Yayoi Kamata

Histamine H4 Receptor Antagonists Ineffective against Itch and Skin Inflammation in Atopic Dermatitis Mouse Model

TO THE EDITOR Histamine is the best known pruritogen in humans and the most commonly used experimental itch-causing substance. It induces increased itch responses in the lesional skin of atopic dermatitis (AD) patients compared with normal skin (Ikoma et al., 2006). However, histamine H1 receptor (H1R) antagonists frequently fail to relieve the itch in AD patients as well as it does in patients with systemic diseases such as kidney and liver diseases. The lack of amelioration by high-potency H1R antagonists of different types in patients with itch suggests that other systems are involved (Ikoma et al., 2006; Ständer and Weisshaar, 2012). Involvement of histamine H4 receptor (H4R) in histamine-evoked itch in animal models has been reported (Dunford et al., 2007; Thurmond et al., 2008). However, the therapeutic efficacy of H4R antagonists on the H1R antagonistresistant itch in AD is poorly understood. We therefore examined the therapeutic effects of H4R antagonists on itch and skin inflammation in AD using NC/Nga mice, a mouse model of AD that has been previously described (Tanaka et al., 2012). Male NC/Nga mice (Charles River Japan, Yokohama, Japan), 10 weeks old, were maintained in clean condition. All animal procedures were approved by the institutional Animal Care and Use Committee of Juntendo University Graduate School of Medicine. It is generally accepted worldwide that AD patients are highly sensitized to house dust mite allergens (Sanda et al., 1992); and that house dust mite Dermatophagoides farinae body (Dfb) and feces are wellknown major environmental allergens (Matsuoka et al., 1995). We used a Dfb ointment-induced AD-like mouse model (Dfb-NC/Nga) to evaluate the therapeutic efficacy of H4R antagonists against itch-related behavior (scratching) and dermatitis in a mouse model of AD. Dermatitis was induced by application of Dfb ointment (Biostir, Kobe, Japan) twice a week for 3 weeks as described (Yamamoto et al., 2009). Severity of skin lesion was graded according to the criteria as described (Matsuda et al., 1997). Animals that received repeated application of Dfb ointment to their skin (Figure 1a) showed higher dermatitis scores than controls after 3 weeks (data not shown). After the induction, transepidermal water loss was measured using a Tewameter TM210 (Courage and Khazawa, Cologne, Germany) and scratching behavior was observed for 2 hours using a MicroAct (Neuroscience, Tokyo, Japan) as described (Inagaki et al., 2003). Dfb-NC/Nga mice showed significant loss of transepidermal water and more scratching bouts (data not shown). We examined effects of H4R antagonists, JNJ7777120 and JNJ28307474, on dermatitis and scratching behavior in Dfb-NC/Nga mice. In mice, antagonist JNJ28307474 shows a longer plasma half-life than JNJ7777120 (Thurmod et al., unpublished observations). Mice that scored over 5 for dermatitis severity were treated by either intraperitoneal injection with a vehicle (20% dimethylsulphoxide and 80% 2-hydroxypropyl-b-cyclodextrin in saline) or H4R antagonists (10 or 30 mg kg ) three times per week for 3 weeks (Figure 1a). Dermatitis score was assessed after each Dfb application, and the data were expressed as fold change values over score of dermatitis before H4R antagonist treatment (baseline) in each group. No significant amelioration of dermatitis followed treatment by either of these H4R antagonists (Figure 1b and c). In addition, scratching behavior was recorded before and after H4R antagonist treatment (# shown in Figure 1a). The data were expressed as fold change values over number of scratching bouts before the treatment (baseline) in each group. Behavior analyses revealed neither treatment inhibited scratching behavior (Figure 1d and e). Moreover, the fold change value of scratching bouts significantly increased by treatment of 30 mg kg 1 JNJ28307474 (Figure 1e). Treatment with JNJ7777120 or JNJ28307474 had no effect on locomotion activity (Kamo et al., unpublished observations). In this study, we found that treatment with H4R antagonist (JNJ7777120 or Accepted article preview online 20 August 2013; published online 19 September 2013 Abbreviations: AD, atopic dermatitis;; Dfb, Dermatophagoides farinae body; H1R, histamine H1 receptor; H4R, histamine H4 receptor A Kamo et al. Effects of H4R Antagonist on Dfb-AD Model

