Response Of Human Keratinocytes Research Articles

Characterization of a Human Skin Equivalent Model to Study the Effects of Ultraviolet B Radiation on Keratinocytes

The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline in both Australia and globally. Hence, the cellular and molecular pathways that are associated with UVR-induced photocarcinogenesis need to be urgently elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular, the highly mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which, to some extent, have limited their relevance to the in vivo situation. To address this, we characterized a three-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53, and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration after photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage.

Metabolic response of human keratinocytes to silver nanoparticles: A metabolomics study

Metabolic response of human keratinocytes to silver nanoparticles: A metabolomics study

Effects of pre- and post-treatment with plant polyphenols on human keratinocyte responses to solar UV

The understanding of the anti-inflammatory mechanisms of action of plant polyphenols (PPs) and clarification of the relationship between their anti-inflammatory and antioxidant properties may result in a new therapeutic approach to skin cancers. To elucidate the underlying mechanism, we analyzed the ability of PPs to attenuate inflammatory, metabolic and oxidative cellular responses to UV irradiation. Normal human epidermal keratinocytes (NHEK) were exposed to physiologically relevant dose of solar-simulated UV irradiation. Effects of pre- and post-treatment with PPs on the overproduction of peroxides and inflammatory mediators (mRNA and protein) were analyzed using real-time RT-PCR, enzyme-linked immunosorbent and fluorometric techniques. Differences between the effectiveness of pre- and post-treatment with polyphenols was found. In particular, PPs post-treatment, but not pretreatment, completely abolished overexpression of Cyp1a1 and Cyp1b1 genes and elevation of intracellular peroxides in NHEK irradiated by UV. Post-treatment with PPs also more efficiently than pretreatment prevented UV-induced overexpression of IL-1 beta, IL-6 and COX2 mRNAs. Our data strongly suggest that PPs predominantly affect delayed molecular and cellular events initiated in NHEK by solar UV rather than primary photochemical reactions. PPs may be important component in cosmetic formulations for post-sun skin care.

Modulation of Human Keratinocyte Responses to Solar UV by Plant Polyphenols As a Basis for Chemoprevention of Non-Melanoma Skin Cancers

Modulation of Human Keratinocyte Responses to Solar UV by Plant Polyphenols As a Basis for Chemoprevention of Non-Melanoma Skin Cancers

Modulation of Human Keratinocyte Responses to Solar UV by Plant Polyphenols As a Basis for Chemoprevention of Non-Melanoma Skin Cancers

Excessive exposure to solar UVA and UVB radiation is widely considered to cause skin cancers such as squamous cell carcinoma and basalioma. Direct UVB damage to skin cell DNA as well as UV-induced chronic skin inflammation, accelerated keratinocyte proliferation, inhibited apoptosis, and immunosuppression seem to underlie the UV-induced carcinogenesis. Also, UVB induces cytochrome P450 subfamilies (CYP1A1 and CYP1B1) involved in metabolic activation of organic pro-carcinogens and their conversion to ultimate carcinogens. Here, the effects of several glycosylated and non-glycosylated plant polyphenols (verbascoside, resveratrol, polydatin, rutin, and quercetin) on the inflammatory, apoptotic, metabolic, and proliferative responses of cultured human epidermal keratinocytes (HEK) to non-cytotoxic doses of solar-simulated UVA+UVB and chemical mediators of UV signalling in HEK, 6-formylindolo[3,2-b]carbazole and squalene isolated from photo-oxidized skin surface lipids (SSL), were evaluated. We showed that the stilbenes and quercetin being exposed to UV were photo-destroyed within a short period of time, while verbascoside and rutin were photo-stable. When SSL were exposed to UV, the stilbenes and quercetin remarkably accelerated photo-oxidation of alpha-tocopherol, squalene, and cholesterol fractions, whilst verbascoside protected them. Verbascoside invariably inhibited molecular pathways in HEK leading to inflammatory cytokine expression (NFkappaB and EGFR/ERK phosphorylation), and cell proliferation (EGFR nuclear translocation), and displayed a stimulus-specific effect on the metabolic axis aryl hydrocarbon receptor (AhR)-CYP1A1/CYP1B1. By contrast, the stilbenes inhibited UV-connected inflammatory cytokines excluding IL-8, but they prevalently stimulated NFkappaB, EGFR nuclear translocation and the AhR-CYP pathway. We conclude that, among the PPs investigated, verbascoside does interfere with multiple UV-sensitive signalling in HEK in a way that it could have a major impact on skin cancer chemoprevention.

