Low temperature and mTOR inhibition favor stem cell maintenance in human keratinocyte cultures.

Adult autologous human epidermal stem cells can be extensively expanded ex vivo for cell and gene therapy. Identifying the mechanisms involved in stem cell maintenance and defining culture conditions to maintain stemness is critical, because an inadequate environment can result in the rapid conversion of stem cells into progenitors/transient amplifying cells (clonal conversion), with deleterious consequences on the quality of the transplants and their ability to engraft. Here, we demonstrate that cultured human epidermal stem cells respond to a small drop in temperature through thermoTRP channels via mTOR signaling. Exposure of cells to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single-cell analysis that long-term inhibition of mTORC1 reduces clonal conversion and favors the maintenance of stemness. Taken together, our results demonstrate that human keratinocyte stem cells can adapt to environmental changes (e.g., small variations in temperature) through mTOR signaling and constant inhibition of mTORC1 favors stem cell maintenance, a finding of high importance for regenerative medicine applications.

A Phenotypic Analysis of Involucrin-Membrane-Bound Ovalbumin Mice after Adoptive Transfer of Ovalbumin-Specific CD8+ T Cells.

To investigate the mechanism of autoimmunity and peripheral tolerance in the skin, several transgenic mouse strains expressing membrane-bound ovalbumin (mOVA) as an epidermal self-antigen under the control of keratinocyte-specific promotors, such as keratin 5 and keratin 14, were employed in combination with adoptive transfer of CD8+ T cells from OT-I mice (OT-I T cells) that recognize an ovalbumin-derived peptide. However, these strains showed bodyweight loss and required additional inflammatory stimuli, such as γ-irradiation and tape-stripping, to induce skin inflammation. In this study, we generated a mouse strain expressing mOVA under the control of human involucrin promoter (involucrin-mOVA mice). In contrast to previous strains, involucrin-mOVA mice spontaneously developed skin inflammation after the transfer of OT-I T cells in the absence of external stimuli without significant bodyweight loss. We focused on the skin infiltration process of OT-I T cells and found that transferred OT-I T cells accumulated around the hair follicles in the early phase of skin inflammation, and in the later phase, the skin inflammation spontaneously resolved despite the remaining OT-I T cells in the skin. Our involucrin-mOVA mice will provide a promising tool to investigate the pathogenesis and the tolerance mechanisms of cytotoxic skin autoimmunity.

Tp63-expressing adult epithelial stem cells cross lineages boundaries revealing latent hairy skin competence.

The formation of hair follicles, a landmark of mammals, requires complex mesenchymal-epithelial interactions and it is commonly believed that embryonic epidermal cells are the only cells that can respond to hair follicle morphogenetic signals in vivo. Here, we demonstrate that epithelial stem cells of non-skin origin (e.g. that of cornea, oesophagus, vagina, bladder, prostate) that express the transcription factor Tp63, a master gene for the development of epidermis and its appendages, can respond to skin morphogenetic signals. When exposed to a newborn skin microenvironment, these cells express hair-follicle lineage markers and contribute to hair follicles, sebaceous glands and/or epidermis renewal. Our results demonstrate that lineage restriction is not immutable and support the notion that all Tp63-expressing epithelial stem cells, independently of their embryonic origin, have latent skin competence explaining why aberrant hair follicles or sebaceous glands are sometimes observed in non-skin tissues (e.g. in cornea, vagina or thymus).

Suprabasin-null mice retain skin barrier function and show high contact hypersensitivity to nickel upon oral nickel loading.

