A CCR4 antagonist attenuates atopic dermatitis-like skin inflammation by inhibiting the recruitment and expansion of Th2 cells and Th17 cells.

CCR4 is a major trafficking receptor for T-helper (Th) 2 cells and Th17 cells and is considered as a potential therapeutic target for atopic dermatitis (AD). The CCR4 ligands CCL17 and CCL22 have been reported to be upregulated in the skin lesions of AD patients. Of note, thymic stromal lymphopoietin (TSLP), a master regulator of the Th2 immune response, promotes the expression of CCL17 and CCL22 in AD skin lesions. Here, we investigated the role of CCR4 in an AD mouse model induced by MC903, a TSLP inducer. Topical application of MC903 to ear skin increased the expression of not only TSLP but also CCL17, CCL22, the Th2 cytokine IL-4, and the Th17 cytokine IL-17A. Consistently, MC903 induced AD-like skin lesions as shown by increased epidermal thickness; increased infiltration of eosinophils, mast cells, type 2 innate lymphoid cells, Th2 cells, and Th17 cells; and elevated serum levels of total IgE. We also found increased expansion of Th2 cells and Th17 cells in the regional lymph nodes (LNs) of AD mice. Compound 22, a CCR4 inhibitor, ameliorated AD-like skin lesions with reduction of Th2 cells and Th17 cells in the skin lesions and regional LNs. We further confirmed that compound 22 diminished the expansion of Th2 cells and Th17 cells in the coculture of CD11c+ dendritic cells (DCs) and CD4+ T cells derived from the regional LNs of AD mice. Collectively, CCR4 antagonists may exhibit anti-allergic effects by inhibiting both the recruitment and expansion of Th2 cells and Th17 cells in AD.

CCR4 plays a pivotal role in Th17 cell recruitment and expansion in a mouse model of rheumatoid arthritis.

T helper 17 (Th17) cells express CC chemokine receptor 4 (CCR4) and secrete cytokines such as interleukin-17A (IL-17A) and granulocyte macrophage colony-stimulating factor (GM-CSF), while dendritic cells (DCs) produce CC chemokine ligand 22 (CCL22), a CCR4 ligand, upon stimulation with GM-CSF. Th17 cells are known to play a critical role in the pathogenesis of rheumatoid arthritis (RA). CCL22 has also been shown to be up-regulated in the synovial tissues of RA patients. Here, we investigated the role of CCR4 in collagen-induced arthritis (CIA), a mouse model of RA. DBA/1J mice efficiently developed CIA as shown by erythema, paw swelling, joint rigidity, and joint destruction. Th17 cells were increased in the arthritic joints and regional lymph nodes (LNs) of CIA mice. A fraction of Th17 cells were also shown to produce GM-CSF. On the other hand, we observed no significant increases of Th2 cells or Treg cells, the T cell subsets also known to express CCR4, in these tissues. We further observed clusters of CCR4-expressing memory Th17 cells and CCL22-producing DCs in the regional LNs of CIA mice, supporting the role of the CCR4-CCL22 axis in the expansion of Th17 cells in the regional LNs. Compound 22, a CCR4 inhibitor, ameliorated the disease severity with reduction of Th17 cells in the arthritic joints and regional LNs and Th17-DC clusters in the regional LNs. We further confirmed that CCR4-deficient mice in the C57BL/6J background were highly resistant to CIA induction compared with wild-type mice. Collectively, CCR4 contributes to the pathogenesis of CIA and may thus represent a new therapeutic target for RA.

Anti-allergic effect of ascorbic acid derivative DDH-1 in a mouse model of atopic dermatitis.

Atopic dermatitis (AD) is the most common inflammatory skin disease, which is characterized by excessive Th2 immune responses. In AD patients, the expression of the chemokines CCL17 and CCL22 is increased in skin lesions, leading to the infiltration of Th2 cells. In addition, typical pro-inflammatory cytokines, including TNF-α, IL-1ß and IL-6, have also been shown to be associated with the pathogenesis of AD. Recently, DDH-1, an ascorbic acid derivative, has been synthesized and demonstrated to have a more stabilized structure and better skin penetrability. Furthermore, DDH-1 has been shown to suppress pro-inflammatory cytokine expression in vitro and in vivo. Therefore, using an AD mouse model, we evaluated the effect of DDH-1 to reduce allergic skin inflammation. We found that cutaneous administration of DDH-1 significantly reduced the expression levels of TNF-α, IL-1ß and IL-6 in the skin lesions of AD-like mice. Additionally, DDH-1 administration also significantly reduced the expression levels of CCL17 and CCL22, resulting in decreased skin infiltration of Th2 cells. Consequently, DDH-1 reduced ear and epidermal thickness, the serum IgE levels and the number of infiltrating inflammatory cells and mast cells into the AD-like skin lesions. Combination treatment with DDH-1 and corticosteroid more efficiently improved the skin lesions compared with corticosteroid alone. Collectively, our results suggest that DDH-1 has an anti-allergic effect in an AD mouse model by reducing not only the pro-inflammatory cytokine expression but also the Th2-associated chemokine expression. Thus, DDH-1 may be beneficial for AD treatment and prevention as a monotherapy or in combination with corticosteroids.