Exposure to heat-inactivated Trichophyton rubrum resulting in a limited immune response of human keratinocytes

Background Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively. Methods HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×106 and 1.5×105 colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment. Results HaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1. Conclusion The cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.

Modulation of double-stranded RNA- and cytokine- induced responses of human keratinocytes by LL-37.

Background: Besides its microbicidal function, LL-37 can activate different types of cells. Another way, by which LL-37 modulates cellular response, is via forming complexes with the nucleic acids, such as CpG DNA and double-stranded RNA (dsRNA). LL-37 production is high in skin of psoriasis patients but not atopic dermatitis patients. A synthetic dsRNA, polyI:C, stimulates human keratinocytes to produce cytokines/chemokines including a Th2-inducing cytokine-thymic stromal lymphopoetin (TSLP) (Kinoshita et al.

Innate immune response in human keratinocytes infected by a feline isolate of Malassezia pachydermatis

Malassezia pachydermatis is a normal inhabitant of canine and feline skin that can spread to other pets. The outer layer or epidermis is made up primarily of keratinocytes, which are capable of releasing various factors and expressing receptors that are significantly involved in the immune regulation. Little is known about the mechanism by which M. pachydermatis overcomes the natural barrier of the skin. The aim of this study was to evaluate the direct in vitro interaction between human keratinocytes and a clinical strain of live M. pachydermatis isolated as a pure culture from an otitic cat. Human keratinocytes (HaCat) were infected with M. pachydermatis to analyse the modulation of the innate immune response. Gene expression was analysed by real-time PCR. We demonstrated that M. pachydermatis invaded HaCat cells and modulated the expression of TLR2 after 24h infection, while HBD-2, IL-1β TNF-α, IL-6 and IL-8 were modulated both at 24 and 48h. Thus, our results demonstrated that M. pachydermatis is able to stimulate the innate immune response in infected human keratinocytes indicating a possible role of this yeast as a human opportunistic pathogen.

Expression of IL‐24 and IL‐24 receptors in human wound tissues and the biological implications of IL‐24 on keratinocytes

Interleukin (IL)-24, also known as melanoma differentiation-associated gene-7, is a cytokine initially identified from cancerous cells and expressed in a range of cell types. It is a regulator of cell differentiation, growth, and apoptosis, and a promising anticancer agent. IL-24 acts via its heterodimic receptors: the IL-20R1 and IL-20R2 complex and the IL-22R1 and IL-20R2 complex. There is limited information on the effect of IL-24 in wound healing. Human acute and chronic wound tissues were used to analyze the transcript levels and histological staining of IL-24 and the IL-24 receptors. The biological response of human keratinocytes to recombinant human IL-24 was evaluated using electric cell-substrate impedance sensing-based methods in conjunction with inhibitors to candidate signaling pathways. IL-24 significantly slowed the migration of keratinocytes (p = 0.01), with only a marginal effect on cellular adhesion. The inhibitory effect of IL-24 on migration was completed reversed following addition of an AKT inhibitor (p = 0.004) but not an SMAD3 pathway inhibitor. Human chronic wound tissues showed raised levels of both IL-24 (p = 0.003) and its receptor (p = 0.0305) compared with acute wound tissues. We conclude that IL-24 appears to promote wound chronicity via its inhibitory effect on the migratory behavior of human keratinocytes, mediated through an AKT-dependent pathway.

Humanized Mouse Model of Skin Inflammation Is Characterized by Disturbed Keratinocyte Differentiation and Influx of IL-17A Producing T Cells

Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response.As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human β-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin.In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our understanding of the dermal immunopathology in humans and benefit the development of novel therapeutics for controlling inflammatory skin diseases.

Photo-Oxidation Products of Skin Surface Squalene Mediate Metabolic and Inflammatory Responses to Solar UV in Human Keratinocytes

