Single-cell transcriptomics applied to emigrating cells from psoriasis elucidate pathogenic versus regulatory immune cell subsets.

BACKGROUND: In previous human skin single-cell data, inflammatory cells constituted only a small fraction of the overall cell population, such that functional subsets were difficult to ascertain. OBJECTIVE: Our aims were to overcome the aforesaid limitation by applying single-cell transcriptomics to emigrating cells from skin and elucidate ex vivo gene expression profiles of pathogenic versus regulatory immune cell subsets in the skin of individuals with psoriasis. METHODS: We harvested emigrating cells from human psoriasis skin after incubation in culture medium without enzyme digestion or cell sorting and analyzed cells with single-cell RNA sequencing and flow cytometry simultaneously. RESULTS: Unsupervised clustering of harvested cells from psoriasis skin and control skin identified natural killer cells, T-cell subsets, dendritic cell subsets, melanocytes, and keratinocytes in different layers. Comparison between psoriasis cells and control cells within each cluster revealed that (1) cutaneous type 17 T cells display highly differing transcriptome profiles depending on IL-17A versus IL-17F expression and IFN-γ versus IL-10 expression; (2) semimature dendritic cells are regulatory dendritic cells with high IL-10 expression, but a subset of semimature dendritic cells expresses IL-23A and IL-36G in psoriasis; and (3) CCL27-CCR10 interaction is potentially impaired in psoriasis because of decreased CCL27 expression in basal keratinocytes. CONCLUSION: We propose that single-cell transcriptomics applied to emigrating cells from human skin provides an innovative study platform to compare gene expression profiles of heterogenous immune cells in various inflammatory skin diseases.

Progranulin promotes osteogenic differentiation of human periodontal ligament stem cells via tumor necrosis factor receptors to inhibit TNF-α sensitized NF-kB and activate ERK/JNK signaling.

OBJECTIVE: To investigate the molecular mechanism of Progranulin (PGRN) in promoting osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory environment. BACKGROUND: Progranulin is an antagonist of tumor necrosis factor (TNF) receptors (TNFRs) and is known to promote inflammatory periodontal bone defect regeneration. METHODS: TNFR1- and TNFR2-silenced hPDLSCs designed as hPDLSCs-sh-TNFR1 and hPDLSCs-sh-TNFR2 were cultured with osteoinductive medium containing TNF-α and (or) PGRN. Immunofluorescence, quantitative real-time PCR, and western blot were used to, respectively, detect expressions of TNFR1\TNFR2 and osteogenic differentiation markers as well as phosphorylation level in NF-κB\MAPK-related pathways. RESULTS: Immunofluorescence and real-time PCR showed that TNFR1 and TNFR2 positively expressed in hPDLSCs. TNF-α stimulation could significantly decrease the expressions of ALP and RUNX2 in hPDLSCs, whereas PGRN treatment could significantly enhance their expressions, and reverse TNF-α-mediated expression suppression of ALP and RUNX2 in hPDLSCs. In hPDLSCs-sh-TNFR1, TNF-α mediated osteogenic inhibition decreased, but both TNF-α + PGRN and alone PGRN significantly promoted expression of ALP and RUNX2. PGRN significantly enhanced expression of P-ERK1/2 and P-JNK, while corresponding inhibitors eliminated PGRN-stimulated osteogenic differentiation. In hPDLSCs-sh-TNFR2, no significant difference existed in osteogenic markers and P-JNK expression between the PGRN group and the control group. However, PGRN still activated P-ERK1/2 expression. Besides, PGRN antagonized TNF-α-enhanced NF-κB P65 expression. CONCLUSION: Progranulin promotes osteogenic differentiation of hPDLSCs via TNFR1 to inhibit TNF-α-sensitized NF-κB and via TNFR2 to activate JNK signaling. The mechanism by which PGRN activates ERK signaling remains to be explored.

Convergent inactivation of the skin-specific C-C motif chemokine ligand 27 in mammalian evolution.

The appearance of mammalian-specific skin features was a key evolutionary event contributing for the elaboration of physiological processes such as thermoregulation, adequate hydration, locomotion, and inflammation. Skin inflammatory and autoimmune processes engage a population of skin-infiltrating T cells expressing a specific C-C chemokine receptor (CCR10) which interacts with an epidermal CC chemokine, the skin-specific C-C motif chemokine ligand 27 (CCL27). CCL27 is selectively produced in the skin by keratinocytes, particularly upon inflammation, mediating the adhesion and homing of skin-infiltrating T cells. Here, we examined the evolution and coding condition of Ccl27 in 112 placental mammalian species. Our findings reveal that a number of open reading frame inactivation events such as insertions, deletions, and start and stop codon mutations independently occurred in Cetacea, Pholidota, Sirenia, Chiroptera, and Rodentia, totalizing 18 species. The diverse habitat settings and lifestyles of Ccl27-eroded lineages probably implied distinct evolutionary triggers rendering this gene unessential. For example, in Cetacea, the rapid renewal of skin layers minimizes the need for an elaborate inflammatory mechanism, mirrored by the absence of epidermal scabs. Our findings suggest that the convergent and independent loss of Ccl27 in mammalian evolution concurred with unique adaptive roads for skin physiology.

Assessment of CCL27 and IL-11 in Multiple Sclerosis Patients Treated with Interferon-ß and Glatiramer Acetate.