Suprabasin (SBSN) is expressed not only in epidermis but also in epithelial cells of the upper digestive tract where metals such as nickel are absorbed. We have recently shown that SBSN level is decreased in the stratum corneum and serum of atopic dermatitis (AD) patients, especially in intrinsic AD, which is characterized by metal allergy. By using SBSN-null (Sbsn-/-) mice, this study was conducted to investigate the outcome of SBSN deficiency in relation to AD. Sbsn-/- mice exhibited skin barrier dysfunction on embryonic day 16.5, but after birth, their barrier function was not perturbed despite the presence of ultrastructural changes in stratum corneum and keratohyalin granules. Sbsn-/- mice showed a comparable ovalbumin-specific skin immune response to wild type (WT) mice and rather lower contact hypersensitivity (CHS) responses to haptens than did WT mice. The blood nickel level after oral feeding of nickel was significantly higher in Sbsn-/- mice than in WT mice, and CHS to nickel was elevated in Sbsn-/- mice under nickel-loading condition. Our study suggests that the completely SBSN deficient mice retain normal barrier function, but harbor abnormal upper digestive tract epithelium that promotes nickel absorption and high CHS to nickel, sharing the features of intrinsic AD.

Protective role of Galectin-7 for skin barrier impairment in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) patients have a barrier disorder in association with Th2 dominant skin inflammation. Galectin-7 (Gal-7), a soluble unglycosylated lectin, is highly expressed in the stratum corneum of AD patients. However, the biological significance of increased Gal-7 expression in AD skin lesions remains unclear. OBJECTIVE: We aimed to investigate the production mechanism and functional role of Gal-7 in AD patients and IL-4/IL-13-stimulated epidermal keratinocytes. METHODS: We assessed the Gal-7 expression levels in skin lesions and sera from AD patients. Gal-7 levels were also measured in monolayered normal human epidermal keratinocytes (NHEKs) and 3-dimensional (3D)-reconstructed epidermis in the presence or absence of IL-4/IL-13 with or without Stat3, Stat6 or Gal-7 gene silencing. RESULTS: Gal-7 was highly expressed in the stratum corneum or intercellular space of AD lesional epidermis as assessed by the stratum corneum proteome analysis and immunohistochemistry. A positive correlation was noted between serum Gal-7 level and transepidermal water loss in patients with AD. These clinical findings were corroborated by our in vitro data, which showed that IL-4/IL-13 facilitated the extracellular release of endogenous Gal-7 in both monolayered NHEKs and 3D-reconstructed epidermis. This machinery was caused by IL-4/IL-13-induced cell damage and inhibited by knockdown of Stat6 but not Stat3 in NHEKs. Moreover, we performed Gal-7 knockdown experiment on 3D-reconstructed epidermis and the result suggested that endogenous Gal-7 serves as a protector from IL-4/IL-13-induced disruption of cell-to-cell adhesion and/or cell-to-extracellular matrix adhesion. CONCLUSION AND CLINICAL RELEVANCE: Our study unveils the characteristic of Gal-7 and its possible role as an alarmin that reflects the IL-4/IL-13-induced skin barrier impairment in AD.

Potential role of transforming growth factor-beta 1/Smad signaling in secondary lymphedema after cancer surgery.

Secondary lymphedema often develops after cancer surgery, and over 250 million patients suffer from this complication. A major symptom of secondary lymphedema is swelling with fibrosis, which lowers the patient's quality of life, even if cancer does not recur. Nonetheless, the pathophysiology of secondary lymphedema remains unclear, with therapeutic approaches limited to physical or surgical therapy. There is no effective pharmacological therapy for secondary lymphedema. Notably, the lack of animal models that accurately mimic human secondary lymphedema has hindered pathophysiological investigations of the disease. Here, we developed a novel rat hindlimb model of secondary lymphedema and showed that our rat model mimics human secondary lymphedema from early to late stages in terms of cell proliferation, lymphatic fluid accumulation, and skin fibrosis. Using our animal model, we investigated the disease progression and found that transforming growth factor-beta 1 (TGFB1) was produced by macrophages in the acute phase and by fibroblasts in the chronic phase of the disease. TGFB1 promoted the transition of fibroblasts into myofibroblasts and accelerated collagen synthesis, resulting in fibrosis, which further indicates that myofibroblasts and TGFB1/Smad signaling play key roles in fibrotic diseases. Furthermore, the presence of myofibroblasts in skin samples from lymphedema patients after cancer surgery emphasizes the role of these cells in promoting fibrosis. Suppression of myofibroblast-dependent TGFB1 production may therefore represent an effective pharmacological treatment for inhibiting skin fibrosis in human secondary lymphedema after cancer surgery.

