A hámsejtek immunológiai működésében résztvevő gének azonosítása DNS-microarray módszerrel = Identification of genes involved in the immune-function of epithelial cells using DNA microarray.

A hámsejtek immunológiai működésében résztvevő gének azonosítása DNS-microarray módszerrel Az emberi szervezetet a külső környezettől elválasztó legfontosabb határoló szervünk a bőr, amely egyrészt mechanikai védelmet nyújt a külvilág káros hatásai ellen, másrészt aktív, protektív feladatokat is ellát. Munkacsoportunk a bőrnek és a hámsejteknek a veleszületett immunitásban betöltött szerepét kutatja. Vizsgálataink során cDNS microarray módszerrel vizsgáltuk azt a kérdést, hogy a különféle bakteriális és gomba eredetű anyagok (LPS, PGN, hővel elölt Candida albicans, élő Propionibacterium acnes) milyen génexpresszós változásokat okoznak primer tenyésztett keratinocitákban. Eredményeink alapján igazoltuk, hogy a keratinocitákban gyulladásos citokin, kemokin, és antimikrobiális hatású fehérjét kódoló gének kifejeződése megváltozik a patogén eredetű anyagok hatására. A folyamatok szabályozásában a Toll-like receptorok, valamint NF-kB játszik központi szerepet. Igazoltuk, hogy a patogének támadásának kitett testfelületeken számos nem immun-eredetű sejttípus rendelkezik a kórokozók felismerésének képességével, ezek bakteriális és gomba-eredetű anyagok hatására képesek a veleszületett és adaptív immunfolyamatok elindítására és szabályozására. Eredményeink elméleti jelentősége mellett egy olyan tesztrendszer bevezetését is lehetővé tették, mely alkalmas gyulladásos bőrbetegségek esetén alkalmazott lokális immunmoduláló szerek hatásmechanizmusának vizsgálatára. | Identification of genes involved in the immune-function of epithelial cells using DNA microarray. The human skin is the most important barrier of the organism. It can not only passively separate the outer environment from the inner body, but also plays an active protective function. Our main focus is the better understanding of the innate immune functions of epidermal keratinocytes. For this reason we have investigated the global gene expression changes in response to various microbial components (PLS, PGN, heat-killed Candida albicans, and live Propionibacterium acnes) in primary keratinocytes using the microarray technique. Based on our results we could show, that the expression of various pro-inflammatory cytokine, chemokine, and antimicrobial peptide coding genes are changing in response to pathogen derived chemical compounds, and whole pathogen cells. Toll-like receptors and NF-kB are playing an important role in the regulation of these genes. Our results also suggest, that this ability of the keratinocytes is not unique to them, other cell types on various surfaces of the body where pathogen attack happens regularly can also recognize the microbes and initiate and regulate immune processes. Based on our data, we also set up an experimental model system that can be used for the evaluation of the exact function of various topical immunmodulatory chemicals used to treat inflammatory skin diseases

Distinct Strains of Propionibacterium acnes Induce Selective Human β-Defensin-2 and Interleukin-8 Expression in Human Keratinocytes Through Toll-Like Receptors

Acne is a chronic inflammatory disease of the pilosebaceous follicle. One of the main pathogenetic factors in acne is the increased proliferation of Propionibacterium acnes (P. acnes) in the pilosebaceous unit. We investigated whether direct interaction of P. acnes with keratinocytes might be involved in the inflammation and ductal hypercornification in acne. The capacities of different P. acnes strains to activate the innate immune response and the growth of cultured keratinocytes were investigated. We have found that two clinical isolates of P. acnes significantly induced human β-defensin-2 (hBD2) messenger RNA (mRNA) expression; in contrast a third clinical isolate and the reference strain (ATCC11828) had no effect on hBD2 mRNA expression. In contrast, all four strains significantly induced the interleukin-8 (IL-8) mRNA expression. The P. acnes-induced increase in hBD2 and IL-8 gene expression could be inhibited by anti-Toll-like receptor 2 (TLR2) and anti-TLR4 neutralizing antibodies, suggesting that P. acnes-induced secretion of soluble factors in keratinocytes are both TLR2 and TLR4 dependent. In addition, the clinical isolate P. acnes (889) was capable of inducing keratinocyte cell growth in vitro. Our findings suggest that P. acnes modulates the antimicrobial peptide and chemokine expression of keratinocytes and thereby contributes to the recruitment of inflammatory cells to the sites of infections

Expression and Functional Studies on the Noncoding RNA, PRINS.

