Activation of Toll‐like receptors alters the microRNA expression profile of keratinocytes

Keratinocytes recognize invading pathogens by various receptors, among them Toll‐like receptors (TLRs), and provide the first line of defense in skin immunity. The role of microRNAs in this important defense mechanism has not been explored yet. Our aim was to identify microRNAs involved in the innate immune response of keratinocytes. MicroRNA expression profiling revealed that the TLR2 ligand zymosan, the TLR3 ligand poly(I:C) or the TLR5 ligand flagellin significantly altered the microRNA expression in keratinocytes. The regulation of microRNAs was concentration‐dependent and it could be neutralized by siRNAs specific for TLR2, TLR3 and TLR5, respectively, confirming the specificity of the TLR response. Interestingly, one microRNA, miR‐146a, was strongly induced by all studied TLR ligands, while other microRNAs were regulated in a TLR‐ or time point‐specific manner. These findings suggest an important role for microRNAs in the innate immune response of keratinocytes and provide a basis for further investigations.     

角质形成细胞μ-阿片受体表达的研究

目的：研究不同特性的角质形成细胞μ-阿片受体的表达情况。方法：以体外培养的角质形成细胞株HaCaT细胞、人鳞状细胞癌(简称鳞癌)细胞株A431、神经母细胞瘤SK-N-SH细胞株为对象，采用逆转录(RT)-PCR方法研究细胞μ-阿片受体的表达。结果：在常规体外培养条件下，角质形成细胞株HaCaT细胞、人鳞癌细胞株A431的RT-PCR结果显示有μ-阿片受体mRNA的表达，后者的表达水平略高于前者。结论：μ-阿片受体在角质形成细胞的表达，为神经系统和皮肤通过神经肽直接发生作用提供依据。     

Immunostimulatory activity of murine keratinocyte‐derived exosomes

It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble factors. Exosomes, small membrane vesicles of endocytotic origin, have been implicated in intercellular communication processes such as the transfer of tumor cell antigens and the activation of recipient dendritic cells (DC). However, little is known about immunomodulatory functions of keratinocyte‐derived exosomes. To address this question, we analysed exosome secretion of the murine keratinocyte cell line MPEK under steady state as well as inflammatory conditions (+/? IFNγ). These exosomes were readily taken up by bone marrow‐derived DC (BMDC) in vitro resulting in a matured phenotype, as evidenced by increased CD40 expression as well as by the production of large amounts of IL‐6, IL‐10 and IL‐12. When the transfer of antigen‐specific information through exosomes was investigated, it was found that keratinocytes took up antigen (ovalbumin) and transferred it to their exosomes. However, these antigen‐harbouring exosomes failed to induce antigen‐specific T cell responses via BMDC. Together, this novel biological function suggests that keratinocytes are able to direct unspecific immune processes but do not elicit specific immune responses.     

Differential expression and in vivo secretion of the antimicrobial peptides psoriasin (S100A7), RNase 7, human beta‐defensin‐2 and ‐3 in healthy human skin

Antimicrobial peptides (AMP) are key players in the skin's defense system. Previous observations suggest a site‐ and age‐dependent expression of individual AMP. We investigated the expression and secretion patterns of four important AMP in a representative collective of healthy human skin samples. Levels of psoriasin, RNase 7 and hBD‐3 expression – assessed by immunohistochemistry – varied between different body localisations. Older individuals expressed hBD‐2 more frequently. No gender‐related expression was observed. The in vivo secretion of psoriasin, measured in skin washing fluids using ELISA, was related to body localisation and age, whereas RNase 7 secretion showed no significant differences regarding these variables. HBD‐2 and ‐3 secretion could not be detected. Our findings suggest the usage of control samples matching localisation and approximate age (in the case of hBD‐2) for comparative immunohistochemical analysis. To avoid bias through great interindividual differences, sufficient large collectives should be used for in vivo secretion analyses.     

Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers., tolerogens and irritants

Abstract To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensi-tizer (2,4-dinitrofiuorobenzene, 3-n-penladecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Intcrleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, in-terlcukin-8 production was not detectable in unstimulatcd normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immuno-genic or inflammatory signals in man.     

Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis

BACKGROUND: Toll-like receptors (TLRs) are part of the innate immune system involved in the response to microbial pathogens. TLR2 recognizes various ligands expressed by Gram-positive bacteria, while TLR3, TLR4 and TLR5 are specific for double-stranded RNA, Gram-negative lipopolysaccharides and bacterial flagellin, respectively. OBJECTIVES: To determine, firstly, whether epidermal keratinocytes of normal skin express TLRs and, secondly, whether modulation of TLR expression occurs in psoriasis, an inflammatory skin disease associated with certain microorganisms such as streptococci, staphylococci and yeasts. METHODS: Eight samples of normal, and 15 samples of lesional and nonlesional psoriatic skin were stained with polyclonal antibodies specific for TLR1-5 using an avidin-biotin-peroxidase technique. RESULTS: Epidermal keratinocytes in normal skin constitutively expressed TLR1, TLR2 and TLR5, while TLR3 and TLR4 were, in most cases, barely detectable. Cytoplasmic TLR1 and TLR2 were expressed throughout the epidermis, with higher staining of the latter on basal keratinocytes, while TLR5 expression was concentrated in the basal layer. In contrast, in lesional epidermis from patients with psoriasis, TLR2 was more highly expressed on the keratinocytes of the upper epidermis than on the basal layer, while TLR5 was downregulated in basal keratinocytes compared with corresponding nonlesional psoriatic epidermis. In addition, nuclear TLR1 staining was observed in the upper layers of both nonlesional and lesional psoriatic epidermis, but not in that of normal skin. CONCLUSIONS: These findings suggest that TLRs expressed by epidermal keratinocytes constitute part of the innate immune system of the skin. The relevance of altered keratinocyte TLR expression in psoriasis remains to be determined.     

Interferon‐γ induces upregulation and activation of the interleukin‐31 receptor in human dermal microvascular endothelial cells

Abstract: Interleukin‐31 (IL‐31), a recently discovered cytokine derived from T helper cells, is involved in chronic dermatitis and pruritus. This study demonstrates for the first time that the IL‐31 receptor complex for IL‐31 is substantially upregulated in human dermal microvascular endothelial cells after stimulation with interferon‐γ (IFN‐γ). Activation of the IL‐31 receptor complex results in the induction of the intracellular ERK1/2 signaling pathway and downregulation of IFN‐γ‐induced monokine induced by IFN‐γ expression. Inhibitor studies revealed that the IFN‐γ‐induced IL‐31RA upregulation is processed via JNK and PI3 kinase activation. In sum, our study points toward an interaction between the T_H1‐derived cytokine IFN‐γ and the T_H2‐derived cytokine IL‐31 on endothelial cells.     

Increased expression of glucagon‐like peptide‐1 receptors in psoriasis plaques

Recent case reports suggest that treatment with glucagon‐like peptide‐1 (GLP‐1) agonists results in clinical improvement of psoriasis. The purpose of this study was to determine whether GLP‐1 receptors (GLP‐1Rs) are found in the skin of healthy volunteers and psoriasis patients and if so, whether GLP‐1Rs are located on keratinocytes or immune cells. Three mm‐punch skin biopsies were taken for gene expression analysis from six healthy volunteers and from affected and unaffected skin of six psoriasis patients. In addition, a blood sample was obtained from all participants. Cultured human keratinocytes were either untreated or incubated with tumor necrosis factor‐ α (TNF‐α), interferon‐γ (IFN‐γ) or a combination of TNF‐α and IFN‐γ for 48 h. Total RNA was extracted from all the samples, reversely transcribed and analysed for the expression of GLP‐1R using real‐time PCR. Gene expression analysis showed expression of GLP‐1Rs in five of six skin biopsies from psoriasis plaques, in one of six biopsies from unaffected psoriatic skin and in one of six biopsies from healthy skin. GLP‐1R expression was found in the blood of both healthy volunteers and psoriasis patients. No GLP‐1R expression was found in either stimulated or unstimulated cultured human keratinocytes. Our results show increased presence of GLP‐1Rs in psoriasis plaques and that this most likely is due to infiltration with immune cells. This offers a possible explanation for the positive effect of treatment with GLP‐1R agonists in patients with psoriasis.     

Alpha-MSH reduces the internalization of Staphylococcus aureus and down-regulates HSP 70, integrins and cytokine expression in human keratinocyte cell lines

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuropeptide predominantly produced by the pituitary gland, but it is also generated by many extra-pituitary cells including keratinocytes of the skin. This neuropeptide has anti-inflammatory and antimicrobial effects and probably contributes in innate immunity. Staphylococcus aureus is the aetiological agent of a wide range of infections in humans. Colonization of human skin by S. aureus is a characteristic feature of several skin diseases and is often followed by tissue invasion and severe cell damage. The aim of our study was to detect a possible role of alpha-MSH during the early infection stages in the adhesion and penetration of keratinocytes before cell damage. Our data demonstrated that alpha-MSH precociously down-regulates the production of integrins such as beta1 and heat shock surface protein 70, essential molecules for the entry of S. aureus. Moreover, in our experimental model, alpha-MSH induces the down-regulation of the pro-inflammatory cytokine expression and of the adhesion molecules in keratinocytes activated by S. aureus. Our data suggest that alpha-MSH plays a protective role in the skin by reducing infection and the inflammatory process.     

