UNASSIGNED: Atopic dermatitis (AD) is a chronic, non-infectious inflammatory dermatosis. Chloroquine (CQ) has long been proven to possess anti-inflammatory properties.
UNASSIGNED: This paper aims to investigate the impact of CQ on type 2 inflammatory response in MC903-induced AD mice.
UNASSIGNED: An AD mouse model was established via MC903 induction. After CQ treatment, AD mice were intraperitoneally injected with polyinosinic: polycyclic acid [poly (I:C)] or Nigericin. Dermatitis severity was scored, and the thickness of the left ear was measured. The pathological changes in mouse skin tissues were observed by H&E staining. The number of mast cells was counted via TB staining. The content of peripheral blood T-helper 2 (Th2) cells and levels of immunoglobulin E (IgE), thymic stromal-derived lymphopoietin (TSLP), interleukin (IL)-4, IL-13, interferon (IFN)-γ, IL-1β, and IL-18 were assessed by flow cytometry and ELISA. The levels of toll-like receptor 3 (TLR3), NLRP3, ASC, and cleaved caspase-1 proteins in skin tissues were determined by Western blot.
UNASSIGNED: CQ treatment abated dermatitis severity and left ear thickness in AD mice, alleviated skin damage, reduced mast cell number, diminished IgE, TSLP, IL-4, and IL-13 levels, and peripheral blood Th2 cell content, with no significant changes in IFN-γ level. CQ alleviated type 2 inflammatory response in AD mice by inhibiting the activation of TLR3. CQ suppressed NLRP3 inflammasome activation. Activating TLR3/NLRP3 annulled CQ-mediated alleviation on type 2 inflammatory response in AD mice.
UNASSIGNED: CQ alleviated type 2 inflammatory response in AD mice by inhibiting TLR3 activation and NLRP3 inflammasome activation.

Therapeutic strategies that enhance regulatory T (Treg) cell proliferation or suppressive function hold promise for the treatment of autoimmune and inflammatory diseases. We previously reported that the topical application of the vitamin D3 analog MC903 systemically expands Treg cells by stimulating the production of thymic stromal lymphopoietin (TSLP) from the skin. Using mice lacking TSLP receptor expression by dendritic cells (DCs), we hereby show that TSLP receptor signaling in DCs is required for this Treg expansion in vivo. Topical MC903 treatment of ear skin selectively increased the number of migratory DCs in skin-draining lymph nodes (LNs) and upregulated their expression of co-stimulatory molecules. Accordingly, DCs isolated from skin-draining LNs but not mesenteric LNs or spleen of MC903-treated mice showed an enhanced ability to promote Treg proliferation, which was driven by co-stimulatory signals through CD80/CD86 and OX40 ligand. Among the DC subsets in the skin-draining LNs of MC903-treated mice, migratory XCR1- CD11b+ type 2 and XCR1- CD11b- double negative conventional DCs promoted Treg expansion. Together, these data demonstrate that vitamin D3 stimulation of skin induces TSLP expression, which stimulates skin migratory DCs to expand Treg cells. Thus, topical MC903 treatment could represent a convenient strategy to treat inflammatory disorders by engaging this pathway.

