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A GPR40 Agonist GW9508 Suppresses CCL5, CCL17, and CXCL10 Induction in and Attenuates Cutaneous Immune Inflammation Tomoko Fujita1,2, Toshiyuki Matsuoka1,2, Tetsuya Honda3, Kenji Kabashima1,3, Takako Hirata1,2 and Shuh Narumiya1,2

G- coupled receptors (GPCR) exert diverse physiological functions, many of which are exploited therapeutically. The roles of GPCR in keratinocytes in immune response in the skin, however, remain poorly defined. In this study, we focused on Gi-coupled GPCR in keratinocytes and defined their actions in immunoactivation of cultured keratinocytes in vitro and immune reaction in the skin in vivo. We first activated HaCaT cells by (TNF)-a and IFN-g and examined effects of various ligands for GPCR on production of CCL17 and CCL5. Agonists for Gi-coupled receptors, particularly GW9508 for GPR40, inhibited CCL17 and CCL5 expression in a pertussis toxin-sensitive manner. The inhibitory effect by GW9508 was abrogated by depletion of GPR40 with RNA interference. GW9508 further suppressed expression of IL-11, IL-24, and IL-33 induced in HaCaT cells by TNF-a and IFN-g. GW9508 also inhibited CCL5 and CXCL10 production by normal human epidermal keratinocytes. Administration of GW9508 topically to the skin in the challenging phase suppressed ear swelling in a repeated hapten application model and contact hypersensitivity with downregulation of CCL5 and CXCL10, respectively. Thus, in the skin, stimulation of Gi-coupled receptors attenuates induction of critical and by proinflammatory cytokines in keratinocytes and suppresses allergic inflammation in the skin. Journal of Investigative Dermatology (2011) 131, 1660–1667; doi:10.1038/jid.2011.123; published online 19 May 2011

INTRODUCTION such as taste or odor perception. Notably, about half of all G- consisting of a, b, and g subunits are signal drugs currently used clinically target GPCR in various transducers and mediate actions of G-protein coupled systems from the central nervous system to the cardiovascular receptors (GPCR) with seven transmembrane domains in system. However, the role of GPCR in the immune system the plasma membrane (Neves et al., 2002). Each GPCR is remains obscure and their therapeutic application in this area coupled to either a different class of G-proteins, Gs, Gi, Gq, has been limited to those for chemokines and other or G12/13, or a combination thereof. Gsa activates adenylate chemotactic substances such as sphingosine-1-phosphate cyclase, and raises the intracellular concentration of cAMP. (Rosen et al., 2007). GPCR have been generally viewed as On the other hand, Gia inhibits adenylate cyclase and mediating immunosuppression through elevation of intra- decreases intracellular cAMP. Among more than 1,000 GPCR cellular cAMP in cells such as and T cells encoded by mammalian genomes, up to 400–500 GPCR (Nance and Sanders, 2007). However, contrary to this recognize neurotransmitters, hormones, and paracrine oversimplified view, recent studies have indicated that GPCR factors, whereas others are involved in sensory functions have diverse roles in immunity depending on the context of immune response. For example, we have recently shown that stimulation of the cAMP pathway by prostaglandin 1Center for Innovation in Immunoregulative Technology and Therapeutics, E receptors EP2 and EP4 exerts significant facilitation of Th17 2 Kyoto University, Kyoto, Japan; Department of Pharmacology, Faculty of expansion by regulating IL-23 expression by dendritic cells Medicine, Kyoto University, Kyoto, Japan and 3Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto, Japan and enhancing the IL-23-induced Th17 amplification Correspondence: Shuh Narumiya, Department of Pharmacology, Faculty of (Yao et al., 2009). These findings have questioned the simple Medicine, Kyoto University, Yoshida Konoecho, Kyoto, 606-8501 Japan. paradigm of the immunosuppression by cAMP and suggest E-mail: [email protected] that the action of a GPCR in the immune system has to be Abbreviations: AD, atopic dermatitis; CHS, contact hypersensitivity; GPCR, defined in the contexts of target cells and tissues, timing of G-protein coupled receptors; NHEK, normal human epidermal ; stimulation, types of other combined stimuli, and output of PTX, pertussis toxin; QRT-PCR, quantitative RT-PCR; TNF, tumor necrosis factor; TNF-a þ IFN-g, TNF-a and IFN-g in combination the response at various levels. Received 17 November 2010; revised 24 February 2011; accepted 21 March The incidence and severity of allergic skin diseases such as 2011; published online 19 May 2011 contact hypersensitivity (CHS) and atopic dermatitis (AD)