Plasmacytoid dendritic cells as a possible key player to initiate alopecia areata in the C3H/HeJ mouse.

BACKGROUND: Alopecia areata (AA) is a tissue-specific autoimmune disease, and interferon (IFN)-γ has been regarded as the key cytokine in the pathogenesis of AA. The clinical observation that AA can occur after viral infection or IFN-α administration implies that IFN-α-producing plasmacytoid dendritic cells (pDCs) may be involved in the AA pathogenesis. METHODS: We generated AA in C3H/HeJ mice by intradermal injection of T cells derived from lymph nodes of AA-bearing syngeneic mice and stimulated IL-2, IL-7, and IL-15. Distribution of IFN-γ producing pDCs were immunohistochemically analyzed. Realtime PCR were also demonstrated to detect the expression of IFN-γ mRNA. Hair follicles were cultured with IFN-α in order to calculate the hair elongation. Imiquimod was employed to induce catagen stage. PDCs were injected into C3H/HeJ mice to initiate AA. RESULTS: In this mouse, IFN-α-producing pDCs densely infiltrated around HFs in not only AA lesional but also vicinity of AA lesion. Importantly, intradermal injection of pDCs induced AA lesions. Finally, IFN-α inhibited hair elongation of murine vibrissae and upregulated MHC class I and CXCL10 levels in vitro. CONCLUSIONS: These findings suggest that IFN-α-producing pDCs initiate AA by inducing apoptosis and increasing Th1/Tc1 chemokine production such as CXCL10, that accumulates Th1/Tc1 cells and result in autoimmune reactions against hair follicles.

Decreased expression of suprabasin induces aberrant differentiation and apoptosis of epidermal keratinocytes: Possible role for atopic dermatitis.

BACKGROUND: Suprabasin (SBSN), a secreted protein, is expressed in various epithelial tissues. The role of SBSN in epidermal differentiation and atopic dermatitis (AD) pathology remains largely unknown. OBJECTIVE: To evaluate the effects of SBSN on epidermal keratinocytes and its role in AD. METHODS: We examined the SBSN expression levels in the stratum corneum and the epidermis by proteome analysis and immunohistochemistry, respectively. The serum SBSN concentration was measured by ELISA. These values were compared between AD and healthy control. Morphological changes in the epidermis were investigated in SBSN-knockdown three-dimensional human living skin equivalent (LSE) model with or without IL-4/IL-13. RESULTS: Epidermal SBSN expression was decreased in AD lesional skin compared to healthy skin, as assessed by the stratum corneum proteome analysis and immunohistochemistry. The SBSN serum levels were significantly lower in AD patients than in normal subjects (P<0.05). The SBSN-deficient LSE exhibited compact stratum corneum, immature stratum granulosum, and increased keratinocyte apoptosis. Th2 cytokines, IL-4 and IL-13, did not affect SBSN expression in LSE. There were no differentiation-associated makers that were affected by the SBSN knockdown. SBSN deficiency-induced apoptosis of keratinocytes was exaggerated by IL-4/IL-13, and accordingly, the addition of recombinant SBSN induced significant keratinocyte proliferation (P<0.05). CONCLUSION: Our data demonstrated that SBSN regulates normal epidermal barrier. Th2 cytokines unaffect SBSN expression in keratinocytes, but promote SBSN deficiency-induced apoptosis. It is suggested that SBSN has an anti-apoptotic activity, and its deficiency is involved in the pathogenesis of AD.

Palmar hyperlinearity in early childhood atopic dermatitis is associated with filaggrin mutation and sensitization to egg.