Differences in therapeutic effects of topically applied corticosteroid and tacrolimus on atopic dermatitis-like symptoms in NC/Nga mice

BACKGROUND Topical corticosteroid and calcineurin inhibitor have similar therapeutic benefits in atopic dermatitis (AD), but the differences in therapeutic mechanisms of action of these agents against AD symptoms are not fully understood. OBJECTIVE This study was performed to examine the different effects of topical betamethasone valerate (BMV), clobetasol propionate (CBP), and tacrolimus (TAC) on itch-related behavior and dermatitis in NC/Nga mice with AD-like symptoms. METHODS AD-like dermatitis was induced in the dorsal skin of NC/Nga mice by repeated topical application of Dermatophagoides farinae body (Dfb) ointment twice weekly for three weeks. Mice with dermatitis scores over 5 were divided into five groups with equal dermatitis scores and treated with BMV, CBP, TAC, or Vaseline (Vas) once daily for two consecutive days, or were not treated (NT). Scratching behavior was analyzed using a SCLABA®-Real system. Transepidermal water loss (TEWL) before and after treatment was measured using a Tewameter® TM210. Skin collected from each group was analyzed histologically. RESULTS After the second treatment, dermatitis showed significantly greater improvement in the CBP and TAC-treated groups than in the Vas-treated and NT groups. The numbers of scratching bouts were significantly lower in CBP and TAC-treated mice than in Vas-treated mice. TEWL was significantly lower in TAC-, but not in CBP-, treated mice than in Vas-treated mice. Immunohistochemical examination showed that BMV, CBP and TAC did not reduce the increased densities of epidermal protein gene product 9.5- and substance P-immunoreactive fibers. The numbers of dermal CD4-immunoreactive T cells were significantly lower in BMV and CBP-treated mice than in Vas-treated and NT mice. The numbers of dermal eosinophils were significantly lower in BMV, CBP and TAC-treated mice than in Vas-treated and NT mice, with CBP showing the strongest effect. CBP significantly reduced epidermal thickness compared with Vas and NT. There were no significant differences in the numbers of interleukin-31-immunoreactive cells and mast cells, or in expression of epidermal thymic stromal lymphopoietin among all five groups. CONCLUSION The therapeutic potency of TAC against AD-like symptoms, including pruritus, is equal to that of the corticosteroid CBP. Epidermal innervation of sensory nerves itself might not be related to the therapeutic effects of topical tacrolimus and corticosteroids in its early phase.

Relationships among plasma granzyme B level, pruritus and dermatitis in patients with atopic dermatitis

BACKGROUND Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by skin barrier dysfunction, allergic inflammation and intractable pruritus resistant to conventional antipruritic treatments, including H1-antihistamines. Granzymes (Gzms) are a family of serine proteases expressed by cytotoxic T lymphocytes and natural killer cells that have been shown to modulate inflammation. However, the relationship between Gzms and pathology in AD remains unclear. OBJECTIVE This study assessed the correlation between plasma GzmB levels and severity of pruritus and dermatitis, in AD patients. METHODS Plasma was collected from 46 patients with AD, 24 patients with psoriasis, and 30 healthy controls. AD severity was assessed with the scoring atopic dermatitis (SCORAD) index, psoriasis severity with the psoriasis area and severity index (PASI), and degree of pruritus by visual analogue scale (VAS) score. GzmA, GzmB and gastrin releasing peptide (GRP) levels were measured by enzyme-linked immunosorbent assays. RESULTS Plasma GzmB concentrations were significantly higher in patients with AD and psoriasis than in healthy controls. Correlation analyses showed that plasma GzmB concentrations positively correlated with SCORAD and serum levels of severity markers such as thymus and activation-regulated chemokine, and lactate dehydrogenase in AD patients. Moreover, plasma levels of GRP, an itch-related peptide, were higher in patients with AD, positively correlating with VAS score and plasma GzmB level. In addition, plasma GzmB concentration was significantly lower in the treatment group than the untreated group with AD. Meanwhile, there were no correlations among GzmB levels, VAS score and PASI score in patients with psoriasis. In contrast to the results of plasma GzmB, plasma GzmA levels were unchanged among AD, psoriasis and healthy groups, and showed no correlations with VAS score and SCORAD index in patients with AD. CONCLUSION Plasma GzmB levels may reflect the degree of pruritus and dermatitis in patients with AD.

