264
Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271
Acknowledgments The authors are grateful to the late Dr. T. Saeki for the discussions and helpful advice and to K. Miyazaki, Y. Takase and O. Asano for the synthesis of E6005. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci.2014. 09.010.
Fig. 2. NGF increased the sensitivity to itch stimulation in NC/Nga mice. Intradermally administered NGF enhanced the scratching behavior induced in mice. NC/Nga mice were sensitized and challenged with oxazolone. On day 7, saline or NGF was applied to the ear. After 30 min, mice were challenged with 0.005% oxazolone and their scratching behavior was assessed for 2 h using the MicroAct system. The results are expressed as mean SEM (n = 23 or 24). *p < 0.05 versus saline-treated group (unpaired t-test).
decreased NGF production in the damaged skin to 8.0 0.3 and 6.5 0.6 pg/mg protein at doses of 0.03% and 0.3%, respectively (Fig. 1E). In addition, intradermal injection of NGF in our mouse AD model before itch provocation significantly augmented the scratching counts by approximately 1.4-fold compared with that in the salinetreated group (896.3 94.2 for NGF group vs. 648.5 69.6/2 h for saline group, Fig. 2), as in patients with AD [4]. Our data demonstrated that allergic and mechanical irritation of the skin upregulated NGF, which was inhibited by the topically applied PDE4 inhibitor E6005. Moreover, intradermally administered NGF increased the itch response. Taken together, inhibition of NGF production may partly contribute to an antipruritic mechanism of E6005. Currently, the anti-NGF strategy is a potentially promising strategy for treating chronic itch in AD. The potential of E6005 to inhibit NGF production may provide relief from chronic itch by reducing sensitization and epidermal innervation. In fact, we found that repeatedly applied E6005 reduced the itch response over time in a chronic itch model (Supplemental Fig. 3). The mechanism through which E6005 reduces NGF levels remains unknown. PDE4 may play a role in the response of keratinocytes to skin trauma [9,10] and up-regulated NGF level was observed in keratinocyte of AD patient [5], suggesting that keratinocytes may be one of target of E6005. Further studies are required to confirm the effect of E6005 on keratinocytes. In conclusion, E6005 suppressed up-regulation of skin NGF levels in AD-like animal models. Inhibition of NGF is a new function for PDE4 inhibitors, which may partly account for the antipruritic effect of E6005. Thus, E6005 may be a safe approach for targeting NGF and a beneficial treatment for itch in a variety of skin disorders.
References [1] Raap U, Kapp A. Neurotrophins in healthy and diseased skin. G Ital Dermatol Venereol 2010;145:205–11. [2] Ikezawa Z, Komori J, Ikezawa Y, Inoue Y, Kirino M, Katsuyama M, et al. A role of Staphyococcus aureus, interleukin-18, nerve growth factor and semaphorin 3A, an axon guidance molecule, in pathogenesis and treatment of atopic dermatitis. Allergy Asthma Immunol Res 2010;2:235–46. [3] Tominaga M, Takamori K. Itch and nerve fibers with special reference to atopic dermatitis: therapeutic implications. J Dermatol 2014;41:205–12. [4] Rukwied RR, Main M, Weinkauf B, Schmelz M. NGF sensitizes nociceptors for cowhage- but not histamine-induced itch in human skin. J Invest Dermatol 2013;133:268–70. [5] Roggenkamp D, Falkner S, Sta¨b F, Petersen M, Schmelz M, Neufang G. Atopic keratinocytes induce increased neurite outgrowth in a coculture model of porcine dorsal root ganglia neurons and human skin cells. J Invest Dermatol 2012;132:1892–900. [6] Takano N, Sakurai T, Kurachi M. Effects of anti-nerve growth factor antibody on symptoms in the NC/Nga mouse, an atopic dermatitis model. J Pharmacol Sci. 2005;99:277–86. [7] Ishii N, Shirato M, Wakita H, Miyazaki K, Takase Y, Asano O, et al. Antipruritic effect of the topical phosphodiesterase 4 inhibitor E6005 ameliorates skin lesions in a mouse atopic dermatitis model. J Pharmacol Exp Ther 2013;346: 105–112. [8] Furue M, Kitahara Y, Akama H, Hojo S, Hayashi N, Nakagawa H. Safety and efficacy of topical E6005, a phosphodiesterase 4 inhibitor, Japanese adult patients with atopic dermatitis: results of a randomized, vehicle-controlled, multicenter clinical trial. J Dermatol. 2014;41:577–85. [9] Chujor CS, Hammerschmid F, Lam C. Cyclic nucleotide phosphodiesterase 4 subtypes are differentially expressed by primary keratinocytes and human epidermoid cell lines. J Invest Dermatol 1998;110:287–91. [10] Schafer PH, Parton A, Gandhi AK, Capone L, Adams M, Wu L, et al. Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis. Br J Pharmacol 2010;159:842–55.