Spontaneous keratinocyte cell lines representing early and advanced stages of malignant transformation of the epidermis

A unique series of epidermal cell lines representing different stages of malignant transformation were spontaneously derived from a single adult immunosuppressed individual. Four keratinocyte lines (PM1-4) established from forehead skin are here compared with 4 squamous cell carcinoma (SCC) lines (MET1-4) derived respectively from a primary cutaneous tumour, two local recurrences and a distant metastasis of invasive SCC. Despite altered growth properties, the PM lines retained many features of normal keratinocytes including keratin phenotype, differentiation capacity and non-tumorigenicity in athymic mice. In contrast, from early passage, the MET lines displayed markedly reduced growth requirements, abnormal differentiation, aberrant K18 expression and tumorigenicity in athymic mice. The abnormal keratin profile of individual MET lines closely reflected the keratin phenotype of the tumour of origin. Although unusual HPV types were identified in the original tissue, there was no evidence of persistent virus within any cell line and it appears that HPV is not critical for maintenance of the immortal phenotype. The PM lines were distinctly different from invasive SCC lines and are likely to be useful for studies of mutations important early in neoplastic progression. The SCC series represent primary, recurrent and metastatic carcinoma. Availability of such a series from the same individual will facilitate genetic analysis of the malignant process.     

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT

Abstract: In contrast to extracellular, long chain ceramides which comprise a structural component of the epidermal water barrier, intracellular ceramides originating from sphingomyelin hydrolysis have been shown to inhibit proliferation and to induce apoptosis in different cell populations. To further elucidate the possible role of intracellular ceramides in human epidermis, two new cell-permeable ceramide analogues, N -thioacetylsphingosine (C₂-Cer=S) and 4-dodecanoylamino-decan-5-ol (FS-5), were synthesized and tested for their ability to suppress cell growth and to induce apoptosis in immortalized human keratinocytes. It was shown that the well-investigated ceramide analogue N -acetylsphingosine (C₂-Cer=O), as well as the new compound C₂-Cer=S inhibited proliferation of HaCaT cells with half-inhibitory concentrations (IC₅₀) of 20 μg/ml and 10 μg/ml, respectively, whereas FS-5 has been potent with an IC₅₀>40 μg/ml. Overall, all three ceramide analogues induced apoptosis in HaCaT cells as assessed by DNA-fragmentation using ELISA technique and in situ nick end labelling, thereby confirming the importance of ceramide signalling in keratinocytes.     

Efficacy of quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro

Please cite this paper as: Efficacy quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro. Experimental Dermatology 2010; 19 : e282–e285. Abstract: In this study, we describe a simple, specific, reproducible and quantitative assay system to assess melanosome transfer. We first established a co‐culture model of normal human epidermal melanocytes and HaCaT keratinocytes. The cells were co‐cultured for 72 h in a serum‐free keratinocyte growth media and double labelled with Fluorescein isothiocyanate (FITC)‐conjugated antibody against the melanosome‐specific protein gp100, and with Phycoerythrin (PE)‐conjugated antibody against the keratinocyte‐specific marker cytokeratin. Then, the cells were examined using co‐focal microscope and flow cytometry. The increased melanosome transfer from melanocytes to HaCaT keratinocytes was observed in a time‐dependent manner. To verify the accessibility of this method, two known melanosome transfer inhibitors and two known melanosome transfer stimulators were applied. Consistent with previous investigation, soybean trypsin inhibitor (STI), niacinamide inhibited melanosome transfer, α‐melanocyte stimulating hormone (α‐MSH) and keratinocyte growth factor (KGF) increased melanosome transfer, respectively, in a dose‐dependent manner. The model used in this study could thus represent a rapid and reliable tool to identify modulators of human melanosome transfer.     

Retinal and retinol are potential regulators of gene expression in the keratinocyte cell line HaCaT

Please cite this paper as: Retinal and retinol are potential regulators of gene expression in the keratinocyte cell line HaCaT. Experimental Dermatology 2010. Abstract: Vitamin A is a pivotal regulator of differentiation and growth of developing and adult skin. Retinoic acid is the major physiologically active form of vitamin A regulating the expression of different genes through retinoic acid nuclear receptors. Here, we present evidence that other vitamin A derivates – retinol and retinal – are also capable of functioning as regulators of gene expression in the keratinocyte cell line HaCaT. We have shown that all‐trans retinol (ATRol) and all‐trans retinal (ATRal) are capable of modulating gene expression in keratinocytes, which is not because of vitamin A metabolism in the cells, and retinol and retinal modulate gene expression through nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Based on the data, we propose that ATRol and all‐trans retinal, in addition to all‐trans retinoic acid, can function as important regulators of gene expression manifesting their effect through the nuclear receptors RARs and RXRs.