1660 Journal of Investigative Dermatology (2011), Volume 131 & 2011 The Society for Investigative Dermatology T Fujita et al. A GPR40 Agonist Suppresses Skin Inflammation

have increased extensively over the last 20 years in keratinocytes (NHEKs), and mouse models of AD and CHS, developed countries, and drugs targeted to these diseases and addressed these issues. are urgently demanded. In these allergic diseases, keratino- cytes have a crucial role in disease progression (Girolomoni RESULTS et al., 2004; Pastore et al., 2006). We previously reported that GPR40 agonist GW9508 suppresses CCL17 production through administration of an agonist of prostaglandin E receptor EP3, pertussis toxin-sensitive Gi protein a Gi-coupled receptor, attenuates allergic asthma (Kunikata We first used HaCaT human epidermal keratinocyte cell line. et al., 2005), CHS (Honda et al., 2009), and allergic Previous reports showed that HaCaT cells produce CCL17 in conjunctivitis (Ueta et al., 2009). Stimulation of cannabinoid response to stimulation with tumor necrosis factor (TNF)-a CB1 and CB2 receptors, both coupled to Gi, has also been or IFN-g alone, or TNF-a and IFN-g in combination shown to induce attenuation of DNFB-induced CHS (Karsak (TNF-a þ IFN-g; Saeki and Tamaki, 2006). We exploited this et al., 2007). Although EP3 is expressed on epithelial cells in finding and reproduced synergistic production of CCL17 by the airway and conjunctiva and keratinocytes in the skin TNF-a þ IFN-g in HaCaT cells, both at mRNA and protein and EP3 in these cells is suggested to mediate the above levels (Figure 1a). We also found that TNF-a and IFN-g therapeutic action, a direct proof of the involvement of synergistically enhance CCL5 production by HaCaT cells Gi-coupled receptors in keratinocytes in such action is (Figure 1b). CCL17 and CCL5 are produced by keratinocytes lacking. In this study, we first sought to define the actions in inflammatory states and have crucial roles in Th2-type of Gi-coupled receptors in cultured keratinocytes and then to immune responses such as AD (Pastore et al., 2006; Saeki analyze the effects of such pathway in elicitation of immune and Tamaki, 2006). To screen GPCR ligands that inhibit the inflammation in the skin. Here, we used HaCaT, a human TNF-a þ IFN-g-induced expression, we examined epidermal keratinocyte cell line, normal human epidermal the activity of compounds in the Tocriscreen Mini library in

*** ** * * * ) 70 * 35 * ) 1,250 ** –1 125 –1 60 30 1,000 50 100 25 40 75 20 750 15 30 50 500 20 10 250 25 5

10 Ccl5/Gapdh (a.u.) Ccl17/Gapdh (a.u.) 0 CCL5 protein (pg ml 0 0 CCL17 protein (pg ml 0 None TNF-α IFN-γ TI None TNF-α IFN-γ TI None TNF-α IFN-γ TI None TNF-α IFN-γ TI

90 125 80 ) 70 ) –1 1,250

70 100 60 –1 1,000 60 50 75 50 *** *** 40 * 750 40 ** ** * ** 30 30 50 ** 500 20 20 25

Ccl17/Gapdh (a.u.) 250

10 Ccl5/Gapdh (a.u.) 10 CCL17 protein (pg ml 0 0 0 CCL5 protein (pg ml 0 TI TI+veh –8 –7 –6 TI TI+veh –8 –7 –6 TI TI+veh –8 –7 –6 TI TI+veh –8 –7 –6 TI + GW9508 (log[M]) TI + GW9508 (log [M]) TI + GW9508 (log [M]) TI + GW9508 (log [M])

250 ** 70 *** 150

*** ) 1,250 200 60 * –1 * 50 1,000 150 100 ** 40 750 100 ** 30 500 20 50 50

Ccl17/Gapdh (a.u.) 10 250 Ccl5/Gapdh (a.u.) 0 CCL17 protein (pg/ml)

0 0 CCL5 protein (pg ml TI+veh 0 1 10 TI+veh 0 1 10 0 TI+veh 0 1 10 TI+veh 0 1 10 μ –1 TI + 1 M GW9508 + PTX (ng ml ) TI + 1 μM GW9508 + PTX (ng ml–1) –1 TI + 1 μM GW9508 + PTX (ng ml ) TI + 1 μM GW9508 + PTX (ng ml–1)

150 *** 125 *** 100 100 75

50 50 25 Ccl5/Gapdh (a.u.) Ccl17/Gapdh (a.u.)