The study aimed to identify endogenous lipid mediators of metabolic and inflammatory responses of human keratinocytes to solar UV irradiation. Physiologically relevant doses of solar simulated UVA+UVB were applied to human skin surface lipids (SSL) or to primary cultures of normal human epidermal keratinocytes (NHEK). The decay of photo-sensitive lipid-soluble components, alpha-tocopherol, squalene (Sq), and cholesterol in SSL was analysed and products of squalene photo-oxidation (SqPx) were quantitatively isolated from irradiated SSL. When administered directly to NHEK, low-dose solar UVA+UVB induced time-dependent inflammatory and metabolic responses. To mimic UVA+UVB action, NHEK were exposed to intact or photo-oxidised SSL, Sq or SqPx, 4-hydroxy-2-nonenal (4-HNE), and the product of tryptophan photo-oxidation 6-formylindolo[3,2-b]carbazole (FICZ). FICZ activated exclusively metabolic responses characteristic for UV, i.e. the aryl hydrocarbon receptor (AhR) machinery and downstream CYP1A1/CYP1B1 gene expression, while 4-HNE slightly stimulated inflammatory UV markers IL-6, COX-2, and iNOS genes. On contrast, SqPx induced the majority of metabolic and inflammatory responses characteristic for UVA+UVB, acting via AhR, EGFR, and G-protein-coupled arachidonic acid receptor (G2A).Conclusions/SignificanceOur findings indicate that Sq could be a primary sensor of solar UV irradiation in human SSL, and products of its photo-oxidation mediate/induce metabolic and inflammatory responses of keratinocytes to UVA+UVB, which could be relevant for skin inflammation in the sun-exposed oily skin.

Organotypic Modeling of Human Keratinocyte Response to Peroxisome Proliferators

Peroxisome proliferators (PPs) are a diverse chemical group including hypolipidemic drugs and some fatty acids. Their stimulation of PP-activated receptors (PPARs) and subsequent control of gene expression regulates metabolism and differentiation in many cells. PPs have multiple opportunities to target human epidermal keratinocytes because of delivery through dietary, clinical, and/or topical exposure routes. PPAR knockout mice and PP treatment of mouse skin or human keratinocytes in monolayer culture have established some effects for PPs in cutaneous differentiation. However, incomplete epidermal maturation characteristic of monolayer keratinocytes and rodent-specific effects may limit our full understanding of human keratinocyte responses to PPs. To address these issues, we investigated PP influence on primary human keratinocytes in organotypic cultures that recapitulate biochemical markers of epidermis. We found that the PPARα agonists clofibrate, docasohexaenoic acid, and WY-14,643 produced mild to moderate keratinocyte hyperplasia, increased stratification (particularly of granular and cornified layers), and enhanced levels of the differentiation markers filaggrin, ABCA12, and phosphorylated HSP27. Several PP effects generated in the organotypic system, however, were distinct from those previously reported for rodent skin and human keratinocyte monolayer cultures, suggesting that the species and growth context of target cells can impact exposure outcomes. Given the utility of organotypic cultures for modeling the epidermis, studies in this system may bridge the gap between the rodent assays and clinical studies of human epidermal responses to PPs.

A time-lapse approach to examine chromium and nickel effects on wound healing in vitro

Chromium and nickel cause allergic contact dermatitis, a common biological skin response to sensitizing agents. This study used a conventional in vitro wounding model to study the impact of sensitizing agents on the innate immune response of human keratinocytes. Experiments were designed to evaluate the involvement of specific Toll-like receptors and metalloproteinases as effectors molecules downstream, at a molecular level. Further, keratinocytes were co-cultured with monocytes (THP-1 cells) to reproduce an inductive stimulus on monocytes made by metals. Human keratinocytes (HaCat) were grown on plates covered with collagen type I, chemically treated, and then mechanically injured with a sterile pipette tip. Restoration of the monolayer integrity was monitored by time-lapse video microscopy. Effector gene expression was evaluated by real-time PCR. The presence of chromium significantly dropped the rate of wound closure, while nickel-induced hyper-proliferation ended in an acceleration of the healing process, an event that does not occur in vivo. This latter outcome led to considering nickel as an unsuitable example for use in the experimental model. Focusing solely on the chromium aspect of this study, RNA profiles of selected molecular markers were generated to ascertain if the detrimental stimulus from chromium was eliminated or persisted both in keratinocytes alone and/or during co-cultures of keratinocytes and monocytes. Monocytes accelerated the process of wound repair. This in vitro experimental model highlighted the involvement of innate immunity in response to chromium and might be useful for test molecules of therapeutic interest for the treatment of skin lesions. However, the experience with nickel reveals that there are limitations to the utility of this wound model system after all.

Increased hydrophobicity inMalasseziaspecies correlates with increased proinflammatory cytokine expression in human keratinocytes

Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and β-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not β-1,3-glucan expression.

Inflammatory response of human keratinocytes triggered by Sporothrix schenckii via toll-like receptor 2 and 4

Sporotrichosis is the most common cutaneous and subcutaneous mycosis caused by the dimorphic fungus Sporothrix schenckii of which conidia and/or hyphae could convert into yeast form at the site of infection after traumatic inoculation [1]. Histopathological findings of early cutaneous sporotrichosis showed an infiltration of neutrophils and other inflammatory cells within the epidermis and dermis [2]. Recently Sassá et al. reported that activation of macrophage TLR4 played an important role in host defense against S.

