Yusuke Honma

Involvement of IL‐31 on scratching behavior in NC/Nga mice with atopic‐like dermatitis

Abstract:  Pruritus is an important symptom in atopic dermatitis (AD), but the major pruritogen have not been identified. NC/Nga mice, spontaneously develop an eczematous AD‐like skin lesion when kept under conventional conditions, but not under specific pathogen‐free (SPF) conditions, have been thought to be an animal model for AD. In this study, to determine whether newly identified cytokine, IL‐31, may be involved in pruritus of AD, we examined the IL‐31 expression in spontaneous dermatitis model which showed itch‐associated long‐lasting (over 1.5 s duration) scratching behavior and compared with that of hapten‐induced contact dermatitis model without itch‐associated long‐lasting scratching behavior, using NC/Nga mice. In NC/Nga mice cohabited with NC/Nga mice which developed severe dermatitis for 2 weeks (conventional NC/Nga mice), the numbers of long‐lasting scratching counts were significantly increased. Yet in 2,4,6‐trinitrochlorobenzene (TNCB)‐sensitized and challenged mice (TNCB‐applied NC/Nga mice), no significant increase in long‐lasting scratching counts was observed. In conventional NC/Nga mice with long‐lasting scratching behavior, expression of IL‐31 mRNA was increased, while in TNCB‐applied NC/Nga mice without long‐lasting scratching behavior, the expression of IL‐31 mRNA were unchanged. There was a good correlation between the scratching counts and expression of IL‐31 mRNA in conventional NC/Nga mice, but not so in TNCB‐applied NC/Nga mice. These results suggest that IL‐31 causes the itch‐associated scratching behavior in conventional NC/Nga mice, an experimental animal model for AD.

Modulation of scratching behavior by silencing an endogenous cyclooxygenase-1 gene in the skin through the administration of siRNA

RNA interference (RNAi) is rapidly becoming a major tool that is revolutionizing research in the bioscience and biomedical fields. To apply the RNAi technique in vivo, it is crucial to develop appropriate methods of guiding the short interfering RNA (siRNA) molecules to the right tissues and cells. Here, we demonstrate an efficient method for performing gene knockdown in the body skin using the in vivo electro‐transduction of siRNA. Using this method, we examined whether the targeted silencing of the cyclooxygenase (COX) gene in the skin could modulate the scratching behavior of an atopic dermatitis mouse model.

Scratching behavior in spontaneous- or allergic contact-induced dermatitis in NC/Nga mice.

Abstract:  NC/Nga mice have pathological and behavioral features similar to those seen in human atopic dermatitis. There are two known dermatitis models in NC/Nga mice, one being spontaneous‐induced dermatitis under conventional conditions and the other 2,4,6‐trinitrochlorobenzene (TNCB)‐induced allergic contact dermatitis. However, there are significant differences in time course on development of dermatitis. We studied the role of scratching behavior (sign of itch) on the development of dermatitis on spontaneous‐ and TNCB‐induced dermatitis. We measured scratching counts, transepidermal water loss (TEWL), and skin inflammation score, under conventional conditions or by applying 5% TNCB once a week for 6 weeks in NC/Nga mice. In spontaneous‐induced dermatitis, scratching counts increased with the passage of time. The scratching counts were significantly increased only 1 week after housing the mice under conventional conditions, but no changes were observed in cases of TNCB‐induced dermatitis. In spontaneous‐induced dermatitis, TEWL and skin‐inflammation score were gradually increased, time‐dependently. On the other hand, in TNCB‐induced dermatitis, these dependent values rapidly increased and reached a maximum only after 24 h TNCB application. These data suggest that pathogenesis of spontaneous‐ and allergic contact‐induced dermatitis was clearly different. It will be of major interest to identify the pruritic mediators causing profound scratching behavior and scratching‐induced aggravation of inflammation in the spontaneous‐induced dermatitis, as opposed to the inflammatory mediators that cause contact allergic dermatitis without major scratching.

Role of scratch-induced cutaneous prostaglandin D2 production on atopic-like scratching behaviour in mice