0 0 TI+veh –8 –7 –6 TI+veh –8 –7 –6 TI + forskolin (log[M]) TI + forskolin (log [M])

Figure 1. Inhibition by GW9508 of tumor necrosis factor (TNF)-a þ IFN-c-induced production of CCL17 and CCL5 in HaCaT cells. CCL17 (a) and CCL5 (b) production by TNF-a and/or IFN-g. Inhibitory effect of GW9508 on TNF-a þ IFN-g-induced CCL17 (c) and CCL5 (d) production. Effect of pertussis toxin (PTX) (e and f) and forskolin (g) on TNF-a þ IFN-g-induced CCL17 and CCL5 production. TI, TNF-a þ IFN-g; veh, vehicle. Quantitative real-time RT-PCR data are shown as arbitrary units (a.u.) where the value for unstimulated sample is set at 1. Results show a representative of three independent experiments. Data show mean±SEM (n ¼ 3). *Po0.05, **Po0.01, ***Po0.001 versus TI þ veh in (c–f).

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this system with CCL17 as a parameter. This screening GPR40 (–) GPR120 (–) GAPDH (–) revealed that several agonists for putative Gi-coupled receptors, such as ST91 for a2 adrenergic receptor, GP1a and GP2a for CB2, 30-fluorobenzyl-spiperone for dopamine

D2, and GW9508 for GPR40 potently suppressed the 1.0 TNF-a þ IFN-g-induced CCL17 production (Supplementary Table S1 online). The presence of these receptors in HaCaT cells was confirmed by GPCR array analysis and indicated as 0.5 DCt (Supplementary Table S1 online). We chose GW9508 for ** further analysis, because it most potently suppressed CCL17 expression. To verify the finding in the screening, we Gpr40/Gapdh (a.u.) 0.0 examined the dose dependency of the inhibitory effect of Control siRNA this compound on CCL17 and CCL5 by quantitative real-time siRNA siRNA RT-PCR (QRT-PCR) and ELISA. We found the concentration- 125 70 dependent suppression of both CCL17 and CCL5 mRNA and 100 60 Control Control 50 protein production by GW9508 in HaCaT cells (Figure 1c 75 40 and d) without any significant effect on their viability 50 * 30 *** 20 (Supplementary Figure S1 online). This inhibitory action of 25 10 Ccl5/Gapdh (a.u.) Ccl17/Gapdh (a.u.) GW9508 was reversed by low doses of pertussis toxin (PTX; 0 0 Figure 1e,), verifying that GPR40 stimulation exerts such TI – ++– ++ TI – ++– ++ effects through Gi. Conversely, forskolin, an adenylate GW9508 ––+ ––+ GW9508 ––+ ––+ cyclase activator, enhanced TNF-a þ IFN-g-induced Figure 2. GPR40 expression in HaCaT cells and GPR40 knockdown production of CCL17 and CCL5 (Figure 1g). On the other experiments. (a) GPR40 and GPR120 mRNA. Data of two different batches hand, inhibitors of AKT, PI3 kinase, p38 MAPK, MEK, and Src of HaCaT cells are shown. (), Control without template. (b) Reduction of did not reverse the effect of GW9508 (data not shown). These GPR40 mRNA by small interfering RNA (siRNA) treatment. (c) Reversal of GW9508-mediated inhibition of CCL17 (left) and CCL5 (right) mRNA results collectively suggest that PTX-sensitive Gi signal- expression by GPR40 depletion. Control, control siRNA. Bars ¼ 50 mm. ing suppresses CCL17 and CCL5 production induced by Results show a representative of three independent experiments. Data show TNF-a þ IFN-g through decrease in cAMP. mean±SEM (n ¼ 3). *Po0.05, **Po0.01, ***Po0.001 versus control RNA Although GW9508 is an agonist of GPR40, it also acts interference (b)orTIþ veh (c). on GPR120 at higher concentrations (pEC50 ¼ 7.32±0.03 GPR40 GPR120 GAPDH and pEC50 ¼ 5.46±0.09, respectively; Briscoe et al, 2006). (–) (–) (–) Reverse transcription PCR analysis detected expression of both GPR40 and GPR120 mRNA in HaCaT cells (Figure 2a). To verify that the immunosuppressive effect is

) 200 mediated by GPR40, we knocked down GPR40 with 30 –1 RNA interference. Small interfering RNA for GPR40 signifi-

cantly decreased the amount of GPR40 mRNA compared (a.u.) 20 * * 100 with control small interfering RNA after 72 hours (Figure 2b). 10 * Depletion of GPR40 by this procedure reversed the