BACKGROUND: Atopic dermatitis is a common skin disease caused by genetic susceptibility, environmental factors, immune response, and skin barrier dysfunction. Kaempferol is a natural flavonoid widely found in tea, vegetables, and fruits and has been reported to have excellent anti-inflammation activity. However, the therapeutic effect of kaempferol on atopic dermatitis is unclear.
OBJECTIVE: This study aimed to elucidate the effect of kaempferol on skin inflammation in atopic dermatitis.
METHODS: The suppressive effect of kaempferol administration on skin inflammation was examined using MC903-induced atopic dermatitis-like skin inflammation mouse model. Quantification of skin dermatitis and transepidermal water loss was performed. A histopathological study was performed to examine thymic stromal lymphopoietin expression, cornified envelope proteins such as filaggrin, loricrin, and involucrin, and the numbers of infiltrating inflammatory cells, including lymphocytes, macrophages, and mast cells in the dermatitis area. The expressions of IL-4 and IL-13 were investigated by qPCR and flow cytometry analysis using skin tissues. The expression of HO-1 was investigated by western blot and qPCR.
RESULTS: Kaempferol therapy significantly suppressed MC903-induced dermatitis, TEWL, TSLP, and HO-1 expression, and infiltration of inflammatory cells. Kaempferol therapy improved the decreased expressions of filaggrin, loricrin, and involucrin in MC903-induced dermatitis skin site. The expressions of IL-4, and IL-13 were partially decreased in kaempferol-treated mice.
CONCLUSIONS: Kaempferol might improve MC903-induced dermatitis via suppression of type 2 inflammation and improvement of barrier dysfunction by inhibition of TSLP expression and oxidative stress. Kaempferol might have the potential to be a new treatment for atopic dermatitis.

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.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by eczema and itching. Recently, mTORC, a central regulator of cellular metabolism, has been reported to play a critical role in immune responses, and manipulation of mTORC pathways has emerged as an effective immunomodulatory drug. In this study, we assessed whether mTORC signaling could contribute to the development of AD in mice. AD-like skin inflammation was induced by a 7-day treatment of MC903 (calcipotriol), and ribosomal protein S6 was highly phosphorylated in inflamed tissues. MC903-induced skin inflammation was ameliorated significantly in Raptor-deficient mice and exacerbated in Pten-deficient mice. Eosinophil recruitment and IL-4 production were also decreased in Raptor deficient mice. In contrast to the pro-inflammatory roles of mTORC1 in immune cells, we observed an anti-inflammatory effect on keratinocytes. TSLP was upregulated in Raptor deficient mice or by rapamycin treatment, which was mediated by hypoxia-inducible factor (HIF) signaling. Taken together, these results from our study indicate the dual roles of mTORC1 in the development of AD, and further studies on the role of HIF in AD are warranted.

Atopic dermatitis (AD) is a multifactorial disease with underlying barrier disruption and altered microbial flora, resulting in dry skin and eczematous inflammation with persistent pruritis. Mouse models have been heavily used to investigate AD pathophysiology. Among various AD mouse models, AD-like inflammation induced by topical calcipotriol, a vitamin D3 analog referred to as MC903 in experimental settings, is a versatile model that can be applied to any strain of mice, which can be used for immunologic and morphologic studies. Herein, we provide basic protocols for the topical application of MC903 and approaches to assess phenotypes. After inducing AD-like inflammation, the skin is harvested for flow cytometry analysis, as well as for histologic and immunofluorescence microscopy analyses. The combination of these approaches enables accurate characterization of the degree of inflammation, type of inflammatory infiltrate, and localization of immune infiltrates. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Application of MC903 and gross phenotype assessment Basic Protocol 2: Processing skin for flow cytometry analysis Support Protocol: Skin immune cell surface staining and flow cytometry analysis Basic Protocol 3: Harvesting skin for histologic analysis Basic Protocol 4: Immunofluorescence staining to identify immune cell infiltrates.

Atopic dermatitis (AD) is a chronic, relapsing, and extremely pruritic inflammatory skin disease with a particular impact on children. AD pathogenesis is not yet fully understood, and there is no curative treatment for this disease. Therefore, several genetically or chemically-induced AD mouse models have been developed. These preclinical mouse models are an indispensable research tool for studying AD pathogenesis and evaluating the efficacy of new candidate AD therapeutics. A commonly used mouse model of AD has been developed using the topical application of a low-calcemic analog of vitamin D3, MC903, to induce AD-like inflammatory phenotypes that closely resemble human AD. Moreover, this model shows a minimal effect on systemic calcium metabolism that is observed in the vitamin D3-induced AD model. Thus, an expanding number of studies use the MC903-induced AD model to interrogate AD pathobiology in vivo and to test new candidate small molecule and monoclonal antibody therapies. This protocol describes in detail functional measurements including the measurement of skin thickness, which is a surrogate marker for ear skin inflammation, as well as itch assessment, histological evaluation to assess the structural changes associated with AD skin inflammation, and preparation of single-cell suspensions from ear skin and draining lymph nodes for the assessment of inflammatory leukocyte subset infiltration in these tissues using flow cytometry. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Topical application of MC903 induces AD-like skin inflammation Support Protocol 1: Measurement of ear skin thickness Support Protocol 2: Itch assessment Support Protocol 3: Dissection of ear skin and ear draining lymph nodes Support Protocol 4: Histological evaluation and quantification Support Protocol 5: Preparation of single-cell suspension from ear skin and draining lymph nodes for the assessment of inflammatory immune cell infiltration using flow cytometry.