P2X receptor agonist enhances tumor-specific CTL responses through CD70+ DC-mediated Th17 induction.

Extracellular ATP is known to promote Th17 cell differentiation in the intestinal lamina propria by stimulating CD70+CD11clow dendritic cells (DCs) via P2X receptors (P2XRs). Recent studies have also shown that Th17 cells enhance antitumor immunity by directly promoting proliferation of cytotoxic T lymphocytes (CTLs). These finding led us to test a P2XR agonist, αß-methylene ATP (αß-ATP), as a mucosal vaccine adjuvant to promote CTL responses through Th17 induction. We demonstrated that (i) CD70+CD11clow DCs were present in the nasal lamina propria and expressed P2X1R, P2X2R and P2X4R; (ii) CD70+CD11clow DCs isolated from the nasal lamina propria enhanced Th17 cell differentiation of cocultured splenic CD4+ T cells upon stimulation with αß-ATP; (iii) mice intranasally immunized with ovalbumin (OVA) and αß-ATP had increased OVA-specific Th17 cells and CTLs in the nasal lamina propria and regional lymph nodes; (iv) mice intranasally immunized with OVA and αß-ATP also had elevated resistance to E.G7-OVA tumor growth compared with those intranasally immunized with OVA alone; (v) suramin, a broad-range inhibitor of P2 receptors, suppressed the increases of OVA-specific Th17 cells and CTLs in mice intranasally immunized with OVA and αß-ATP; and (vi) suramin also abrogated the enhanced antitumor immunity of mice intranasally immunized with OVA and αß-ATP against E.G7-OVA. Collectively, αß-ATP may be a promising mucosal adjuvant that promotes antigen-specific CTL responses via CD70+CD11clow DC-mediated Th17 induction.

Transcutaneous immunization with a highly active form of XCL1 as a vaccine adjuvant using a hydrophilic gel patch elicits long-term CD8+ T cell responses.

Memory CD8+ cytotoxic T-lymphocytes (CTLs) play a key role in protective immunity against infection and cancer. However, the induction of memory CTLs with currently available vaccines remains difficult. The chemokine receptor XCR1 is predominantly expressed on CD103+ cross-presenting dendritic cells (DCs). Recently, we have demonstrated that a high activity form of murine lymphotactin/XCL1 (mXCL1-V21C/A59C), a ligand of XCR1, can induce antigen-specific memory CTLs by increasing the accumulation of CD103+ DCs in the vaccination site and the regional lymph nodes. Here, we combined a hydrophilic gel patch as a transcutaneous delivery device and mXCL1-V21C/A59C as an adjuvant to further enhance memory CTL responses. The transcutaneous delivery of ovalbumin (OVA) and mXCL1-V21C/A59C by the hydrophilic gel patch increased CD103+ DCs in the vaccination site and the regional lymph nodes for a prolonged period of time compared with the intradermal injection of OVA and mXCL1-V21C/A59C. Furthermore, the hydrophilic gel patch containing OVA and mXCL1-V21C/A59C strongly induced OVA-specific memory CTLs and efficiently inhibited the growth of OVA-expressing tumors more than the intradermal injection of OVA and mXCL1-V21C/A59C. Collectively, this type of hydrophilic gel patch and a high activity form of XCL1 may provide a useful tool for the induction of memory CTL responses.

Ascorbic acid derivative DDH-1 ameliorates psoriasis-like skin lesions in mice by suppressing inflammatory cytokine expression.

Psoriasis is a chronic inflammatory skin disease in which inflammatory cytokines play a major role in its pathogenesis. Because DDH-1, a novel amphipathic ascorbic acid derivative, has been recently shown to reduce inflammatory cytokine expression in human keratinocytes in vitro, we investigated its effect on imiquimod-induced psoriasis-like skin lesions in C57BL/6 mice. We first found that IL-1ß and TNF-α mRNA expression was significantly decreased in the skin lesions treated with DDH-1. Furthermore, cutaneous administration of DDH-1 ameliorated psoriasis-like skin lesions. These results suggest that DDH-1 may be effective in the prevention and supplemental treatment of psoriasis.