Ccl5/Gapdh 0 0 protein (pgCCL5 ml suppressive effect of 1m M GW9508 on CCL17 and CCL5 –8 –7 –6 –8 –7 –6 +veh None γ production (Figure 2c), corroborating that GW9508 exerts None γ IFN-γ + GW9508 (log [M]) - +vehIFN-γ + GW9508 (log [M]) its inhibitory effect through GPR40 and not GPR120 in IFN- IFN

HaCaT cells. ) 15,000 –1 40

30 GW9508 inhibits CCL5 and CXCL10 production by NHEK (a.u.) 10,000 * * * 20 To further corroborate the findings in HaCaT cells, we next * * * 5,000 used primary culture of NHEKs. NHEK cells expressed 10

GPR40 but not GPR120 (Figure 3a). Consistent with a Cxcl10/Gapdh 0 0 CXCL10 protein (ngCXCL10 ml h previous report (Tsuda et al., 2003), we did not find –8 –7 –6 ne –8 –7 –6 No γ+ve significant production of CCL17 by NHEK on stimulation None γ+vehIFN-γ + GW9508 (log [M]) IFN-γ + GW9508 (log[M]) IFN- with TNF-a, IFN-g, or TNF-a þ IFN-g (data not shown). IFN- On the other hand, we noted induction of CCL5 by IFN-g Figure 3. Effects of GW9508 on CCL5 and CXCL10 production in NHEK. alone, and found that this induction was concentration- Reverse transcription PCR analysis of GPR40 and GPR120 mRNA (a)and dependently inhibited by GW9508 (Figure 3b). Under the inhibition by GW9508 of CCL5 (b) and CXCL10 (c) production in normal human epidermal keratinocyte (NHEK). In (a), data of two different batches of NHEK are same conditions, we also noted production of CXCL10, shown. (), Control without template. QRT-PCR data are shown as arbitrary units which was also inhibited by GW9508 (Figure 3c). Again, (a.u.) where the value for unstimulated sample is set at 1 (b and c, left). Veh, GW9508 was without effect on viability of NHEK (Supple- vehicle. Results show a representative of three independent experiments. Data mentary Figure S1 online). Therefore, GW9508 suppresses show mean±SEM (n ¼ 3). *Po0.05 versus IFN-g þ veh.

1662 Journal of Investigative Dermatology (2011), Volume 131 T Fujita et al. A GPR40 Agonist Suppresses Skin Inflammation

IFN-g-induced CCL5 and CXCL10 production through GPR40 GAPDH GPR40.

(–) BALB/c C57BL/6 (–) BALB/c C57BL/6 Changes in expression pattern after GW9508 treatment Previous studies reported that besides CCL17, many chemo- kines and cytokines are also markedly elevated in HaCaT cells after combined stimulation with TNF-a þ IFN-g (Xiao et al., 2003; Lee et al., 2009; Lo et al., 2010). Therefore, we speculated that many other proinflammatory proteins are upregulated by TNF-a þ IFN-g and that their expression GPR120 GAPDH might be differentially regulated by GPR40 stimulation. We conducted comparative microarray analyses for gene (–) BALB/c C57BL/6Lung (–) BALB/c C57BL/6 Lung expression and noted that 16 cytokines and 13 chemokines were upregulated more than 2-fold 24 hours after the TNF-a þ IFN-g stimulation (Supplementary Table S2 online). GW9508 treatment resulted in no further upregulation of any but induced reduction of expression of five genes, CCL17, CCL5, IL-11, IL-24, and IL-33 (Supplementary Table S3 online). IL-33 is a Th2-related , which activates basophils and acts on dendritic cells to promote Th2 immunity (Kakkar and Lee, 2008) and its expression is elevated in lesional skin of AD patients (Pushparaj et al., 2009).

