An Altered Skin and Gut Microbiota Are Involved in the Modulation of Itch in Atopic Dermatitis.

Skin and gut microbiota play an important role in the pathogenesis of atopic dermatitis (AD). An alteration of the microbiota diversity modulates the development and course of AD, e.g., decreased microbiome diversity correlates with disease severity, particularly in lesional skin of AD. Itch is a hallmark of AD with unsatisfying treatment until now. Recent evidence suggests a possible role of microbiota in altering itch in AD through gut-skin-brain interactions. The microbial metabolites, proinflammatory cytokines, and impaired immune response lead to a modulation of histamine-independent itch, disruption of epidermal barrier, and central sensitization of itch mechanisms. The positive impact of probiotics in alleviating itch in AD supports this hypothesis, which may lead to novel strategies for managing itchy skin in AD patients. This review summarizes the emerging findings on the correlation between an altered microbiota and gut-skin-brain axis in AD, especially in modulating itchy skin.

The Pathology of Type 2 Inflammation-Associated Itch in Atopic Dermatitis.

Accumulated evidence on type 2 inflammation-associated itch in atopic dermatitis has recently been reported. Crosstalk between the immune and nervous systems (neuroimmune interactions) is prominent in atopic dermatitis research, particularly regarding itch and inflammation. A comprehensive understanding of bidirectional neuroimmune interactions will provide insights into the pathogenesis of itch and its treatment. There is currently no agreed cure for itch in atopic dermatitis; however, increasing numbers of novel and targeted biologic agents have potential for its management and are in the advanced stages of clinical trials. In this review, we summarize and discuss advances in our understanding of type 2 inflammation-associated itch and implications for its management and treatment in patients with atopic dermatitis.

Regulatory T Cells Exhibit Interleukin-33-Dependent Migratory Behavior during Skin Barrier Disruption.

Regulatory T cells (Tregs) suppress immune responses and maintain immunological self-tolerance and homeostasis. We currently investigated relationships between skin barrier condition and Treg behavior using skin barrier-disrupted mice. Skin barrier disruption was induced by repeated topical application of 4% sodium dodecyl sulfate (SDS) on mice. The number of CD4+ forkhead box protein P3 (Foxp3)+ Tregs was higher in 4% SDS-treated skins than in controls. This increasing was correlated with the degree of acanthosis. The numbers of interleukin (IL)-10+ and transforming growth factor (TGF)-ß+ Tregs also increased in 4% SDS-treated skins. Localization of IL-33 in keratinocytes shifted from nucleus to cytoplasm after skin barrier disruption. Notably, IL-33 promoted the migration of Tregs in chemotaxis assay. The skin infiltration of Tregs was cancelled in IL-33 neutralizing antibody-treated mice and IL-33 knockout mice. Thus, keratinocyte-derived IL-33 may induce Treg migration into barrier-disrupted skin to control the phase transition between healthy and inflammatory conditions.

Mechanisms and Management of Itch in Dry Skin.

Chronic itch is a burdensome clinical problem that often accompanies pathological dry skin-based conditions, such as atopic dermatitis, and systemic disorders, such as kidney diseases, with an unclear pathomechanism and treatments. One of the basic mouse models to investigate mechanisms of itch associated with dry skin is a mixture of acetone and ether followed by water. Animal studies using the acetone and ether followed by water model have revealed that many mediators and receptors, e.g. mas-related G protein-coupled receptor family, transient receptor potential, and chemokines, are responsible for itch and its hypersensitivity, supporting the hypothesis that dry skin-induced itch is a histamine-independent pathway. New insights have been acquired into the interplay between neurones and non-neuronal cells in the initiation, modulation, and sensitization of itch. Several thera-peutic options for itching have thus been developed. This review summarizes the updated pathogenesis and therapeutic strategies for itch in dry skin conditions.

Calcium-Inducible MAPK/AP-1 Signaling Drives Semaphorin 3A Expression in Normal Human Epidermal Keratinocytes.