Abstract:  NC/Nga mice are known to develop scratching dermatitis akin to atopic dermatitis, under conventional (Conv), but not under the specific‐pathogen‐free (SPF) condition. In this study, we examined the effects of mechanical‐scratching on the spontaneous scratching counts (sign of itching), in relation to the cutaneous prostaglandin D2 (PGD2) levels in NC/Nga or BALB/c mice. Mechanical‐scratching increased the cutaneous barrier damage and PGD2 levels in both strain mice under the SPF condition. By 4 weeks of cohabitation with the skin‐lesioned NC/Nga mice, both the increase in the spontaneous scratching and development of dermatitis score were higher in the Conv‐NC/Nga than in the Conv‐BALB/c mice. At this time‐point, the cutaneous PGD2 level induced by mechanical‐scratching was significantly lower in the Conv‐NC/Nga when compared with that in the SPF‐NC/Nga mice, and that in the Conv‐BALB/c was almost equal to that in the SPF‐BALB/c mice. With mechanical scratches, the cohabitation‐induced scratching was suppressed in the Conv‐BALB/c, but not in the Conv‐NC/Nga mice. These results suggest that the scratch‐induced cutaneous PGD2 inhibits scratching and the subsequent development of dermatitis in BALB/c, while the impaired scratch‐induced cutaneous PGD2 production in the NC/Nga mice resulted in no suppression of scratching, and aggravated the dermatitis.

Itch‐associated scratching contributes to the development of dermatitis and hyperimmunoglobulinaemia E in NC/Nga mice

Abstract:  Atopic dermatitis (AD) is related to immunoglobulin E (IgE) production, and a type‐1 and type‐2 helper T cell (Th1/Th2) imbalance has been hypothesized as the aetiology. While itching and scratching are important factors in the development of dermatitis, the mechanisms underlying these phenomena are poorly understood. We investigated the relationship between scratching, transepidermal water loss (TEWL), signs of dermatitis and serum Ig levels in NC/Nga mice, a model of AD. We also sensitized specific pathogen‐free (SPF)‐NC/Nga mice and BALB/c mice to mite antigen to determine the effects of IgE overproduction on scratching and investigated the involvement of mast cells and T/B cells in the induction of scratching using WBB6F1‐W/Wv mice and C.B.17/Icr‐scid mice. Under conventional conditions, the scratch counts increased, followed by increases in TEWL and the inflammation score in NC/Nga mice that were not kept under SPF conditions. However, no change was observed in scratching, TEWL, or signs of dermatitis in mite antigen‐sensitized SPF‐NC/Nga and BALB/c mice, although the serum total IgE, IgG1 and IgG2a levels increased. The scratch count increased significantly in both the WBB6F1‐W/Wv mice and C.B.17/Icr‐scid mice when they were co‐housed with skin‐lesioned NC/Nga mice, raised under conventional conditions. These results show that IgE overproduction results from itch‐associated scratching‐induced dermatitis in NC/Nga mice.

COX-1 inhibition enhances scratching behaviour in NC/Nga mice with atopic dermatitis

Abstract:  NC/Nga (NC) mice, spontaneously develop an eczematous atopic dermatitis (AD)‐like skin lesion when kept under conventional condition (Conv), but not under specific pathogen‐free (SPF) conditions, have been thought to be an animal model of AD. We have previously shown that PGD2 and arachidonic acid inhibited the scratching behaviour of NC mice, while indomethacin enhanced it. This study was designed to assess the role of cyclooxygenase (COX)‐1 and COX‐2 in the itch‐related scratching behaviour of NC mice. We examined the expression of COX in the skin using real‐time PCR and Western blotting and the effects of SC‐560 (a COX‐1 selective inhibitor) or NS‐398 (a COX‐2 selective inhibitor) on scratching behaviour in relation to skin prostaglandin (PG) levels in NC mice. COX‐1 mRNA expression was unchanged and protein expression decreased in Conv NC mice compared with that of SPF mice. By contrast, COX‐2 mRNA and protein expression increased in Conv NC mice. SC‐560 increased scratching behaviour and significantly reduced skin PGD2, PGE2 and PGF2α levels, but NS‐398 did not have effects on scratching and skin PG level. Moreover, the topical application of PGD2, which might be the endogenous inhibitor of itching, suppressed the SC‐560‐induced enhancement of scratching behaviour by NC mice. These results suggest COX‐1‐coupled skin PGD2 biosynthesis plays a physiological role in inhibiting regulation of pruritus in NC mice with AD.

Time course changes of scratching counts, dermatitis symptoms, and levels of cutaneous prostaglandins in NC/Nga mice

Abstract:  NC/Nga (NC) mice are known to develop dermatitis resembling atopic dermatitis (AD) in conventional (Conv) conditions, but not in specific pathogen‐free (SPF) conditions. We reported that the ability of skin prostaglandin D2 (PGD2) production, which might be the endogenous inhibitor of itching, was attenuated in skin‐lesioned Conv‐NC mice. We examined the age‐related change in scratching, dermatitis symptoms, and skin PGs of SPF‐ and Conv‐NC mice. In Conv‐NC, PGD2 increased at 7 weeks, at which scratching counts increased, but dermatitis did not develop. PGE2, PGI2 and PGF2α increased at 10 and 13 weeks, at which dermatitis developed. The ability to produce skin PGs was examined by measuring PGs after application of arachidonic acid or after mechanical scratching using a wire brush. In Conv‐NC, PGD2 production at 13 weeks was lower than at 7 weeks. In Conv‐NC, hematopoietic PGD synthase (hPGDS) expression in the skin at 13 weeks was lower than at 7 weeks by Western blotting and immunohistochemical analysis. The increase of skin PGD2 level in the early phase of the development of dermatitis is due to the stress of extensive scratching, but did not increase in spite of the stress of extensive scratching in the late phase, due to decreasing capacity of PGD2 production attributable to decreasing hPGDS expression in Conv‐NC mice. These results suggest that a decreased ability to produce skin PGD2 production could enhance scratching and aggravate dermatitis in Conv‐NC mice.