Suppression by GW9508 of ear inflammation by repeated DNFB treatment The above results demonstrate that stimulation of GPR40 suppresses the cytokine-induced expression of CCL17 and CCL5 in HaCaT cells and that of CCL5 and CXCL10 in NHEK. To assess the in vivo effects of GPR40 stimulation, we examined whether treatment with GW9508 could exert effects expected from the in vitro experiments in cutaneous immune inflammation models. We first examined GPR40 expression in keratinocytes of the mouse ear. Reverse transcription PCR analysis of the whole ear showed a similar Figure 4. GPR40 and GPR120 expression in mouse epidermis. (a) Reverse extent of GPR40 mRNA expression in this tissue of BALB/c transcription PCR (RT-PCR) of GPR40 mRNA expression in the ear and C57BL/6 mice (Figure 4a). In situ hybridization for (upper row) and GPR120 mRNA expression in the epidermis (lower row) of GPR40 mRNA further showed that GPR40 was expressed in BALB/c and C57BL/6 mice. Mouse lung cDNA was used as a positive control for PCR of GPR120 (Hirasawa et al., 2005). (), Control without template. the epidermis of BALB/c mice (Figure 4b) and in the (b,c) In situ hybridization for GPR40 mRNA in unstimulated ears of BALB/c epidermis and the dermis of C57BL/6 mice (Figure 4c). mice (b) and C57BL/6 mice (c). Left, hybridization with antisense probe; Reverse transcription PCR did not detect GPR120 mRNA right, hybridization with sense probe. Positive signals are indicated expression in mouse epidermis (Figure 4a). with arrows. Bars ¼ 100 mm. A representative of three independent To examine effects of GW9508 on a Th2-type inflamma- experiments is shown. tion, we adopted repeated DNFB treatment in BALB/c mice. Repeated elicitation with hapten results in a shift from non-immunized, non-challenged group, CCL5 expression Th1-mediated to Th2-mediated inflammation (Kabashima was significantly elevated in the immunized, challenged et al., 2003; Inagaki et al., 2006), which mimics AD. From and vehicle-treated group (the vehicle-treated group) and the the fifth to the ninth elicitation, we applied 20 ml of 200 mM GW9508 treatment significantly attenuated the expression of GW9508 or vehicle topically to the ear 1 h before each this chemokine compared with that found in the vehicle- DNFB application. Treatment with GW9508 resulted in treated group (Figure 5c). Immunostaining for CCL5 revealed significant reduction of ear swelling compared with the that the CCL5 signal was enriched in the cytoplasm of control mice (Figure 5a). Histological examination revealed keratinocytes of the skin of the vehicle-treated animals, and diminished epidermal thickening and attenuated cell infiltra- that the GW9508 treatment markedly attenuated the signal tion in the dermis of the GW9508-treated group compared (Figure 5d). Consistent with this suppression, the number with the vehicle-treated group (Figure 5b). We next examined of eosinophils infiltrating in the skin was significantly the expression of CCL17 or CCL5 in the epidermis. QRT-PCR suppressed in the GW9508-treated group compared with analysis revealed that, although expression of CCL17 showed the control, vehicle-treated group (Figure 5b and e). no elevation in either group compared with the Attenuation of mast cell infiltration was also noted in the

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Veh GW9508 1,500 * * 1,000 * *

500 Ear thickness ( μ m)

5th Pre 7th6th 8th 9th 10th 0

90 80 70 60 50 Veh GW9508 40 ** 30 20

Ccl5/Gapdh (a.u.) 10 0 None Veh GW9508

90 80 70 Veh GW9508 60 50 40 30 (per field) 20 ***

Eosinophil count 10 0 Veh GW9508

Figure 5. Inhibition by GW9508 of immune inflammation with repeated DNFB treatment, a model of atopic dermatitis. (a) Time course of ear swelling of control- (broken line) and GW9508-treated (solid line) mice. (b) Hematoxylin–eosin staining. (c) QRT-PCR analysis of CCL5 mRNA expression. Data are shown as arbitrary units (a.u.) where the value for unstimulated group is set at 1. (d) Immunohistochemistry for CCL5 in inflamed ears. (e) Eosinophil count per field. (f) Toluidine blue staining. None, non-immunized and unstimulated group; veh, vehicle-treated group; GW9508, GW9508-treated group. *Po0.05, **Po0.01 and ***Po0.001 versus vehicle. Data show mean±SEM of four to five mice. Bars ¼ 100 mm. A representative of three independent experiments is shown.