Epidermal keratinocytes are primarily involved in the expression of semaphorin (Sema) 3A, which is involved in the regulation of cutaneous innervation. However, the mechanisms underlying the intracellular signaling of Sema3A expression in keratinocytes remain unknown. We herein investigated the signaling mechanisms for the induction of Sema3A expression in normal human epidermal keratinocytes (NHEKs). Sema3A expression is transiently increased in calcium-stimulated NHEKs, whereas it is markedly decreased in terminally differentiated NHEKs. Sema3A mRNA is mainly localized in the stratum basale and stratum suprabasale of the epidermis. We cloned the 5'-flanking region of the Sema3A gene and identified a critical region for Sema3A promoter activity within -134 base pairs of the start codon. We found transcription factor binding sites, including that for activator protein (AP)-1, in this region. Sema3A expression was increased by the co-overexpression of JunB and Fra-2 in the presence of 0.1 or 1.4 mM calcium. The calcium-mediated transient upregulation of Sema3A expression was significantly suppressed by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 or AP-1 inhibitors. These results demonstrate that the calcium-mediated transient upregulation of Sema3A in NHEKs is involved in the MEK/ERK and AP-1 signaling axis. Therefore, Sema3A mRNA may be expressed in the lower epidermis under controlled conditions by calcium via the MAPK-AP-1 axis.

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes.

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined. Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity. In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin. Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.

Aquaporin-9 facilitates membrane transport of hydrogen peroxide in mammalian cells.

Aquaporin (AQP) 9, a member of the transmembrane water channel family, is defined as a water/glycerol transporting protein. Some AQPs including AQP3 and AQP8 have been recently found to transport hydrogen peroxide (H2O2). Here we show that AQP9 facilitates the membrane transport of H2O2 in human and mice cells. Enforced expression of human AQP9 in Chinese hamster ovary-K1 potentiated the increase in cellular H2O2 after adding exogenous H2O2. In contrast, AQP9 knockdown by siRNA in human hepatoma HepG2 cells reduced the import of extracellular H2O2. In addition, the uptake of extracellular H2O2 was suppressed in erythrocytes and bone marrow-derived mast cells from AQP9 knockout mice compared with wild-type cells. Coincidentally, H2O2-induced cytotoxicity was attenuated by AQP9 deficiency in human and mice cells. Our findings implicate the involvement of AQP9 in H2O2 transport in human and mice cells.

Hepatic stellate cells relay inflammation signaling from sinusoids to parenchyma in mouse models of immune-mediated hepatitis.

UNLABELLED: Hepatic stellate cells (HSCs) constitute the liver sinusoid with Kupffer cells and liver sinusoidal endothelial cells. While the sinusoid functions as the gateway to liver inflammation, whether HSCs contribute to liver inflammation and, if so, how they exert such functions remain elusive. Here, we found that mouse as well as human HSCs expressed DP1 receptor for prostaglandin D2 selectively in the liver. Pharmacological stimulation of DP1 by BW245C, a DP1-selective agonist, suppressed the activation of cultured HSCs by tumor necrosis factor-α at least in part through down-regulation of nuclear factor kappa-light-chain-enhancer of activated B cells signaling and inhibition of c-Jun N-terminal kinase phosphorylation. DP1 deficiency or BW245C administration in mice significantly enhanced or suppressed concanavalin A (ConA)-induced hepatitis, respectively. ConA injection induced tumor necrosis factor-α and interferon-γ expression in the sinusoid, which was suppressed by administration of BW245C. Coculture of spleen cells and liver nonparenchymal cells showed that ConA first activated spleen cells and that this activation led to activation of nonparenchymal cells to secondarily produce tumor necrosis factor-α and interferon-γ. Microarray analysis revealed ConA-induced expression of endothelin-1, tissue factor, and chemokines in the liver and inducible nitric oxide synthase in hepatocytes, resulting in flow stagnation, leukocyte adherence and migration to the parenchyma, and hepatocyte death. DP1 stimulation inhibits all these events in the liver. Therefore, HSCs mediate amplification of ConA-induced liver inflammation in the sinusoid, causing direct and indirect hepatocyte injury, and DP1 stimulation inhibits this HSC activation. CONCLUSIONS: HSCs integrate cytokine-mediated inflammatory responses in the sinusoids and relay them to the liver parenchyma, and these HSC actions are inhibited by DP1 stimulation.

Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity.

Aquaporin-9 (AQP9), a water/glycerol channel protein, is expressed in several immune cells including neutrophils; however, its role in immune response remains unknown. Here we show the involvement of AQP9 in hapten-induced contact hypersensitivity (CHS), as a murine model of skin allergic contact dermatitis, using AQP9 knockout (AQP9(-/-)) mice. First, the CHS response to hapten dinitrofluorobenzene (DNFB) was impaired in AQP9(-/-) mice compared with wild-type (WT) mice. Adoptive transfer of sensitized AQP9(-/-) draining lymph node (dLN) cells into WT recipients resulted in a reduced CHS response, indicating impaired sensitization in AQP9(-/-) mice. Second, administration of WT neutrophils into AQP9(-/-) mice during sensitization rescued the impaired CHS response. Neutrophil recruitment to dLNs upon hapten application was attenuated by AQP9 deficiency. Coincidentally, AQP9(-/-) neutrophils showed a reduced CC-chemokine receptor 7 (CCR7) ligand-induced migration efficacy, which was attributed to the attenuated recruitment of neutrophils to dLNs. Furthermore, we found that neutrophil deficiency, observed in AQP9(-/-) or neutrophil-depleted mice, decreased IL-17A production by dLN cells, which might be responsible for T cell activation during a subsequent CHS response. Taken together, these findings suggest that AQP9 is required for the development of sensitization during cutaneous acquired immune responses via regulating neutrophil function.

IL-17A as an inducer for Th2 immune responses in murine atopic dermatitis models.

Atopic dermatitis (AD) is generally regarded as a type 2 helper T (Th2)-mediated inflammatory skin disease. Although the number of IL-17A-producing cells is increased in the peripheral blood and in acute skin lesion of AD patients, the role of IL-17A in the pathogenesis of AD remains unclear. To clarify this issue, we used murine AD models in an IL-17A-deficient condition. In a repeated hapten application-induced AD model, skin inflammation, IL-4 production in the draining lymph nodes (LNs), and hapten-specific IgG1 and IgE induction were suppressed in IL-17A-deficient mice. Vγ4(+) γδ T cells in the skin-draining LNs and Vγ5(-) dermal γδ T cells in the skin were the major sources of IL-17A. Consistently, in flaky-tail (Flg(ft/ft) ma/ma) mice, spontaneous development of AD-like dermatitis and IgE induction were attenuated by IL-17A deficiency. Moreover, Th2 differentiation from naive T cells was promoted in vitro by the addition of IL-17A. Taken together, our results suggest that IL-17A mediates Th2-type immune responses and that IL-17A signal may be a therapeutic target of AD.

Filaggrin in atopic dermatitis: flaky tail mice as a novel model for developing drug targets in atopic dermatitis.

The barrier abnormality, a loss-of-function mutation in the gene encoding filaggrin (FLG), which is linked to the incidence of atopic dermatitis (AD), is a recently discovered but important factor in the pathogenesis of AD. To investigate this issue in greater detail, mice that have a genetic defect (FLG) in barrier function will provide a model of AD closer to the human disease. Flaky tail (Flg(ft)) mice, essentially deficient in filaggrin, recently have been introduced to investigate the role of filaggrin on AD. These mice showed eczematous skin lesion in the steady state in line with increased of total IgE and Th17 expression in the skin. There is also an altered skin barrier function as a key element of AD either outside-to-inside barrier function or vice versa in Flg(ft) mice. Moreover, like human AD, these mice showed enhanced percutaneous allergen priming or response to cutaneous stimulants. Application of mite allergen in Flg(ft) mice, even without prior barrier disruption, remarkably enhanced both the clinical manifestations and the laboratory findings that correspond to indicators of human AD. These features of Flg(ft) mice allow us to investigate further the role of filaggrin in AD, and the knowledge obtained using these mice will be quite useful to develop a new therapeutic target for AD.