Increased scratching counts depend on a decrease in ability of cutaneous prostaglandin D2 biosynthesis in NC/Nga mice with atopic dermatitis

Abstract:  Spontaneous and 2,4,6‐trinitrochlorobenzene (TNCB)‐induced dermatitis models using NC/Nga mice have been recognized as animal models of atopic dermatitis. We reported that scratching behavior leads to dermatitis in a spontaneous dermatitis but not in a TNCB‐induced dermatitis. Prostaglandin D2 (PGD2) suppressed the scratching behavior of NC/Nga mice, suggesting that PGD2 plays a physiological role on inhibiting pruritus. We studied whether there was a difference in skin PG contents between spontaneous and TNCB‐induced dermatitis. Spontaneous dermatitis was induced by cohabitation with NC/Nga mice having severe skin lesions. TNCB‐induced dermatitis was caused by applications of TNCB. PGD2, PGE2, 6keto‐PGF1α, and PGF2α contents in the skin were examined using enzyme‐immunoassay kits. For studying ability to produce skin PGs, PG contents were evaluated after topical treatment of arachidonic acid (AA) or mechanical scratching. In spontaneous dermatitis, PGE2, 6keto‐PGF1α, and PGF2α contents increased with dermatitis, but only PGD2 did not do so. In TNCB‐induced dermatitis, PGD2, PGE2, 6keto‐PGF1α, and PGF2α increased. Determination of skin PG contents after AA treatment or mechanical scratching revealed that skin PGD2 production of conventional group of spontaneous dermatitis was lower than the specific pathogen‐free group. It seemed that ability of skin PGD2 production was attenuated in spontaneous dermatitis. These results suggest that enhancement of scratching behavior in spontaneous dermatitis was caused by the defect of ability to produce PGD2, which plays a physiological role in inhibiting pruritus, resulting in development of dermatitis.

Effects of indomethacin and dexamethasone on mechanical scratching-induced cutaneous barrier disruption in mice.

Abstract:  Effects of indomethacin and dexamethasone on recovery of cutaneous barrier disruption induced by mechanical scratching were examined. Cutaneous barrier was disrupted by scratching using a stainless‐steel wire brush (mechanical scratching) and compared to cutaneous application of acetone/ether (1 : 1) mixture (AE) and tape‐stripping. Increase of transepidermal water loss (TEWL), as an indicator of a broken skin barrier, and recovery period for mechanical scratching were higher and longer than those for AE treatment and tape‐stripping and we also confirmed the severity of skin damage in a histological study. Topical application of moisturizers showed a temporal effect, rapidly decreased TEWL on mechanical scratching‐ or AE treatment‐induced cutaneous barrier disruption, and gradually increased base levels from 4 to 12 h after treatment. Topical application of indomethacin or dexamethasone prolonged the recovery period for the cutaneous barrier, and concomitant use further worsened the status of the barrier. Additionally, we examined the effects of prostaglandins (PGs) and inflammatory cytokine on mechanical scratching‐induced cutaneous barrier disruption pretreated with indomethacin and dexamethasone. As a results, PGD2 and interleukin (IL)‐1β significantly accelerated the recovery of cutaneous barrier disruption by mechanical scratching but such was not the case with PGE2, IL‐1α, and tumor necrosis factor‐α treatment. These results suggest that indomethacin and dexamethasone prolonged the recovery period caused by inhibition of PGD2 and IL‐1β. Mechanical scratching‐induced cutaneous barrier disruption may be a useful method for evaluating means of recovery from skin damage.

The importance of brain PGE2 inhibition versus paw PGE2 inhibition as a mechanism for the separation of analgesic and antipyretic effects of lornoxicam in rats with paw inflammation.

Objectives Lornoxicam is a non‐selective cyclooxygenase inhibitor that exhibits strong analgesic and anti‐inflammatory effects but a weak antipyretic effect in rat models. Our aim was to investigate the mechanism of separation of potencies or analgesic and antipyretic effecls of lornoxicam in relatioin to its effect on prostaglandin E2 (PGE2) production in the inflammatory paw and the brain.