GW9508-treated group (Figure 5f). These data collectively which was markedly attenuated by GW9508 treatment suggest that GW9508 reduces CCL5 production in keratino- (Figure 6d). Taken together, these results suggest that cytes and infiltration of eosinophils and mast cells, GW9508 attenuates CHS at least in part by suppressing and attenuates ear swelling in repeated DNFB treatment, a CXCL10 expression by keratinocytes. model of AD. DISCUSSION Suppression by GW9508 of elicitation of CHS Keratinocytes are a major cellular component of the skin and We next examined whether GPR40 stimulation has a have a key role in establishing local inflammatory milieu in suppressive effect also in Th1-type skin inflammation, various cutaneous diseases (Pastore et al., 2006). In this because GPR40 stimulation inhibited CXCL10 production study, we screened Gi-coupled GPCR expressed in HaCaT in NHEK. CXCL10 recruits Th1 cells and is known to have cells by chemical library screening and GPCR array, and important roles in Th1-type immune responses (Nakae et al., found GPR40 as one of them. Stimulation of GPR40 2003). To this end, we subjected wild-type C57BL/6 mice suppresses expression of CCL17 and CCL5 in vitro in HaCaT to DNFB-induced CHS and treated mice with different cells stimulated with TNF-a þ IFN-g and CCL5 and CXCL10 concentrations of GW9508 topically to the ear 30 minutes expression in NHEK stimulated with IFN-g. Furthermore, before the challenge. Application of GW9508 suppressed ear stimulation of GPR40 suppresses immune inflammation swelling in a concentration-dependent manner (Figure 6a). in vivo in the skin in the mouse. Histological examination revealed marked attenuation of CCL17 is a ligand for CCR4, which is mainly expressed epidermal thickening and infiltration of cells in the dermis in on Th2 cells. CCL17 is produced by diverse cell types, the GW9508-treated group (Figure 6b). QRT-PCR analysis of including bronchial epithelial cells, fibroblasts, endothelial the whole ear showed marked elevation of CXCL10 mRNA cells, and dendritic cells in addition to HaCaT cells (Saeki expression in mice subjected to CHS, and this increase was and Tamaki, 2006). Although we have not detected elevation significantly suppressed by GW9508 treatment (Figure 6c). of CCL17 in our mouse model for AD, keratinocytes in skin Changes in expression of CCL5 and CCL17 could not be lesion of AD patients are stained positive for CCL17. Further, detected, probably because the mouse CHS is Th1-dependent the serum concentration of CCL17 is significantly higher in and not Th2-dependent immune inflammation. Immuno- AD patients than in psoriasis vulgaris subjects or normal staining for CXCL10 exhibited strong accumulation of this healthy donors, and correlates well with the severity of AD chemokine in the epidermis of the vehicle-treated group, (Kakinuma et al., 2001). These findings suggest that CCL17

1664 Journal of Investigative Dermatology (2011), Volume 131 T Fujita et al. A GPR40 Agonist Suppresses Skin Inflammation

250 epidermis with repeated DNFB treatment in the mouse 200 model of AD, and that GW9508 treatment attenuated ear swelling in this model with concomitant decrease of the 150 * * CCL5 production in the epidermis. Suppression of infiltration 100 of eosinophils and mast cells was also observed with 50 GW9508 treatment. Our results are thus consistent with the Ear swelling ( μ m) 0 above genomic study and suggest that GPR40 stimulation

10 30 inhibits Th2-type skin inflammation in part by inhibiting Veh 100 CCL5 expression by keratinocytes. GW9508 (μM) No sens. We also examined effects of GPR40 stimulation in CHS, a Vehicle GW9508 Th1-dependent inflammation. In CHS, keratinocytes produce many chemokines to attract inflammatory cells to the site of inflammation. Among them, CXCL10 is one of the most important chemokines in CHS pathogenesis (Nakae et al., 2003; Mori et al., 2008). CXCL10 is a ligand for CXCR3, which is primarily expressed on Th1 cells. CHS response was decreased in TNF-a deficient mice, which was reversed by CXCL10 injection during the elicitation phase. Moreover, 350 CHS was suppressed by treatment with anti-CXCL10 mAb, 300 suggesting a critical role for CXCL10 in CHS (Nakae et al., 250 * 2003). We found that GW9508 suppressed ear swelling 200 and dermal infiltration of inflammatory cells in CHS with 150 100 concomitant suppression of CXCL10 expression in keratino- 50 cytes. In conclusion, GPR40 stimulation inhibits chemokine