A CCR4 antagonist enhances DC activation and homing to the regional lymph node and shows potent vaccine adjuvant activity through the inhibition of regulatory T-cell recruitment.

CCR4 is a major chemokine receptor expressed by Treg cells that downregulate immune responses. Here, we investigated the role of CCR4-mediated Treg cell recruitment in antigen-specific immune responses. CCR4-deficient mice immunized intramuscularly with ovalbumin (OVA) showed enhanced OVA-specific IgG responses. Furthermore, intramuscular administration of OVA induced the expression of MDC/CCL22, a ligand for CCR4, in macrophages of the muscle tissues, and enhanced the recruitment of CCR4+ Treg cells in wild-type mice, whereas this recruitment of Treg cells was severely impaired in CCR4-deficient mice. Furthermore, OVA-loaded dendritic cells (DCs) derived from the muscle injection site of CCR4-deficient mice had an upregulated expression of the DC activation marker CD40 and 86, and the lymphoid organ homing receptor CCR7 resulting in an increased number of migratory DCs in the regional lymph node. Compound 22, a CCR4 antagonist, also inhibited the recruitment of Treg cells to the muscle tissue, and further enhanced DC activation and homing to the regional lymph node. Consequently, Compound 22 enhanced OVA-specific IgG responses, and the expression levels of IL-4 and IFN-γ in CD4+ T cells and the levels of IFN-γ in CD8+ T cells. Finally, intramuscular administration of OVA and Compound 22 significantly inhibited the growth of OVA-expressing tumors. Collectively, CCR4 plays a pivotal role in Treg cell recruitment to the muscle tissue, and intramuscular administration of CCR4 antagonists may be a promising approach for enhancing vaccine efficacy.

Class switch recombination and somatic hypermutation of virus-neutralizing antibodies are not essential for control of friend retrovirus infection.

Toll-like receptor 7 and Myd88 are required for antiretroviral antibody and germinal center responses, but whether somatic hypermutation and class-switch recombination are required for antiretroviral immunity has not been examined. Mice deficient in activation-induced cytidine deaminase (AID) resisted Friend virus infection, produced virus-neutralizing antibodies, and controlled viremia. Passive transfer demonstrated that immune IgM from AID-deficient mice contributes to Friend virus control in the presence of virus-specific CD4+ T cells.

Aging rather than sun exposure is a major determining factor for the density of miR-125b-positive epidermal stem cells in human skin.

Sunlight exposure and aging are two major factors in the deterioration of skin function. In the present study, we used eighty formalin-fixed human skin samples from sun-exposed and unexposed areas from old and young individuals to evaluate the presence of miR-125b-positive epidermal stem cells (ESCs) by in situ hybridization. miR-125b-positive ESCs were detected in the basal layer of the epidermis. The density of miR-125b-positive ESCs was significantly associated with age rather than sun exposure, whereas the density of miR-125b-positive ESCs tended to decrease in the sun-exposed area. These data suggest the potential use of miR-125b as a surrogate marker for the quality of epidermal cells.

Alopecia areata: infiltration of Th17 cells in the dermis, particularly around hair follicles.

BACKGROUND: The precise pathogenesis of alopecia areata remains unknown, although this disease seems to be triggered by helper T cell infiltration in hair follicles. Recent studies of psoriasis and vitiligo have demonstrated the involvement of Th17 cells. Psoriasis and vitiligo occasionally develop concomitantly or inversely in patients with alopecia areata. OBJECTIVE: The aim of this study was to determine whether Th17 cells are present in the affected lesions of alopecia areata. METHODS: We performed immunofluorescent staining of representative immunocompetent cells that had infiltrated into the skin of the scalp in 4 individuals with alopecia areata (single patchy alopecia areata, multiple patchy alopecia areata, alopecia totalis and alopecia universalis). RESULTS: We found the infiltration of CD4(+)IL-17A(+) Th17 cells in the dermis, particularly around hair follicles, in all 4 cases. CONCLUSIONS: These findings suggest the possibility that alopecia areata is induced by a Th17 cell-associated autoimmune mechanism.

Development of new in vivo imaging methodology and system for the rapid and quantitative evaluation of the visual appearance of facial skin firmness.