Rho-mDia1 pathway is required for adhesion, migration, and T-cell stimulation in dendritic cells.

Dendritic cells (DCs) are essential for the initiation of acquired immune responses through antigen acquisition, migration, maturation, and T-cell stimulation. One of the critical mechanisms in this response is the process actin nucleation and polymerization, which is mediated by several groups of proteins, including mammalian Diaphanous-related formins (mDia). However, the role of mDia in DCs remains unknown. Herein, we examined the role of mDia1 (one of the isoforms of mDia) in DCs. Although the proliferation and maturation of bone marrow-derived DCs were comparable between control C57BL/6 and mDia1-deficient (mDia1(-/-)) mice, adhesion and spreading to cellular matrix were impaired in mDia1(-/-) bone marrow-derived DCs. In addition, fluorescein isothiocyanate-induced cutaneous DC migration to draining lymph nodes in vivo and invasive migration and directional migration to CCL21 in vitro were suppressed in mDia1(-/-) DCs. Moreover, sustained T-cell interaction and T-cell stimulation in lymph nodes were impaired by mDia1 deficiency. Consistent with this, the DC-dependent delayed hypersensitivity response was attenuated by mDia1-deficient DCs. These results suggest that actin polymerization, which is mediated by mDia1, is essential for several aspects of DC-initiated acquired immune responses.

Prostaglandin I2-IP signaling promotes Th1 differentiation in a mouse model of contact hypersensitivity.

PGI(2), which exerts its actions via its specific Gs-coupled I prostanoid receptor (IP), is known to be present in the lymph nodes, but its roles in acquired cutaneous immune responses remain unclear. To investigate the role of PGI(2)-IP signaling in cutaneous immune responses, we applied IP-deficient (Ptgir(-/-)) mice to contact hypersensitivity as a model of acquired immune response and found that Ptgir(-/-) mice exhibited a significantly decreased contact hypersensitivity response. Lymph node cells from sensitized Ptgir(-/-) mice exhibited decreased IFN-gamma production and a smaller T-bet(+) subset compared with control mice. PGI synthase and IP expression were detected in dendritic cells and T cells, respectively, by quantitative real-time PCR analysis, suggesting that PGI(2) produced by dendritic cells acts on IP in T cells. In fact, in vitro Th1 differentiation was enhanced by an IP agonist, and this enhancement was nullified by protein kinase A inhibitor. These results suggest that PGI(2)-IP signaling promotes Th1 differentiation through a cAMP-protein kinase A pathway and thereby initiates acquired cutaneous immune responses.

Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.

The barrier abnormality, a loss-of-function mutation in the gene encoding filaggrin (FLG), which is linked to the incidence of atopic dermatitis (AD), is a recently discovered but important factor in the pathogenesis of AD. Flaky tail (Flg(ft)) mice, essentially deficient in filaggrin, have been used to investigate the role of filaggrin on AD. However, the relevancy of Flg(ft) mice to human AD needs to be determined further. In this study, we observed the clinical manifestations of Flg(ft) mice in the steady state and their cutaneous immune responses against external stimuli, favoring human AD. Under specific pathogen-free conditions, the majority of Flg(ft) mice developed clinical and histological eczematous skin lesions similar to human AD with outside-to-inside skin barrier dysfunction evaluated by newly devised methods. In addition, cutaneous hapten-induced contact hypersensitivity as a model of acquired immune response and a mite extract-induced dermatitis model physiologically relevant to a human AD were enhanced in Flg(ft) mice. These results suggest that the Flg(ft) mouse genotype has potential as an animal model of AD corresponding with filaggrin mutation in human AD.