels of suppression were also observed with 100 nM CCK8S (Fig. 9D). Moreover, when NHEKs were stimulated with IL-17,

(data not shown). CCK8S also decreased basal IL-6 production by the mRNA expression of CCK was significantly decreased. It is http://www.jimmunol.org/ the unstimulated NHEKs. When NHEKs were pretreated with lor- thus suggested that CCK reduction by IL-17 relieves CCK-based glumide, the effect of CCK8S was completely abrogated, indicating self-regulation of keratinocytes and subsequently leads to con- that CCK8S suppresses IL-6 production through CCKAR on NHEKs tinuous epidermal inflammation as seen in psoriasis. (Fig. 9C). Interestingly, lorglumide enhanced IL-6 production even when it was added to the cultures of the unstimulated keratinocytes Discussion as well as the IMQ- or IL-17–stimulated keratinocytes, leading us We previously showed that CCK8S downregulates substance to the idea that IL-6 production by keratinocytes is physiologically P–induced pruritus by suppressing degranulation of mast cells, downregulated by endogenous CCK in an autocrine and/or para- indicating a significant role of CCK in skin disease (21). In the crine manner. In contrast, YM022, a CCKBR antagonist (25), did current study, we demonstrated that CCK8 was constitutively by guest on September 23, 2021 not enhance IL-6 production from unstimulated keratinocytes, expressed in the epidermis of normal healthy subjects, but its which confirms that the effect of endogenous CCK is mediated via expression was markedly reduced in skin lesions of psoriasis. CCKAR rather than CCKBR (Supplemental Fig. 2). To address Because CCK8 was normally expressed in the skin lesions of this mechanism, we investigated the expression of CCK in atopic dermatitis, the impaired epidermal expression of CCK unstimulated NHEKs by real-time PCR and found that steady- is a characteristic feature of psoriasis rather than a nonspecific state NHEKs expressed a detectable amount of mRNA for CCK finding in inflammatory skin diseases. These results prompted

FIGURE 7. Expression levels of ear cytokine mRNA. Total RNA was puriﬁed from untreated or IMQ-treated ears harvested on day 2 or day 4, and expression of IL-17 (A), IL-22 (B), IL-6 (C), and IL-23 (D) mRNA was determined by real-time PCR. Data are expressed as mean 6 SE of three independent experiments. *p , 0.05, **p , 0.01. 6 DOWNREGULATORY ROLE OF CHOLECYSTOKININ IN PSORIASIS

FIGURE 8. A CCKAR antagonist restrains the suppressive effect of CCK8S on IMQ-induced psoriatic epidermal inﬂammation. (A) Experimental protocol. (B) The net increase at day 6 in ear thickness of mice treated with IMQ in combination with i.p. injection of CCK8S and lorglumide. Data are expressed as mean 6 SE of three independent experiments. *p , 0.05, **p , 0.01.

us to investigate a role of CCK in the pathogenesis of psoriasis. completely depressed by the administration of CCK8S. Although We found that CCK8S exerted a suppressive effect on the devel- IL-6 is also produced by DCs (7), it is more likely that CCK8S opment of IMQ-induced psoriatic epidermal inflammation. It is targeted keratinocytes rather than DCs in the IL-6 depression well-known that IL-17 and IL-22 are highly expressed in psoriasis because CCK8S did not affect the elevation of another DC-derived Downloaded from skin lesions and play an indispensable role in its pathogenesis cytokine, IL-23. In vitro study confirmed that CCK8S suppressed (1, 2, 26, 27). Administration of CCK8S decreased the expression IL-6 production by NHEKs. Likewise, the suppressive effect of of local IL-17 and IL-22 and improved the epidermal inflammation. CCK8S on Th17 cytokine expression might be caused by decreased Epidermal keratinocytes and T cells mutually interact with each keratinocyte production of Th17 chemokines such as CCL20. It is other in the development of inflammatory skin diseases, as typically also likely that CCK8S inhibits development of Th17 cells through seen in psoriasis. In response to Th17 cytokines, keratinocytes its effect on CD4+ T cells and DCs, as reported previously (16, 18). http://www.jimmunol.org/ proliferate per se to form acanthotic epidermis, but simultaneously, CCK binds to two types of CCK receptors, CCKAR and they produce chemokines for Th17 cells, Th1 cells, and neutro- CCKBR, thereby transducing signals to nucleus (15). To clarify the phils, leading to skin infiltration of these leukocytes (5). IL-6 is one type of receptor mediating the suppressive effect of CCK on the of the major keratinocyte-derived cytokines (28). In systemic psoriatic lesions, we injected CCKAR-specific antagonist lorglumide immunity, IL-6 contributes to differentiation of Th17 cells and before treatments with CCK8S and IMQ and found that lorglumide depresses differentiation of regulatory T cells (29, 30). In cuta- completely abrogated the suppressive action of CCK8S. In the neous inflammation, IL-6 functions as initiator or amplifier, as do in vitro study using CCKAR-expressing NHEKs, the CCK8S- other proinflammatory cytokines such as IL-1 and TNF-a (31). induced IL-6 suppression was restored by lorglumide. These by guest on September 23, 2021 We found that the expression of IL-6 was increased in the IMQ- results suggest that CCK downregulates psoriatic epidermal treated psoriatic skin lesions, and this IL-6 elevation was almost inflammation through CCKAR.

FIGURE 9. CCK8S suppresses IL-6 release from human keratinocytes through CCKAR. (A) Flow cytometric analysis of CCK receptors on NHEKs. Representative histograms obtained by staining with anti-CCKAR Ab (left, red line), anti-CCKBR Ab (right, red line), or fluorescence-conjugated anti- rabbit IgG Ab only (filled gray). Mean fluorescence intensity (MFI) was expressed as mean 6 SE of four to seven independent experiments. (B and C) NHEKs were pretreated with CCK8S (10 nM) and lorglumide (10 mM) and were stimulated with IMQ or IL-17 for 24 h. The amount of IL-6 release was determined by ELISA. Data are expressed as mean 6 SE of three to five independent experiments performed in triplicate. (D) mRNA levels of CCK in NHEKs stimulated with IL-17 for 24 h. Data are expressed as mean 6 SE of three independent experiments performed in triplicate. *p , 0.05, **p , 0.01. The Journal of Immunology 7

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