BACKGROUND/OBJECTIVES: Palmar hyperlinearity is a typical clinical feature of Filaggrin gene (FLG) null mutations. There are reports of FLG mutations and allergic sensitization; however, reports on the relationship between palmar hyperlinearity to sensitization are limited. This study aimed to examine the association between palmar hyperlinearity and sensitization in atopic dermatitis (AD) children. METHODS: This cross-sectional, case-control study included children ˂ 6 years old with moderate-severe AD whose parents consented for mutation analysis and photographic documentation. Each child underwent genotyping to detect the eight most prevalent FLG mutations in the Japanese population: R501X, 3321delA, S1695X, Q1701X, S2554X, S2889X, S3296X, and K4022X. Clinical features and parameters including egg-specific IgE were examined, and palm photographs were evaluated by 12 trained dermatologists blinded to genotyping results. RESULTS: Of the 57 patients (age range, 2 months to 5 years; median, 22 months), 16 were heterozygotes and three were compound heterozygotes. Palmar hyperlinearity, as recognized by more than two-thirds of dermatologists, was significantly associated with FLG mutation (P = 0.002, OR = 6.98, 95% CI = 2.1-23.7), and this association was observed especially in children over 2 years. Cross-shaped crease of the thenar eminence, as known in previous reports, also demonstrated significant correlation with FLG mutation. When the children were divided according to the presence or absence of palmar hyperlinearity, the egg white-specific IgE was significantly higher in the hyperlinearity group (55.9 vs 18.3 IU/mL, P < 0.05). CONCLUSIONS: Palmar hyperlinearity indicates possible inherited barrier abnormalities of the skin in early childhood. Its identification may help to predict a more accurate prognosis, such as sensitization.

Voriconazole-induced photocarcinogenesis is promoted by aryl hydrocarbon receptor-dependent COX-2 upregulation.

Voriconazole (VRCZ) induces the development of UV-associated skin cancers. The mechanism underlying the VRCZ-induced carcinogenesis has been largely unknown. Here, we showed that VRCZ metabolites plus UVA generated reactive oxygen species and resultant DNA damage of the epidermis, but did not induce substantial apoptosis in human keratinocytes (KCs). Furthermore, VRCZ per se stimulates aryl hydrocarbon receptor (AhR) and upregulates COX-2, which is a pivotal enzyme for the promotion of UV-associated tumors, in an AhR-ARNT dependent manner of the classical (genomic) pathway. Our findings suggest that the phototoxic moieties of VRCZ metabolites may participate in the initiation phase of VRCZ skin cancer, while VRCZ per se promotes the tumor development. Therefore, during VRCZ therapy, sun exposure protection is essential to prevent photocarcinogenesis caused by VRCZ metabolites plus UV. Chemoprevention with selective COX-2 inhibitors may be helpful to repress the development of skin cancers derived from DNA-damaged KCs.

Sensitive skin is highly frequent in extrinsic atopic dermatitis and correlates with disease severity markers but not necessarily with skin barrier impairment.

BACKGROUND: Sensitive skin is a condition of cutaneous hypersensitivity to environmental factors. Lactic acid stinging test (LAST) is commonly used to assess sensitive skin and composed of four distinct sensations (pain, burning sensation, itch, and crawly feeling). A link between sensitive skin and barrier dysfunction has been proposed in atopic dermatitis (AD) patients. However, clinical and laboratory factors that are associated with sensitive skin remain unelucidated. OBJECTIVE: To investigate relationship between sensitive skin and AD-associated markers. METHODS: Forty-two Japanese AD patients and 10 healthy subjects (HS) were enrolled. AD patients were divided into extrinsic (EAD; high IgE levels) and intrinsic (IAD; normal IgE levels) types. We conducted 1% LAST by assessing the four distinct sensations and calculated the frequencies of sensitive skin in EAD, IAD, and HS. We also performed clinical AD-related tests, including transepidermal water loss (TEWL), visual analogue scale (VAS) of pruritus, and quality of life, and measured laboratory markers, including blood levels of IgE, CCL17/TARC, lactate dehydrogenase (LDH) and eosinophil counts, and concentration levels of serum Th1/Th2 cytokines. Filaggrin (FLG) mutations were examined in 21 patients. These values were subjected to correlation analyses with each of the four sensation elements. RESULTS: According to the standard criteria for LAST positivity, the frequencies of LAST-positive subjects were 54.8% and 10.0% in AD and HS, respectively (P=0.014). EAD patients showed a significantly (P=0.026) higher frequency of positive LAST (65.6%) than did IAD patients (20.0%). Among the four LAST sensation elements, the crawly feeling and pain scores positively correlated with VAS of pruritus, total serum IgE, mite-specific IgE, CCL17/TARC, and/or LDH. There was no association of the LAST scores with serum Th1/Th2 cytokine levels. Notably, neither TEWL nor FLG mutations correlated with LAST positivity or any sensation scores. CONCLUSIONS: The frequency of sensitive skin is higher in EAD than in IAD. Sensitive skin is associated with AD severity, but not necessarily with barrier condition.