The Excimer Lamp Induces Cutaneous Nerve Degeneration and Reduces Scratching in a Dry-Skin Mouse Model

Epidermal hyperinnervation, which is thought to underlie intractable pruritus, has been observed in patients with atopic dermatitis (AD). The epidermal expression of axonal guidance molecules has been reported to regulate epidermal hyperinnervation. Previously, we showed that the excimer lamp has antihyperinnervative effects in nonpruritic dry-skin model mice, although epidermal expression of axonal guidance molecules was unchanged. Therefore, we investigated the antipruritic effects of excimer lamp irradiation and its mechanism of action. A single irradiation of AD model mice significantly inhibited itch-related behavior 1 day later, following improvement in the dermatitis score. In addition, irradiation of nerve fibers formed by cultured dorsal root ganglion neurons increased bleb formation and decreased nerve fiber expression of nicotinamide mononucleotide adenylyl transferase 2, suggesting degenerative changes in these fibers. We also analyzed whether attaching a cutoff excimer filter (COF) to the lamp, thus decreasing cytotoxic wavelengths, altered hyperinnervation and the production of cyclobutane pyrimidine dimer (CPD), a DNA damage marker, in dry-skin model mice. Irradiation with COF decreased CPD production in keratinocytes, as well as having an antihyperinnervative effect, indicating that the antipruritic effects of excimer lamp irradiation with COF are due to induction of epidermal nerve degeneration and reduced DNA damage.

Importance of tryptophan nitration of carbonic anhydrase III for the morbidity of atopic dermatitis

The nitration of proteins results from the vigorous production of reactive nitrogen species in inflammatory disease. We previously reported the proteomic analysis of nitrated tryptophan residues in in vitro model cells for inflammatory diseases using a 6-nitrotryptophan-specific antibody. In this paper, we applied this method to the analysis of a disease model animal and identified the 6-nitrotryptophan-containing proteins in the skin of atopic dermatitis model mice (AD-NC/Nga mice). We found three nitrotryptophan-containing proteins, namely, carbonic anhydrase III (CAIII), α-enolase (α-ENO), and cytoskeletal keratin type II (KTII), and identified the positions of the nitrotryptophan residues in their amino acid sequences: Trp47 and Trp123 in CAIII, Trp365 in α-ENO, and Trp221 in KTII. Among these, the nitration of CAIII was increased not only in the lesional skin of AD-NC/Nga mice but also in the mice that did not present any symptoms. The in vitro nitration of purified CAIII by peroxynitrite reduced its CO2 hydratase activity in a dose-dependent manner. In addition, we found that CAIII was induced during the differentiation of normal human epidermal keratinocytes. Furthermore, we found the presence of CAIII and the formation of 6-nitrotryptophan-containing proteins in both the lesional and the nonlesional sections of the skin of patients with atopic dermatitis through immunohistochemical staining. This study provides the first demonstration of the formation of 6-nitrotryptophan in human tissues and disease.

Itch in Atopic Dermatitis Management

Patients with atopic dermatitis (AD) suffer from chronic inflammatory dermatitis and antihistamine-resistant itch. The management of intractable pruritus in AD is important, requiring the development of new therapeutic approaches. At present, the standard treatments for AD include topical anti-inflammatory drugs such as calcineurin inhibitors and corticosteroids. Topical emollient treatment is recommended to moisten the skin and to restore and maintain barrier function. Phototherapy is also effective in reducing the number of epidermal nerve fibers, normalizing imbalances in the levels of expression of axon guidance molecules, and inhibiting pruritus. Systemic treatments such as cyclosporine A and aprepitant are used to treat severe and intractable pruritus in AD. Clinical trials of dupilumab and CIM331 have displayed a significant reduction of pruritus in patients with AD. New antipruritic approaches are targeted to the central nervous system such as spinal interneurons and glial cells. This chapter describes therapeutic approaches for attenuating intractable itch in AD.

Possible Antipruritic Mechanism of Cyclosporine A in Atopic Dermatitis.

