Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of notch: efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats. J Biol Chem 2012;287:33934e44.

Rosacea is a common chronic inflammation of sebaceous glanderich facial skin characterized by severe skin dryness, elevated pH, transepidermal water loss, and decreased hydration levels. Until now, there has been no thorough molecular analysis of permeability barrier alterations in the skin of patients with rosacea. Thus, we aimed to investigate the barrier alterations in papulopustular rosacea samples compared with healthy sebaceous glanderich skin, using RNA sequencing analysis (n ¼ 8). Pathway analyses by Cytoscape ClueGO revealed 15 significantly enriched pathways related to skin barrier formation. RT-PCR and immunohistochemistry were used to validate the pathway analyses. The results showed significant alterations in barrier components in papulopustular rosacea samples compared with sebaceous glanderich skin, including the cornified envelope and intercellular lipid lamellae formation, desmosome and tight junction organizations, barrier alarmins, and antimicrobial peptides. Moreover, the barrier damage in papulopustular rosacea was unexpectedly similar to atopic dermatitis; this similarity was confirmed by immunofluorescent staining. In summary, besides the well-known dysregulation of immunological, vascular, and neurological functions, we demonstrated prominent permeability barrier alterations in papulopustular rosacea at the molecular level, which highlight the importance of barrier repair therapies for rosacea.

The immunological barrier of the healthy skin is considered to be unified on the whole body surface—however, recent indirect findings have challenged this dogma since microbial and chemical milieu (e.g., sebum, sweat, and pH) exhibit remarkable differences on topographically distinct skin areas. Therefore, in the present study, we performed whole transcriptomic and subsequent pathway analyses to assess differences between sebaceous gland rich (SGR) and sebaceous gland poor (SGP) regions. Here, we provide the first evidence that different skin regions exhibit a characteristic innate and adaptive immune and barrier milieu as we could detect significantly increased chemokine (CCL2, 3, 19, 20, 23, 24) and antimicrobial peptide (S100A7, A8, A9, lipocalin, β-defensin-2) expression, altered barrier (keratin 17, 79) functions, and a non-inflammatory Th17/IL-17 dominance in SGR skin compared to SGP. Regarding pro-inflammatory molecules (IL-1α, IL-6, IL-8, IL-33, TNF-α), similarly low levels were detected in both regions. Our data may explain the characteristic topographical localization of some immune-mediated and autoimmune skin disorders and we also propose that the term “healthy skin control sample,” widely used in experimental Dermatology, should only be accepted if researchers carefully specify the exact region of the healthy skin (along with the site of the diseased sample).

Background Hidradenitis suppurativa (HS) is a chronic, inflammatory disease of the apocrine gland-rich (AGR) skin region. The initial steps of disease development are not fully understood, despite intense investigations into immune alterations in lesional HS skin.Objectives We aimed to systematically investigate the inflammatory molecules involved in three stages of HS pathogenesis, including healthy AGR, non-lesional HS and lesional HS skin, with the parallel application of multiple mRNA and protein-based methods.

Psoriasis is a common inflammatory skin disease and dendritic cells (DCs) play crucial role in the development of skin inflammation. Although the characteristics of skin DCs in psoriasis are well defined, less is known about their peripheral blood precursors. Our aim was to characterize the phenotypic features as well as the cytokine and chemokine production of CD1c myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the blood samples of psoriatic patients. Blood DCs were isolated by using a magnetic separation kit, and their intracytoplasmic cytokine production and CD83/CD86 maturation/activation marker expression were investigated by 8-colour flow cytometry. In CD1c mDCs the intracellular productions of Th1, Th2, Th17, Th22 and Treg polarizing cytokines were examined simultaneously, whereas in pDCs the amounts of IFNα as well as IL-12, IL-23 and IL-6 were investigated. The chemokine production of both DC populations was investigated by flow-cytometry and ELISA. According to our results psoriatic CD1c mDCs were in a premature state since their CD83/CD86 maturation/activation marker expression, IL-12 cytokine, CXCL9 and CCL20 chemokine production was significantly higher compared to control cells. On the other hand, blood pDCs neither produced any of the investigated cytokines and chemokines nor expressed CD83/CD86 maturation/activation markers. Our results indicate that in psoriasis not only skin but also blood mDCs perform Th1 polarizing and Th1/Th17 recruiting capacity, while pDCs function only in the skin milieu.