Naoto Ishii, Hisashi Wakita, Manabu Shirato* Eisai Co., Ltd., Tsukuba Research Laboratories, Ibaraki, Japan *Corresponding author at: Eisai Co., Ltd., Tsukuba Research Laboratories, 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan. Tel.: +81 298 47 5632; fax: +81 298 47 2037 E-mail address: [email protected] (M. Shirato). Received 17 July 2014 http://dx.doi.org/10.1016/j.jdermsci.2014.09.010
Letter to the Editor A line of human keratin 5-Cre transgenic mice harbor a risk of ubiquitous Cre-recombination To the Editor The Cre-loxP system is a site-specific DNA recombination technology and is widely used to carry out conditional mutagenesis in specific cells and tissues, especially when systemic deletion
of the target gene causes embryonic lethality [1]. Limited Cre expression under the control of cell- or tissue-specific promoters enables us the conditional targeting of the loxP-floxed gene. In 1997, Tarutani et al. have generated human keratin 5 (KRT5)Cre mice, in which the Cre protein is expressed under the control of human KRT5 promoter [2]. Because all keratinocytes are derived from KRT5-expressing basal layer cells, KRT5-Cre mice, as well as keratin 14 (KRT14)-Cre mice [3], are fairly useful for
Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271
265
Fig. 1. Two distinct EYFP expression patterns in human KRT5-Cre;R26-stop-EYFP mice. (a–d) EYFP-positive cells in the blood (a, b) and in the lymph node (c, d) of human KRT5-Cre;R26-stop-ETFP mice. The progeny that represents the ubiquitous recombination is shown in (a, c) and keratinocyte-specific recombination is in (b, d). Scale bar = 25 mm. (e) Flow cytometric analysis of the blood, the epidermis, and the lymph node. (f) EYFP-positive cells were observed in the epididymal duct (arrow), whereas no EYFP expression was detected in sperms (arrowhead). Scale bar = 200 mm. High-magnification view of sperms is shown at the upper right inset. Scale bar = 50 mm. (g) Threedimensional imaging of the tail of the epididymis using multi-photon microscopy. Vertical slice image (indicated as yellow box) is shown in lower panel. Scale bar = 150 mm. (h) Two fluorescing unfertilized ova were seen (arrows) out of seven harvested ova. Scale bar = 200 mm. (i) Immunohistochemical analysis of KRT5 expression in ovaries after ovarian stimulation. Arrows represent oocytes in the different stages. Green, KRT5; blue, DAPI. Scale bar = 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
keratinocyte-specific recombination [4]. Therefore, these mice have become essential tools for skin researchers [5]. Subsequently, Ramirez et al. have generated another KRT5-Cre transgenic line utilizing bovine KRT5 promoter and reported unexpected phenomena that both keratinocyte-specific and generalized Cre-recombination were inducible depending on the breeding scheme [6]. They demonstrated that all of the progenies
derived from the bovine KRT5-Cre mother showed ubiquitous recombination independent of the transmission of the Cre transgene, while the progenies from the bovine KRT5-Cre father represented keratinocyte-specific recombination. It is presumed that bovine KRT5 promoter was constitutively activated in the latestage oocytes, with the Cre protein persisting into the developing embryo, leading to ubiquitous recombination.
Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271
266
Table 1a The frequency of ubiquitous recombination in the human KRT5-Cre progeny. Mating pattern Cre Mice Ubiquitous %
C, p.N47S, p.R48 W, p.P76L, p.E94X, p.R102Q, p.102Y, p.S113L, p.T123R and p.T123M [1,8,9], and four IL36RN mutations p.R10X, c.115 + 6T > C: p.R10RfsX1, p.T123R and p.T123M, have been reported in a Japanese population [1,5,6]. In this study, two SNVs, p.N47S and p.P82L, were observed in both PV cases and controls with allele frequencies > 1% (Supplementary Table 1); however, no statistically significant differences were observed in these two genotypes between PV cases and controls (data not shown). Both p.N47S and p.P82L were predicted to be deleterious by SIFT (http:// sift.jcvi.org/) but neutral by PROVEAN (http://sift.jcvi.org/). One control subject was homozygous for p.N47S and the possibility that this subject might develop GPP throughout life could not be completely excluded. c.334G > A (rs143724424) was not observed in the patients with PV or controls. In this study, we identified a novel IL36RN missense mutation, c.334G > A in exon 5, that caused p.E112K substitution and was located adjacent to a 113 amino acid residue. The missense c.338C > T mutation causing p.S113L substitution is the most prevalent allele in European GPP patients, and this variant is associated with an elevated proinflammatory response following
Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271
Acknowledgments The authors are grateful to the late Dr. T. Saeki for the discussions and helpful advice and to K. Miyazaki, Y. Takase and O. Asano for the synthesis of E6005. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci.2014. 09.010.