Cxcl10/Gapdh (a.u.) 0 production by keratinocytes in not only Th2-dependent but No sens. Veh GW9508 also Th1-dependent skin inflammation, and suppresses Vehicle GW9508 progression of the inflammation. GPR40, a Gi- and Gq-coupled GPCR, is a recently deorphanized long-chain free fatty acid receptor (Itoh et al., 2003). It is abundantly expressed on pancreatic b cells and stimulates insulin secretion. The actions of GPR40 in the immune system are currently unknown. Our findings suggest that GPR40 expressed in the skin regulates cutaneous immune responses. Keratinocytes proliferate at the basal Figure 6. Inhibition of contact hypersensitivity by topical application of layer of the epidermis and differentiate as they migrate GW9508. (a) Effect of GW9508. No sens., group without sensitization toward the surface. The outermost layer of the epidermis, the and treated with 0.3% DNFB; veh, vehicle-treated group; GW9508, stratum corneum, serves as a rigid barrier, which prevents GW9508-treated group. (b) Hematoxylin–eosin staining. (c) QRT-PCR water loss and the entry of pathogens into the dermis (Proksch analysis of CXCL10 mRNA. Data are shown as arbitrary units (a.u.) where the et al., 2008). Intracellular components of the barrier function value for the group without sensitization is set at 1. (d) Immunostaining for of stratum corneum consist of keratins and filaggrin, whereas m CXCL10. Bars ¼ 100 m. Results are representative of three independent the extracellular components consist of long-chain free fatty experiments. Data show mean±SEM of five mice. *Po0.05 compared with vehicle. acids, cholesterol, and ceramide. Essential free fatty acid deficiency (Feingold, 2007) or defective fatty acid transport has a key role in the pathogenesis of AD in humans. Given (Klar et al., 2009) causes skin barrier defects characterized by the suppression of CCL17 expression by GPR40 stimulation red and scaly skin, severe bacterial infection, and impaired in HaCaT cells, it may be worthwhile to test the effect of a wound healing. Our present findings may indicate that long- GPR40 agonist on CCL17 expression in the skin of AD chain free fatty acids in the skin not only form a barrier, but patients. also suppress abnormal immune responses by stimulating CCL5 is a chemokine secreted by platelets, fibroblasts, GPR40 to protect terrestrial organisms from water loss and airway epithelial cells (Castellani et al., 2007), and epidermal noxious stimuli. In the mouse ear, GPR40 is expressed in the keratinocytes (Yamada et al., 1997; Park et al., 2005). CCL5 epidermis. Quite recently, GPR120, a GPCR related to promotes migration of T cells, dendritic cells, eosinophils, GPR40, functions as a receptor for o-3 unsaturated fatty natural killer cells, mast cells, and basophils (Levy, 2009). acids and exerts anti-inflammatory actions (Oh da et al., Recently, single-nucleotide polymorphism was found in the 2010). Although GW9508 can act also on GPR120 at higher promoter region of CCL5 gene, and significant association of concentrations (Briscoe et al, 2006), our small interfering the variant with enhanced promoter activity with AD was RNA experiments as well as expression study suggest that reported (Nickel et al., 2000; Tanaka et al., 2006). In the GW9508 acts on GPR40 and not GPR120 to exert immuno- present work, we found the elevation of CCL5 in the suppressive effect on keratinocytes both in vitro and in vivo.

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Our present findings that stimulation of a Gi-coupled collected 6 hours after the last elicitation. Mice treated with vehicle receptor, GPR40, suppresses immune inflammation in the without DNFB were used as control. CHS was performed essentially skin are consistent with previous reports by our laboratories as described (Honda et al., 2009). GW9508 was administered and others on other Gi-coupled GPCR such as PGE receptor topically to the ear 30 minutes before elicitation. Ear thickness was EP3 subtype and CB1 and CB2 cannabinoid receptors. measured with a micrometer before and 24 hours after challenge to Stimulation of EP3 inhibits experimental allergic conjuncti- assess inflammation. Ears were then collected for histology and vitis (Ueta et al., 2009), allergic airway inflammation QRT-PCR analysis. (Kunikata et al., 2005), and CHS (Honda et al., 2009) in For other methods including GPCR array, QRT-PCR, RT-PCR, mice, and stimulation of CB1 and CB2 suppresses CHS ELISA, microarray experiments, MTT assay, RNA interference, (Karsak et al., 2007). These results suggest that Gi-coupled histological studies, and in situ hybridization, see Supplementary GPCR including GPR40 can be a good candidate for drug Materials and Methods online. Primers used for QRT-PCR and RT- development against allergic diseases targeted to the epithe- PCR are indicated in Supplementary Table S4 online. lium. Statistical analysis MATERIALS AND METHODS Data were expressed as means±SEM, and statistical analyses were Mice performed by means of Student’s t-test. A P value o0.05 was Female BALB/c mice and C57BL/6NCrSlc mice, 7–11 weeks of age, considered to be statistically significant. were obtained from Japan SLC (Hamamatsu, Japan). They were housed at the Institute of Laboratory Animals of Kyoto University on CONFLICT OF INTEREST a 12-hours light/dark cycle under specific pathogen-free conditions. TF, TM, KK, and TaH are employed by the Coordination Fund from the Japan All experimental procedures were approved by the Committee on Science and Technology Agency and Astellas Pharma, and SN received a grant from this Fund. TeH declares no conflict of interest. Animal Research of Kyoto University Faculty of Medicine.