BACKGROUND/AIMS: Lack of firmness is a key sign of skin aging, but there are no methods for quantifying the firmness of facial skin as perceived visually. The objective of this study was to develop a convenient and accurate method for this purpose. METHODS: A compact imaging system (Magic Ring) was developed to capture images of facial skin. By using an image-analysis algorithm, the number of facial lines and their direction were analyzed to give an index termed the 'Ageless Vector'. Correlations between the Ageless Vector and visually perceived facial skin firmness, mechanical skin firmness (R5), and actual age were examined for 108 Asian females. The technique was also used to assess the effects of a 14-day skin-moisturization regimen in 47 Asian female volunteers. RESULTS: The Ageless Vector showed highly significant correlation with visually perceived skin firmness (r = 0.816) and significant correlation with mechanical skin firmness (R5) (r = -0.775). Skin moisturization significantly improved both the Ageless Vector (P < 0.05) and the visual grading of apparent skin firmness. CONCLUSIONS: We confirmed by two clinical studies that the imaging methodology using the Ageless Vector and the Magic Ring facial-imaging system was sufficiently sensitive to permit the measurement of apparent visible skin firmness and that it is an excellent method suitable for practical in vivo applications.

Localised giant haematoma and subsequent widespread purpura as a sign of acquired haemophilia A.

Patients with acquired haemophilia A usually show widespread subcutaneous bleeding. We describe an 86-year-old man with acquired haemophilia A associated with prostate carcinoma, showing initial localised giant haematoma and subsequent widespread subcutaneous bleeding. A localised giant haematoma may present as a first and important sign of acquired haemophilia A.

Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1.

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.

Clinical impact of switching to a second EGFR-TKI after a severe AE related to a first EGFR-TKI in EGFR-mutated NSCLC.

OBJECTIVE: Somatic mutations in the epidermal growth factor receptor gene are associated with a therapeutic response to epidermal growth factor receptor tyrosine kinase inhibitors such as gefitinib and erlotinib in patients with non-small cell lung cancer. Although the safety profile of these drugs is favorable, a small proportion of patients with EGFR mutation-positive non-small cell lung cancer must discontinue treatment because of adverse events such as interstitial lung disease and hepatotoxicity. Subsequent chemotherapy has not been optimized in such patients. METHODS: We performed a retrospective analysis of EGFR mutation-positive non-small cell lung cancer patients who received both gefitinib and erlotinib at our institution. Patients received the second epidermal growth factor receptor-tyrosine kinase inhibitor after experiencing an adverse event or progressive disease on the first epidermal growth factor receptor-tyrosine kinase inhibitor. RESULTS: We identified 14 patients who received both gefitinib and erlotinib in the course of their treatment. Three patients initially treated with gefitinib and two with erlotinib discontinued epidermal growth factor receptor-tyrosine kinase inhibitor therapy because of severe non-hematologic toxicity (one because of gefitinib-induced interstitial lung disease, one because of erlotinib-induced lupus erythematosus-like eruption and three because of hepatotoxicity). All five of these patients were able successfully to continue therapy with the second epidermal growth factor receptor-tyrosine kinase inhibitor with no evidence of a recurrent adverse event. Progression-free survival was significantly longer in these five patients than in the nine patients who discontinued treatment with the first epidermal growth factor receptor-tyrosine kinase inhibitor because of disease progression. CONCLUSIONS: EGFR mutation-positive non-small cell lung cancer patients who discontinue treatment with a first epidermal growth factor receptor-tyrosine kinase inhibitor because of an adverse event benefit substantially from switching to a second epidermal growth factor receptor-tyrosine kinase inhibitor before the development of drug resistance.

Freckling promoted by topical tacalcitol in a Japanese boy with left eyelid vitiligo.

Vitiligo is an acquired disorder in which the loss of melanocytes results in depigmented patches. Topical tacalcitol, a vitamin D(3) analog, is occasionally used to treat vitiligo. We report a case of a 7-year-old Japanese boy who showed moderate repigmentation and worsened freckles after using topical tacalcitol to treat a vitiligo lesion on and around the left eyelid. Topical tacalcitol might have induced and worsened the freckles, because the changes occurred during treatment. Dermatologists should be aware that topical application of tacalcitol to the cheeks can cause freckling and worsen existing freckles, in predisposed children, as described here.

Erythema dyschromicum perstans in a Japanese child.

Erythema dyschromicum perstans (EDP) is asymptomatic slate-gray to blue-brown macules. Idiopathic eruptive macular pigmentation is asymptomatic brown nonconfluent macules. We describe electron microscopic studies of a 9-year-old Japanese girl with EDP. The ultrastructural figures indicated that the production of immature, small, irregular-shaped melanosomes in melanocytes and peripheral localization of melanosomes in keratinocytes caused the clinical appearance of EDP. The ultrastructural evidence distinguishes EDP from idiopathic eruptive macular pigmentation and suggests a distinct pathogenesis of the disease.