Fig. 2. NGF increased the sensitivity to itch stimulation in NC/Nga mice. Intradermally administered NGF enhanced the scratching behavior induced in mice. NC/Nga mice were sensitized and challenged with oxazolone. On day 7, saline or NGF was applied to the ear. After 30 min, mice were challenged with 0.005% oxazolone and their scratching behavior was assessed for 2 h using the MicroAct system. The results are expressed as mean SEM (n = 23 or 24). *p < 0.05 versus saline-treated group (unpaired t-test).
decreased NGF production in the damaged skin to 8.0 0.3 and 6.5 0.6 pg/mg protein at doses of 0.03% and 0.3%, respectively (Fig. 1E). In addition, intradermal injection of NGF in our mouse AD model before itch provocation significantly augmented the scratching counts by approximately 1.4-fold compared with that in the salinetreated group (896.3 94.2 for NGF group vs. 648.5 69.6/2 h for saline group, Fig. 2), as in patients with AD [4]. Our data demonstrated that allergic and mechanical irritation of the skin upregulated NGF, which was inhibited by the topically applied PDE4 inhibitor E6005. Moreover, intradermally administered NGF increased the itch response. Taken together, inhibition of NGF production may partly contribute to an antipruritic mechanism of E6005. Currently, the anti-NGF strategy is a potentially promising strategy for treating chronic itch in AD. The potential of E6005 to inhibit NGF production may provide relief from chronic itch by reducing sensitization and epidermal innervation. In fact, we found that repeatedly applied E6005 reduced the itch response over time in a chronic itch model (Supplemental Fig. 3). The mechanism through which E6005 reduces NGF levels remains unknown. PDE4 may play a role in the response of keratinocytes to skin trauma [9,10] and up-regulated NGF level was observed in keratinocyte of AD patient [5], suggesting that keratinocytes may be one of target of E6005. Further studies are required to confirm the effect of E6005 on keratinocytes. In conclusion, E6005 suppressed up-regulation of skin NGF levels in AD-like animal models. Inhibition of NGF is a new function for PDE4 inhibitors, which may partly account for the antipruritic effect of E6005. Thus, E6005 may be a safe approach for targeting NGF and a beneficial treatment for itch in a variety of skin disorders.
References [1] Raap U, Kapp A. Neurotrophins in healthy and diseased skin. G Ital Dermatol Venereol 2010;145:205–11. [2] Ikezawa Z, Komori J, Ikezawa Y, Inoue Y, Kirino M, Katsuyama M, et al. A role of Staphyococcus aureus, interleukin-18, nerve growth factor and semaphorin 3A, an axon guidance molecule, in pathogenesis and treatment of atopic dermatitis. Allergy Asthma Immunol Res 2010;2:235–46. [3] Tominaga M, Takamori K. Itch and nerve fibers with special reference to atopic dermatitis: therapeutic implications. J Dermatol 2014;41:205–12. [4] Rukwied RR, Main M, Weinkauf B, Schmelz M. NGF sensitizes nociceptors for cowhage- but not histamine-induced itch in human skin. J Invest Dermatol 2013;133:268–70. [5] Roggenkamp D, Falkner S, Sta¨b F, Petersen M, Schmelz M, Neufang G. Atopic keratinocytes induce increased neurite outgrowth in a coculture model of porcine dorsal root ganglia neurons and human skin cells. J Invest Dermatol 2012;132:1892–900. [6] Takano N, Sakurai T, Kurachi M. Effects of anti-nerve growth factor antibody on symptoms in the NC/Nga mouse, an atopic dermatitis model. J Pharmacol Sci. 2005;99:277–86. [7] Ishii N, Shirato M, Wakita H, Miyazaki K, Takase Y, Asano O, et al. Antipruritic effect of the topical phosphodiesterase 4 inhibitor E6005 ameliorates skin lesions in a mouse atopic dermatitis model. J Pharmacol Exp Ther 2013;346: 105–112. [8] Furue M, Kitahara Y, Akama H, Hojo S, Hayashi N, Nakagawa H. Safety and efficacy of topical E6005, a phosphodiesterase 4 inhibitor, Japanese adult patients with atopic dermatitis: results of a randomized, vehicle-controlled, multicenter clinical trial. J Dermatol. 2014;41:577–85. [9] Chujor CS, Hammerschmid F, Lam C. Cyclic nucleotide phosphodiesterase 4 subtypes are differentially expressed by primary keratinocytes and human epidermoid cell lines. J Invest Dermatol 1998;110:287–91. [10] Schafer PH, Parton A, Gandhi AK, Capone L, Adams M, Wu L, et al. Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis. Br J Pharmacol 2010;159:842–55.