ACKNOWLEDGMENTS Reagents and cytokines We thank Drs Gozoh Tsujimoto, Akira Hirasawa, Naoki Inagaki, Kiminori Tocriscreen Mini library containing GW9508 was from Tocris Hosoda, Tsutomu Tomita, and Kimiko Nakajima for valuable advice Cookson (Avonmouth, UK). PTX was from List Biological Labora- and comments, and Dr Toru Oga for providing mouse lung cDNA. We also tories (Campbell, CA). Forskolin was from Sigma-Aldrich (St Louis, thank Masashi Mizutani for animal care, and Tae Arai for assistance. This work was supported by the Special Coordination Funds for Promoting Science MO). Human TNF-a and IFN-g were from Peprotech (Rocky Hill, and Technology of the Japanese Government and Astellas Pharma in the NJ). DNFB was from Nacalai Tesque (Kyoto, Japan). Formation of Innovation Center for Fusion of Advanced Technologies Program. Cell culture HaCaT cells (Boukamp et al., 1988; Cell Lines Service, Eppelheim, SUPPLEMENTARY MATERIAL Germany) were cultured in high glucose DMEM (Gibco, Carlsbad, CA) supplemented with 50 U ml1 penicillin, 50 mgml1 streptomy- Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid cin (Nacalai Tesque) and 10% fetal bovine serum (Gibco). They 4 were seeded at 2 10 cells per well in a 96-well plate in 200 mlof REFERENCES the above medium containing serum. After 24 hours, the medium Boukamp P, Petrussevska RT, Breitkreutz D et al. (1988) Normal keratiniza- was replaced with the serum-free medium and the cells were tion in a spontaneously immortalized aneuploid human keratinocyte cell cultured for another 24 hours. The medium was then changed with line. J Cell Biol 106:761–71 fresh serum-free medium, and drugs were added to the cells. After Briscoe CP, Peat AJ, McKeown SC et al. (2006) Pharmacological regulation of 5 minutes, 10 ng ml1 TNF-a and/or 10 ng ml1 IFN-g were added insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules. Br J Pharmacol and the cells were cultured for indicated times. After incubation, 148:619–28 the medium was saved for ELISA and the cells were lysed for Castellani ML, Bhattacharya K, Tagen M et al. (2007) Anti-chemokine RNA preparation for QRT-PCR. NHEK (KURABO, Osaka, Japan) therapy for inflammatory diseases. Int J Immunopathol Pharmacol were cultured in HuMedia–KG2 (KURABO) supplemented with 20:447–53 insulin, bovine pituitary extract, epidermal , hydro- Feingold KR (2007) Thematic review series: skin lipids. The role of epidermal cortisone, kanamycin, and amphotericin B. They were seeded at lipids in cutaneous permeability barrier homeostasis. J Lipid Res 48:2531–46 2 104 cells per well in 96-well plates. After 24 hours, medium was replaced with HuMedia-KG2 without supplements and cultured Girolomoni G, Pastore S, Cavani A et al. (2004) The role of chemokines in inflammatory skin diseases. Ernst Schering Res Found Workshop for 24 hours. The medium was then changed and treated with 44:191–225 GW9508 and stimulated with cytokines as described for HaCaT Hirasawa A, Tsumaya K, Awaji T et al. (2005) Free fatty acids regulate gut cells. incretin glucagon-like peptide-1 secretion through GPR120. Nat Med 11:90–4

Repeated DNFB treatment and CHS protocol Honda T, Matsuoka T, Ueta M et al. (2009) Prostaglandin E2-EP3 signaling Repeated DNFB treatment was performed essentially as described suppresses skin inflammation in murine contact hypersensitivity. J Allergy Clin Immunol 124:809–18 e802 (Inagaki et al., 2006). From the fifth to the ninth elicitation, 10 mlof Inagaki N, Shiraishi N, Igeta K et al. (2006) Inhibition of scratching behavior 200 mM GW9508 in 99% ethanol was topically applied to each ear associated with allergic dermatitis in mice by tacrolimus, but not by on days of elicitation 1 hour before DNFB treatment. Ears were dexamethasone. Eur J Pharmacol 546:189–96

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