Cyclosporine A is an immunosuppressive agent that suppresses pruritus and is currently used in the treatment of patients with severe atopic dermatitis. The aim of this study was to elucidate the antipruritic mechanism of cyclosporine A using a mouse model of atopic dermatitis. Intraperitoneal injection of cyclosporine A (5 mg/kg) significantly reduced epidermal nerve density, number of scratching bouts, dermatitis scores, and transepidermal water loss, as well as decreasing the numbers of inflammatory cells in the dermis and decreasing epidermal thickness. Intraperitoneal injection of cyclosporine A dose-dependently inhibited increased itch-related receptor gene expression, such as interleukin-31 receptor A and neurokinin-1 receptor, in the dorsal root ganglion of atopic dermatitis model mice. Thus, the antipruritic efficacy of cyclosporine A may involve reduced epidermal nerve density and expression levels of itch-related receptor genes in the dorsal root ganglion, as well as improvement in acanthosis and reduction in cutaneous inflammatory cell number.

Involvement of µ-opioid Receptors and κ-opioid Receptors in Itch-related Scratching Behaviour of Imiquimod-induced Psoriasis-like Dermatitis in Mice

The pathogenesis of psoriatic itch is poorly understood. The aim of this study was to investigate the involvement of opioid receptors in scratching behaviour of imiquimod-induced psoriasis-like dermatitis model mice. Topical application of 5% imiquimod cream to the rostral back skin of mice induced antihistamine-resistant scratching behaviour. The expression of µ-opioid receptor (MOR) protein increased in the epidermis, dorsal root ganglia (DRG) and spinal cord of imiquimod-treated mice. In contrast, the expression of κ-opioid receptor (KOR) protein decreased in the DRG and spinal cord of imiquimod-treated mice, and was undetectable in the epidermis of both groups. Topical or intraperitoneal administration of the MOR antagonist naloxone and oral administration of the centrally acting KOR agonist ICI-199,441 inhibited scratching behaviour, whereas oral administration of the peri-pherally-selective KOR agonist asimadoline did not. These results suggest that peripheral and central MOR and central KOR may be involved in the modulation of scratching behaviour in imiquimod-treated mice.

Tryptophan nitration of immunoglobulin light chain as a new possible biomarker for atopic dermatitis

To reduce the incidence and severity of atopic dermatitis, detection and treatment at an early stage are urgently required, but no effective biomarker has been reported. In this study, we attempted to detect a candidate biomarker of early stage atopic dermatitis by focusing on the levels of nitrated residues in the plasma proteins of atopic dermatitis model mice (NC/Nga mice). We found that the immunoglobulin (Ig) light chain was more highly nitrated in the plasma of the animal model than that of control mice. Western blot analysis showed a statistically significant difference between the 6-nitrotryptophan content of the Ig light chain in the NC/Nga mice before onset of atopic dermatitis symptoms and that of the control mice. LC-ESI-MS/MS analysis demonstrated that these light chains contained nitrotryptophan (Trp56) and nitrotyrosine (Tyr66). Immunofluorescence staining revealed a significant increase in manganese superoxide dismutase and inducible nitric oxide synthase production in the skin lesions of the NC/Nga mice. Furthermore, we found protein-bound 6-nitrotryptophan and 3-nitrotyrosine only in the lesioned skin, where their signals partially overlapped with the IgG signal. Our findings suggest that the 6-nitrotryptophan content of Ig light chains could be a new biomarker for detecting early stage atopic dermatitis.

Oral administration of milk-derived phospholipids inhibits penetration of cutaneous nerve fibres into epidermis in a mouse model of acute dry skin

The density of intraepidermal nerve fibres has been shown to be higher in itchy dry skin than in healthy skin, suggesting that epidermal hyperinnervation is at least partly involved in peripheral itch sensitization. We investigated whether oral administration of milk‐derived phospholipids (MPLs) would inhibit epidermal hyperinnervation in a mouse model of dry skin. We found that the number of intraepidermal nerve fibres was significantly lower in the MPL group than in the control group. Expression of nerve growth factor (NGF) levels in the epidermis was significantly decreased by oral administration of MPLs, whereas expression of semaphorin (Sema)3A, a nerve repulsion factor, was increased in the MPL group. These results suggest that dietary MPLs attenuate the penetration of nerve fibres into the epidermis by reducing epidermal NGF levels and increasing Sema3A level. Thus, dietary MPLs may have beneficial effects in the prevention and/or alleviation of dry skin‐induced itch by reducing intraepidermal nerve fibre density.