The chemical milieu, microbiota composition, and immune activity show prominent differences in distinct healthy skin areas. The objective of the current study was to compare the major permeability barrier components (stratum corneum and tight junction (TJ)), investigate the distribution of (corneo)desmosomes and TJs, and measure barrier function in healthy sebaceous gland-rich (SGR), apocrine gland-rich (AGR), and gland-poor (GP) skin regions. Molecules involved in cornified envelope (CE) formation, desquamation, and (corneo)desmosome and TJ organization were investigated at the mRNA and protein levels using qRT-PCR and immunohistochemistry. The distribution of junction structures was visualized using confocal microscopy. Transepidermal water loss (TEWL) functional measurements were also performed. CE intracellular structural components were similarly expressed in gland-rich (SGR and AGR) and GP areas. In contrast, significantly lower extracellular protein levels of (corneo)desmosomes (DSG1 and CDSN) and TJs (OCLN and CLDN1) were detected in SGR/AGR areas compared to GP areas. In parallel, kallikrein proteases were significantly higher in gland-rich regions. Moreover, gland-rich areas were characterized by prominently disorganized junction structures ((corneo)desmosomes and TJs) and significantly higher TEWL levels compared to GP skin, which exhibited a regular distribution of junction structures. According to our findings, the permeability barrier of our skin is not uniform. Gland-rich areas are characterized by weaker permeability barrier features compared with GP regions. These findings have important clinical relevance and may explain the preferred localization of acantholytic skin diseases on gland-rich skin regions (e.g., Pemphigus foliaceus, Darier’s disease, and Hailey–Hailey disease).

Microbial community is highly diverse on the skin surface and can change in response to environmental challenges or aging. Its mutual relationship with the host and its key role in tissue homeostasis have also been reported. Previously, our workgroup described a non-inflammatory Th17/Treg milieu in sebaceous skin region which can be connected to the different microbiome and chemical milieu of this area. Since these factors also differ in apocrine gland-rich (AGR) areas, we aimed to compare the immune milieu of healthy sebaceous gland-poor (SGP) and AGR skin regions and to study its changes in Hidradenitis Suppurativa (HS), an inflammatory disease characteristically localized on AGR areas. Cytokines, cell surface markers, activation markers and transcription factors of keratinocytes, dendritic cells (DC) and T cells were detected by immunohistochemistry and qPCR in biopsies from healthy AGR and SGP skin and from HS lesional skin. In AGR skin, higher noninflammatory TSLP expression, DC appearance without prominent activation and T cell presence with IL-17/IL-10 cytokine milieu were detected compared to SGP skin. The level of these parameters further increased in HS with the presence of activated DCs and a prominent influx of IL-17+ and IFN-g+ cells. qPCR analyses of the aforementioned factors showed a similar pattern. Similarly to sebaceous region, AGR skin areas represent distinct immune milieu from SGP regions, which can be the result of the different microbiome and chemical milieu. These changes may be connected to the maturation of apocrine glands and change in microbiome during puberty, but it needs further investigation.

In order to suppress self-reactive T cells that escape from central tolerance, additional mechanism in peripheries, so-called peripheral tolerance, is essential; however this mechanism is not fully elucidated. To investigate this issue, here we generated a novel murine transgenic line that expressed membrane-bound ovalbumin (mOVA) under the control of human involucrin (Ivl) promoter; Ivl-mOVA mice. Within one week after the transfer of CD8 + T cells from OT-I mice (OT-I T cells), GVHD-like cutaneous and mucosal eruptions developed. Histological analysis revealed massive infiltration of OT-I T cells to the epidermis. Intriguingly, most (>95%) of the transferred OT-I T cells were deleted in lymph nodes within 24 h, and subsequent extensive expansion of OT-I T cells was observed 48 h post transfer. This expansion was observed not only in skin-draining lymph nodes but also in mesenteric lymph nodes, spleen, and bone marrow. We used bone-marrow chimeric mice reconstituted with b2-microglobulin-deficient mice, which lack functional MHC class I, to identify the responsible cell subset for initial deletion of OT-I T cells. OT-I T cell deletion was observed even in these chimeric mice. This result indicates the essential role of radio-resistant cells, suggestive of stromal cells, in secondary lymphoid organs. Taken together, these results suggest that tissue-specific self-antigens presented by radio-resistant lymph node stromal cells control peripheral tolerance and development of GVHD-like skin lesion. 365Myeloid but not plasmacytoid blood DCs possess Th1 polarizing and Th1/Th17 recruiting capacity in psoriasis A Psoriasis is a common inflammatory skin disease and dendritic cells (DCs) play a crucial role in the development of skin inflammation. Although the characteristics of skin DCs in psoriasis are well defined, less is known about their peripheral blood precursors. Our aim was to characterize the phenotypic features as well as the cytokine and chemokine production of CD1c+ myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the blood samples of psoriatic patients. Blood DCs were isolated by using a magnetic separation kit, and their intracytoplasmic cytokine production and CD83/CD86 maturation/activation marker expression were investigated by 8-colour flow cytometry. In CD1c+ mDCs, the intracellular productions of Th1, Th2, Th17, Th22 and Treg polarizing cytokines were examined simultaneously, whereas in pDCs the amounts of IFNa, as well as IL-12, IL-23, and IL-6, were investigated. The chemokine production of both DC populations was investigated by flow cytometry and ELISA. According to our results, psoriatic CD1c+ mDCs were in a premature state since their CD83/CD86 maturation/activation marker expression, IL-12 cytokine, CXCL9 and CCL20 chemokine production was significantly higher compared to control cells. On the other hand, blood pDCs neither produced any of the investigated cytokines and chemokines nor expressed CD83/CD86 maturation/activation markers. Our results indicate that in psoriasis not only skin but al...