PRINS, a noncoding RNA identified earlier by our research group, contributes to psoriasis susceptibility and cellular stress response. We have now studied the cellular and histological distribution of PRINS by using in situ hybridization and demonstrated variable expressions in different human tissues and a consistent staining pattern in epidermal keratinocytes and in vitro cultured keratinocytes. To identify the cellular function(s) of PRINS, we searched for a direct interacting partner(s) of this stress-induced molecule. In HaCaT and NHEK cell lysates, the protein proved to be nucleophosmin (NPM) protein as a potential physical interactor with PRINS. Immunohistochemical experiments revealed an elevated expression of NPM in the dividing cells of the basal layers of psoriatic involved skin samples as compared with healthy and psoriatic uninvolved samples. Others have previously shown that NPM is a ubiquitously expressed nucleolar phosphoprotein which shuttles to the nucleoplasm after UV-B irradiation in fibroblasts and cancer cells. We detected a similar translocation of NPM in UV-B-irradiated cultured keratinocytes. The gene-specific silencing of PRINS resulted in the retention of NPM in the nucleolus of UV-B-irradiated keratinocytes; suggesting that PRINS may play a role in the NPM-mediated cellular stress response in the skin

Benignus és malignus hiperproliferatív ill. gyulladásos bőrbetegségek pathomechanizmusának vizsgálata funkcionális genomikai módszerekkel = Studies on the pathomechanism of benign and malign hyperproliferative as well as inflammatory skin diseases by means of functional genomics

Pikkelysömörben vizsgáltuk a sejtciklus szabályozásban részt vevő extracelluláris mátrix protein, a fibronektin szerepét, és azonosítottunk egy nem kódoló RNS gént (PRINS), mely szerepet játszik a pikkelysömörre való hajlam, ill. a sejtek stressz válaszának kialakításában. A multifaktoriális etiológiájú lábszárfekély genetikai hátterének vizsgálata során azonosítottunk egy SNP-t, amely magasabb arányban fordul elő a betegekben, mint egészségesekben, feltételezzük, hogy szerepet játszik a betegség pathogenezisében. A serdülőkorban és gyakran még a felnőttkorban is gondot jelentő acne vulgaris gyulladásos bőrbetegség genomikai kutatása során igazoltuk, hogy az interleukin 1 receptor antagonista gén (IL-1RN) egy polimorfizmusa ebben a kórképben magasabb arányban fordul elő, mint az egészséges populációban, amely pathogenetikai szerepére utal. Megmutattuk, hogy az acne pathomechanizmusában kulcsfontosságú Propionibacterium acnes baktérium a keratinocitákban a veleszületett immunitásban szerepet játszó gének expresszióját indukálja. Vizsgáltuk a hisztamin szerepét az allergiás kontakt dermatitisz pathogenezisében. Kísérleteinkhez hisztidin dekarboxiláz knock-out egereket használtunk, és eredményeink alapján feltételezzük, hogy a hisztamin a késői típusú kontakt dermatitiszben negatív reguláló szerepet tölt be. A pályázat keretében a gyulladásos bőrbetegségek ill. a szénanátha kezelésére alkalmas új fototerápiás eljárásokat is fejlesztettünk és vizsgáltuk azok hatásmechanizmusát. | We investigated the role of an extracellular matrix protein, fibronectin (an important regulator of the cell cycle), in psoriasis. We identified a non-coding RNA gene, PRINS playing a role in cell stress, and a key factor of psoriasis susceptibility. The genetic background of the multifactorial leg ulcer was also studied. We discovered an SNP that is more frequent among leg ulcer patients than in controls. This finding suggests that this SNP might play a role in the pathogenesis of this disease. Acne vulgaris is another very common inflammatory skin disorder affecting a significant proportion of the teenager and the adult population. We showed that a polymorphism of the interleukin 1 receptor antagonist gene (IL1-RN) is more frequent among the affected individuals compared to healthy controls. This suggests a role for the IL1-RN gene in the development of this skin condition. Studies on the contribution of the skin colonizing Propionibacterium acne in the pathomechanism of acne showed that this bacterium induces the expression of genes playing a role in innate immunity of human kerationocytes. The role of histamine in allergic contact dermatitis was also investigated. We used histamine decarboxylase knock-out mice, and our results suggest that histamine plays a negative regulatory role in the late-type contact dermatitis. We have developed novel phototherapeutic methods for the treatment of inflammatory skin diseases and rhinitis allergica, and studied their mode of action

RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling

The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Gliomaassociated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway. Copyright © 2013 Landes Bioscience

TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes.

A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea

Chitin Modulates Innate Immune Responses of Keratinocytes

Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood. We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR) TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes. We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined

MicroRNAs: Novel Regulators Involved in the Pathogenesis of Psoriasis?

MicroRNAs are a recently discovered class of posttranscriptional regulators of gene expression with critical functions in health and disease. Psoriasis is the most prevalent chronic inflammatory skin disease in adults, with a substantial negative impact on the patients' quality of life. Here we show for the first time that psoriasis-affected skin has a specific microRNA expression profile when compared with healthy human skin or with another chronic inflammatory skin disease, atopic eczema. Among the psoriasis-specific microRNAs, we identified leukocyte-derived microRNAs and one keratinocyte-derived microRNA, miR-203. In a panel of 21 different human organs and tissues, miR-203 showed a highly skin-specific expression profile. Among the cellular constituents of the skin, it was exclusively expressed by keratinocytes. The up-regulation of miR-203 in psoriatic plaques was concurrent with the down-regulation of an evolutionary conserved target of miR-203, suppressor of cytokine signaling 3 (SOCS-3), which is involved in inflammatory responses and keratinocyte functions. Our results suggest that microRNA deregulation is involved in the pathogenesis of psoriasis and contributes to the dysfunction of the cross talk between resident and infiltrating cells. Taken together, a new layer of regulatory mechanisms is involved in the pathogenesis of chronic inflammatory skin diseases

A Mannose-Binding Receptor is Expressed on Human Keratinocytes and Mediates Killing of Candida albicans

Human keratinocytes are known to kill Candida albicans in vitro, but the mechanism of killing is not yet understood. Here, we demonstrate that spontaneous, ultraviolet-B-light-induced, α-melanocyte-stimulating-hormone-induced, and interleukin-8-induced Candida killing by keratinocytes can be inhibited with mannan and mannosylated bovine serum albumin (Man-BSA). A polyclonal goat serum raised against the human macrophage mannose receptor stained suprabasal keratinocytes, but no staining was observed on keratinocytes with a monoclonal antibody (mAb15) specific for the human macrophage mannose receptor. Mannose-affinity chromatography of keratinocyte extract isolated a 200 kDa protein, and on the Western blot the goat antiserum reacted with a 200 kDa protein. In radioligand binding studies, the binding of 125I-Man-BSA to human keratinocytes was inhibited by mannan in a concentration-dependent manner. Analysis of the binding revealed a single class keratinocyte mannose receptor with a KD of 1.4 × 10-8 M and a Bmax of 1 × 104 binding sites per cell. The binding of 125I-Man-BSA to keratinocytes proved to be time-dependent, acid-precipitable, and Ca2+- and trypsin-sensitive. After trypsinization the receptors underwent a rapid recovery at 37°C. These results demonstrate the presence of mannose receptor on human keratinocytes, and its active involvement in the killing of Candida albicans