Distinctive downmodulation of plasmacytoid dendritic cell functions by vitamin D3 analogue calcipotriol.

BACKGROUND: In relation to Th17 cell actions, interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs) are involved in the pathogenesis of psoriasis. Vitamin D3 analogues are widely used in the treatment of psoriasis, however, their actions on pDCs are not well understood. OBJECTIVE: To investigate the effects of Vitamin D3 analogue calcipotriol (CAL) on pDCs, focusing on the cytokine production and chemotactic activity. METHODS: We compared in mice the effects of CAL, cyclosporine A (CyA), and triamcinolone acetonide (TA) on the cytokine production by pDCs (IFN-α), conventional DCs (TNF-α), and γd T cells (IL-17A). pDCs isolated from mouse spleen cells were stimulated with CpG-ODN in the presence or absence of each drug for 48h. Purified splenic conventional DCs (cDCs) and lymph node γδ T cells were stimulated with CpG-ODN or with anti-CD3/CD28 antibody, respectively. IFN-α, TNF-α and IL-17A in the 48-h culture supernatants were quantified by ELISA. We also studied the ability of CAL to inhibit the chemotaxis of freshly isolated pDCs toward chemerin and VEGF-A, representative chemoattractants of pDCs, by a real-time monitoring method, EZ-Taxiscan. To assess the effect of CAL on pDC accumulation in vivo, we painted CAL ointment to the mouse skin inflamed by topical application of imiquimod cream (IMQ) for 4 consecutive days. In the skin samples, we enumerated 440c+ pDCs by immunohistochemistry and evaluated the mRNA expression of cytokines by real-time PCR. RESULTS: CAL significantly inhibited CpG-enhanced pDC IFN-α production at a comparable level to T cell IL-17A production, whereas its effect on cDC TNF-α production was minimal. Accordingly, CAL suppressed the CpG-augmented expression of TLR9 and MyD88. On the contrary, CyA strongly suppressed the production of TNF-α and IL-17A, but not IFN-α. TA inhibited the production of all the cytokines tested. The effect of CAL on the chemotactic activity of pDCs was also evaluated, demonstrating a significant downmodulation by exposure to the reagent. CAL depressed chemerin receptor CMKLR1 expression in pDCs. The in vivo mouse study showed that simultaneous application of CAL to the imiquimod-applied skin reduce both the recruitment of pDCs and the expression of IFN-α2 in the skin. CONCLUSIONS: Our findings suggest that CAL uniquely downmodulates the cytokine production and chemotactic activity of pDCs. The CAL suppression of the in vivo pDC accumulation to the skin suggests that these actions are therapeutically relevant.

Platelets Regulate the Migration of Keratinocytes via Podoplanin/CLEC-2 Signaling during Cutaneous Wound Healing in Mice.