The AhR–Nrf2 Pathway in Keratinocytes: On the Road to Chemoprevention?

The ligand-activated transcription factor AhR mediates the cutaneous stress response toward a variety of environmental noxae and is therefore currently of interest for modern preventive medicine. In this issue, Tsuji et al. identify the antifungal agent ketoconazole as an inducer of AhR signaling and the Nrf2 antioxidant response in human keratinocytes. Ketoconazole-stimulated nuclear translocation of Nrf2 and its cytoprotective effects against oxidative stress strongly depend on a functional AhR. This newly identified AhR-Nrf2 pathway opens up new opportunities to prevent and treat inflammatory skin diseases.

Tumor Necrosis Factor α-Mediated Induction of Interleukin 17C in Human Keratinocytes Is Controlled by Nuclear Factor κB

IL-17C is a member of the IL-17 family of cytokines. The expression of IL-17C has been demonstrated to be strongly induced by TNFα in human keratinocytes, and recently the level of IL-17C was found to be increased in the inflammatory skin disease psoriasis. However, little is known about the molecular mechanisms involved in the regulation of IL-17C. Here, we show that pretreatment of cultured human keratinocytes with the inhibitor of κB kinase 2 inhibitor, SC-514, resulted in a significant reduction in both IL-17C mRNA and protein expression, indicating the significance of this pathway in the regulation of IL-17C. NF-κB binding sites were identified upstream from the IL-17C gene, and by electrophoretic mobility shift assay NF-κB was shown to bind to all three identified binding sites. Moreover, NF-κB binding to these sites was inducible by TNFα. Supershift analysis revealed binding of the NF-κB subunits p65 and p50 to all three NF-κB binding sites. To determine the contribution of NF-κB in IL-17C expression, we conducted luciferase gene reporter experiments and demonstrated that a 3204-bp promoter fragment of IL-17C containing three putative NF-κB binding sites was strongly activated by TNFα. Interestingly, mutations of the three NF-κB binding sites revealed that one specific NF-κB binding site was crucial for the TNFα-mediated IL-17C induction because mutation of this specific site completely abolished TNFα-induced IL-17C promoter activation. We conclude that the activation of NF-κB (p65/p50) is crucial for the TNFα-induced stimulation of IL-17C expression in human keratinocytes.

Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NFκB and AhR and EGFR–ERK pathway

Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA+UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure, the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems.The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50μM resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR.

Hyaluronan minimizes effects of UV irradiation on human keratinocytes

Exposure to ultraviolet (UV) irradiation has detrimental effects on skin accompanied by the increased metabolism of hyaluronan (HA), a linear polysaccharide important for the normal physiological functions of skin. In this study, the modulation of human keratinocyte response to UVB irradiation by HA (970 kDa) was investigated. Immortalized human keratinocytes (HaCaT) were irradiated by a single dose of UVB and immediately treated with HA for 6 and 24 h. The irradiation induced a significant decrease in the gene expression of CD44 and toll-like receptor 2 6 h after irradiation. The expressions of other HA receptors, including toll-like receptor 4 and the receptor for HA-mediated motility, were not detected in either the control or UVB-irradiated or HA-treated HaCaT cells. UVB irradiation induced a significant decrease in the gene expression of HA synthase-2 and hyaluronidase-2 6 h after irradiation. The expressions of HA synthase-3 and hyaluronidase-3 were not significantly modulated by UV irradiation. Interestingly, HA treatment did not significantly modulate any of these effects. In contrast, HA significantly suppressed UVB-induced pro-inflammatory cytokine release including interleukin-6 and interleukin-8. Similarly, HA treatment reduced the UVB-mediated production of transforming growth factor β1. HA treatment also significantly reduced the UV irradiation-mediated release of soluble CD44 into the media. Finally, HA partially, but significantly, suppressed the UVB-induced decrease in cell viability. Data indicate that HA had significant protective effects for HaCaT cells against UVB irradiation.

Secretion of thymic stromal lymphopoietin from human keratinocytes in response to Malassezia yeasts

Thymic stromal lymphopoietin (TSLP) plays an important role in the pathogenesis of atopic dermatitis (AD) by promoting dendritic cell-mediated activation of the Th2 inflammatory response [1]. The lipophilic yeasts Malassezia globosa and Malassezia restricta are members of the cutaneous microflora and act as exacerbating factors in AD [2]. Despite the in vivo expression and potential importance of keratinocyte-derived TSLP in AD [1], no studies have tested the influence of the presence of Malassezia yeast cells on the production of TSLP by human keratinocytes.