MC903 skin inflammation model is one of well-characterized murine models of atopic dermatitis and driven by TSLP-mediated type 2 inflammation. Since it can be prepared simply by repetitive applications of MC903 and shows consistent clinical results, this model has been widely used. However, in contrast to human atopic dermatitis which is chronic and closely related to TH2 cells, MC903 induces inflammations temporarily and even in the absence of T cells. Here, we modified the MC903 treatment schedule and developed a chronic MC903-induced skin inflammation model. Mice were sensitized with a high dose of MC903 and challenged with a low dose of MC903. Prior to challenge, mice were allowed to recover completely from the inflammation which occurred during the sensitization. The challenge of MC903 induced skin swelling and type 2 inflammations more rapidly, which was dependent on CD4+ T cells and IL-33. We expect that our mouse model will be beneficial for studying the late course of atopic dermatitis.

Atopic dermatitis (AD) is a complex, often lifelong allergic disease with severe pruritus affecting around 10% of both humans and dogs. To investigate the role of mast cells (MCs) and MC-specific proteases on the immunopathogenesis of AD, a vitamin D3-analog (MC903) was used to induce clinical AD-like symptoms in c-kit-dependent MC-deficient Wsh-/- and the MC protease-deficient mMCP-4-/-, mMCP-6-/-, and CPA3-/- mouse strains. MC903-treatment on the ear lobe increased clinical scores and ear-thickening, along with increased MC and granulocyte infiltration and activity, as well as increased levels of interleukin 33 (IL-33) locally and thymic stromal lymphopoietin (TSLP) both locally and systemically. The MC-deficient Wsh-/- mice showed significantly increased clinical score and ear thickening albeit having lower ear tissue levels of IL-33 and TSLP as well as lower serum levels of TSLP as compared to the WT mice. In contrast, although having significantly increased IL-33 ear tissue levels the chymase-deficient mMCP-4-/- mice showed similar clinical score, ear thickening, and TSLP levels in ear tissue and serum as the WT mice, whereas mMCP-6 and CPA3 -deficient mice showed a slightly reduced ear thickening and granulocyte infiltration. Our results suggest that MCs promote and control the level of MC903-induced AD-like inflammation.

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease with pruritus and high prevalence. Indeed, 15-30 % of children and 2-10 % of adults from industrialized countries are affected. Acute AD lesions are characterized by epidermal hyperplasia associated with a dominant Th2/Th17 immune response and dermal inflammatory infiltrates. Moreover, the expression of alarmins such as TSLP, IL-33, and IL-25 is upregulated in acute AD lesions. Topical application of vitamin D3 or of its low-calcemic analog MC903 induces changes in skin morphology and inflammation resembling immune perturbations observed in acute lesions of patients with AD. Mice treated with MC903 or vitamin D3 additionally display increased serum IgE levels, as observed in patients with extrinsic AD. Interestingly, these symptoms are not dependent on mouse gender or on genetic background. Thus, the easiness of this mouse model renders it very attractive to study immunologic abnormalities involved in AD development or maintenance. Furthermore, this model might be useful for preclinical studies aiming at unraveling new therapeutic strategies to treat AD. In this chapter, we describe the induction and major features of MC903 and vitamin D3-induced AD-like inflammation in mice.