Naoto Ishii, Hisashi Wakita, Manabu Shirato* Eisai Co., Ltd., Tsukuba Research Laboratories, Ibaraki, Japan *Corresponding author at: Eisai Co., Ltd., Tsukuba Research Laboratories, 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan. Tel.: +81 298 47 5632; fax: +81 298 47 2037 E-mail address: [email protected] (M. Shirato). Received 17 July 2014 http://dx.doi.org/10.1016/j.jdermsci.2014.09.010
Letter to the Editor A line of human keratin 5-Cre transgenic mice harbor a risk of ubiquitous Cre-recombination To the Editor The Cre-loxP system is a site-specific DNA recombination technology and is widely used to carry out conditional mutagenesis in specific cells and tissues, especially when systemic deletion
of the target gene causes embryonic lethality [1]. Limited Cre expression under the control of cell- or tissue-specific promoters enables us the conditional targeting of the loxP-floxed gene. In 1997, Tarutani et al. have generated human keratin 5 (KRT5)Cre mice, in which the Cre protein is expressed under the control of human KRT5 promoter [2]. Because all keratinocytes are derived from KRT5-expressing basal layer cells, KRT5-Cre mice, as well as keratin 14 (KRT14)-Cre mice [3], are fairly useful for
Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271
265
Fig. 1. Two distinct EYFP expression patterns in human KRT5-Cre;R26-stop-EYFP mice. (a–d) EYFP-positive cells in the blood (a, b) and in the lymph node (c, d) of human KRT5-Cre;R26-stop-ETFP mice. The progeny that represents the ubiquitous recombination is shown in (a, c) and keratinocyte-specific recombination is in (b, d). Scale bar = 25 mm. (e) Flow cytometric analysis of the blood, the epidermis, and the lymph node. (f) EYFP-positive cells were observed in the epididymal duct (arrow), whereas no EYFP expression was detected in sperms (arrowhead). Scale bar = 200 mm. High-magnification view of sperms is shown at the upper right inset. Scale bar = 50 mm. (g) Threedimensional imaging of the tail of the epididymis using multi-photon microscopy. Vertical slice image (indicated as yellow box) is shown in lower panel. Scale bar = 150 mm. (h) Two fluorescing unfertilized ova were seen (arrows) out of seven harvested ova. Scale bar = 200 mm. (i) Immunohistochemical analysis of KRT5 expression in ovaries after ovarian stimulation. Arrows represent oocytes in the different stages. Green, KRT5; blue, DAPI. Scale bar = 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
keratinocyte-specific recombination [4]. Therefore, these mice have become essential tools for skin researchers [5]. Subsequently, Ramirez et al. have generated another KRT5-Cre transgenic line utilizing bovine KRT5 promoter and reported unexpected phenomena that both keratinocyte-specific and generalized Cre-recombination were inducible depending on the breeding scheme [6]. They demonstrated that all of the progenies
derived from the bovine KRT5-Cre mother showed ubiquitous recombination independent of the transmission of the Cre transgene, while the progenies from the bovine KRT5-Cre father represented keratinocyte-specific recombination. It is presumed that bovine KRT5 promoter was constitutively activated in the latestage oocytes, with the Cre protein persisting into the developing embryo, leading to ubiquitous recombination.
Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271
266
Table 1a The frequency of ubiquitous recombination in the human KRT5-Cre progeny. Mating pattern Cre Mice Ubiquitous %
C, p.N47S, p.R48 W, p.P76L, p.E94X, p.R102Q, p.102Y, p.S113L, p.T123R and p.T123M [1,8,9], and four IL36RN mutations p.R10X, c.115 + 6T > C: p.R10RfsX1, p.T123R and p.T123M, have been reported in a Japanese population [1,5,6]. In this study, two SNVs, p.N47S and p.P82L, were observed in both PV cases and controls with allele frequencies > 1% (Supplementary Table 1); however, no statistically significant differences were observed in these two genotypes between PV cases and controls (data not shown). Both p.N47S and p.P82L were predicted to be deleterious by SIFT (http:// sift.jcvi.org/) but neutral by PROVEAN (http://sift.jcvi.org/). One control subject was homozygous for p.N47S and the possibility that this subject might develop GPP throughout life could not be completely excluded. c.334G > A (rs143724424) was not observed in the patients with PV or controls. In this study, we identified a novel IL36RN missense mutation, c.334G > A in exon 5, that caused p.E112K substitution and was located adjacent to a 113 amino acid residue. The missense c.338C > T mutation causing p.S113L substitution is the most prevalent allele in European GPP patients, and this variant is associated with an elevated proinflammatory response following