Podoplanin is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), which is expressed on platelets. Recent evidence indicates that this specific marker of lymphatic endothelial cells is also expressed by keratinocytes at the edge of wounds. However, whether podoplanin or platelets play a role in keratinocyte activity during wound healing remains unknown. We evaluated the effect of podoplanin expression levels on keratinocyte motility using cultured primary normal human epidermal keratinocytes (NHEKs). Down-regulation of podoplanin in NHEKs via transfection with podoplanin siRNA inhibited their migration, indicating that podoplanin plays a mandatory role in this process. In addition, down-regulation of podoplanin was correlated with up-regulation of E-cadherin, suggesting that podoplanin-mediated stimulation of keratinocyte migration is associated with a loss of E-cadherin. Both the addition of platelets and treatment with CLEC-2 inhibited the migration of NHEKs. The down-regulation of RhoA activity and the up-regulation of E-cadherin in keratinocytes were also induced by CLEC-2. In conclusion, these results suggest that podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Altering the regulation of keratinocyte migration by podoplanin might be a novel therapeutic approach to improve wound healing.

Gross cystic disease fluid protein 15 in stratum corneum is a potential marker of decreased eccrine sweating for atopic dermatitis.

It is well known that eccrine sweating is attenuated in patients with atopic dermatitis (AD). We have reported by using proteome analysis that gross cystic disease fluid protein 15 (GCDFP15), a substance secreted from eccrine sweat glands, is decreased in tape-stripped stratum corneum (SC) samples from AD patients. The aim of this study was to evaluate GCDFP15 production by eccrine glands with SC samples and to assess sweating in AD. SC samples were obtained from 51 healthy control (HC) and 51 AD individuals. Sweat samples were from 18 HC and 12 AD subjects. GCDFP15 was quantified by ELISA. By immunohistochemistry, the expression of GCDFP15 in eccrine glands was examined in normal and AD skin specimens. To identify GCDFP15-producing cells, double immunofluorescence staining for GCDFP15 and S100 protein was performed in frozen sections. To address the mechanism underlying the decreased eccrine sweating in AD patients, we examined the expression of cholinergic receptor M3 (CHRM3), a receptor for acetylcholine-induced sweating, in eccrine sweat glands. The amounts of GCDFP15 in the SC extracts were significantly lower in AD than HC (P < 0.0001). The sweat samples from AD patients also had lower levels of GCDFP15 concentration (P < 0.05). Immunohistochemistry showed positive GCDFP15 staining in the eccrine gland secretory cells and the ductal and acrosyringial lumen in normal skin, but AD lacked clear staining. Immunofluorescence staining revealed that GCDFP15 was co-expressed with S100 protein, suggesting that the clear cell of eccrine glands produces GCDFP15. Finally, we found that the expression of CHRM3 was depressed in AD, suggesting contribution to the low sweating. The SC of AD patients contains a low amount of GCDFP15 due to both low sweating and low GCDFP15 concentration in the sweat. GCDFP15 in SC is a potential marker for dysregulated sweating in AD.

Leukoderma in patients with atopic dermatitis.

Atopic dermatitis (AD) is occasionally associated with vitiligo, however, the incidence and conditions of vitiligo or leukoderma, and the characteristics of concurrent AD, remain unclear. We conducted a prospective observational study to investigate the leukoderma-related clinical manifestations and bioparameters of AD. Because vitiligo in AD lesions is occasionally associated with inflammation, we used leukoderma in this study. Enrolled were all AD patients who had been followed up in our AD outpatient clinic and visited within the previous 4 months. During this period, we carefully inspected whether the patients had leukoderma. Eight of 52 patients had leukoderma (15.4%) and were designated as the leukoderma group, and the remaining 44 patients comprised the non-leukoderma group. While the ages were statistically not different between the two groups, female preponderance was significantly observed in the leukoderma group. The leukoderma patients tended to have higher values of SCORAD, CCL17/thymus and activation regulated chemokine and lactate dehydrogenase than the non-leukoderma patients. The leukoderma group was also characterized by a lower frequency of allergic rhinitis and a higher frequency of prurigo lesions. Thus, despite the possession of high AD severity, the leukoderma patients may possibly retain a relatively T-helper 1-skewing state in relation to the development of leukoderma and less association with rhinitis.