INTRODUCTION: Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease which involves the central nervous -system. Although the primary cause of MS is obscure, effects of some cytokine and chemokine patterns in both innate and adaptive immune systems have been described. -Objectives: Since limited studies have examined the role of interleukin (IL)-11 and chemokine CCL27 in MS, we aimed to identify changes in IL-11 and CCL27 gene expression and serum levels in relapsing-remitting MS (RRMS) patients, treated with interferon (IFN)-ß and glatiramer acetate (GA). METHODS: The serum level and gene expression of IL-11 and CCL27 were measured and compared between treatment-naïve MS patients and RRMS patients who were treated with high-dose IFN-ß1a, low-dose IFN-ß1a, IFN-ß1b, and GA via enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction. RESULTS: A significant decrease was observed in the serum level of CCL27 in treatment-naïve patients and IFN-ß1b-treated patients compared to the healthy controls. On the other hand, a significant increase was found in the protein level of CCL27 in low-dose and high-dose IFN-ß1a groups compared to the treatment-naïve group. In addition, CCL27 gene expression was higher in patients treated with GA than in the treatment-naïve group. There were no significant changes in the gene expression or protein level of IL-11 in all experimental groups. Additionally, a positive correlation was found between IL-11 and CCL-27. CONCLUSION: Our results suggest the inflammatory role of CCL27 in MS patients, while IFN-ß1a seems to play a compensatory role for this chemokine.

Hybrid training system-induced myokine secretion in healthy men.

The hybrid training system (HTS) is a special and compact system for effective skeletal muscle training by a combined application of volitional and electrical muscle contraction. Lower limbs' muscle training using HTS has been reported to increase not only muscle strength but also plasma interleukin-6 levels; however, little is known in other cytokines. In this study, we measured 52 cytokines and creatine phosphokinase-MM in the serum of 16 healthy men before and after lower limbs' muscle training by the knee flexion and extension using HTS. Skeletal muscle volume-corrected serum concentrations of cutaneous T-cell-attracting chemokine, erythropoietin, and tumor necrosis factor-related apoptosis-inducing ligand increased immediately after the training. These increased cytokines have been reported to play important roles in wound healing, neuroprotection, and cardiovascular protection.

Oral keratinocytes synthesize CTACK: A new insight into the pathophysiology of the oral mucosa.

The skin-associated chemokine CTACK plays a key role in many inflammatory conditions and could be instrumental in the pathophysiology of tissue-specific immunological diseases such as oral lichen planus (OLP). In this study, we investigated, by RT-PCR, ELISA, chemotaxis assays, and fluorescence-activated cell sorting (FACS), the production of CTACK in oral keratinocytes, its expression in tissues from normal and OLP patients, and its role in T-cell recruitment.CTACK was produced by the oral epithelium, and it affects chemotaxis of memory CLA+ cells to the oral epithelium. CTACK mRNA was expressed constitutively in primary oral epithelium and was increased during pro-inflammatory IFN-γ treatment. We found a constitutive production of CTACK at a protein level in oral primary cells that increased after IFN-γ treatment. Moreover, we confirmed that CTACK attracts memory T cells and those T cells that express CLA above the level of basal migration.

Acteoside relieves mesangial cell injury by regulating Th22 cell chemotaxis and proliferation in IgA nephropathy.

The existing therapies of IgA nephropathy are unsatisfying. Acteoside, the main component of Rehmannia glutinosa with anti-inflammatory and anti-immune effects, can improve urinary protein excretion and immune disorder. Th22 cell is involved in IgA nephropathy progression. This study was determined to explore the effect of acteoside on mesangial injury underlying Th22 cell disorder in IgA nephropathy. Serum Th22 cells and urine total protein of patients with IgA nephropathy were measured before and after six months treatment of Rehmannia glutinosa acteoside or valsartan. Chemotactic assay and co-culture assay were performed to investigate the effect of acteoside on Th22 cell chemotaxis and differentiation. The expression of CCL20, CCL22 and CCL27 were analyzed. To explore the effect of acteoside on mesangial cell injury induced by inflammation, IL-1, IL-6, TNF-α and TGF-ß1 were tested. Results showed that the proteinuria and Th22 lymphocytosis of patients with IgA nephropathy significantly improved after combination treatment of Rehmannia glutinosa acteoside and valsartan, compared with valsartan monotherapy. In vitro study further demonstrated that acteoside inhibit Th22 cell chemotaxis by suppressing the production of Th22 cell attractive chemokines, i.e., CCL20, CCL22 and CCL27. In addition, acteoside inhibited the Th22 cell proliferation. Co-culture assay proved that acteoside could relieve the overexpression of pro-inflammatory cytokines, and prevent the synthesis of TGF-ß1. TGF-ß1 level in mesangial cells was positively correlated with the Th22 cell. This research demonstrated that acteoside can alleviate mesangial cell inflammatory injury by modulating Th22 lymphocytes chemotaxis and proliferation.

Ionizing radiation promotes CCL27 secretion from keratinocytes through the cross talk between TNF-α and ROS.

The skin-associated chemokine CCL27 and its receptor CCR10 mediate the immune response of skin-homing T cells. The CCL27 secreted from keratinocytes was reportedly involved in inflammatory skin diseases such as atopic dermatitis, contact dermatitis, and psoriasis. However, whether ionizing radiation increases the levels of CCL27 secretion still remains unclear. In HaCaT cells, a human keratinocyte cell line, CCL27 secretion was markedly increased after X-ray irradiation. We further found that irradiation boosted the generation of reactive oxygen species (ROS), which was concomitant with the release of tumor necrosis factor-alpha (TNF-α). Moreover, alteration of ROS in irradiated HaCaT cells correlated with TNF-α secretion, indicating a positive loop of TNF-α secretion and ROS generation. This positive loop regulated the secretion of CCL27 from irradiated cells. We therefore concluded that the cross talk between TNF-α and ROS after keratinocytes was exposed to radiation, triggered CCL27 secretion for subsequent inflammation response.

A novel molecular disease classifier for psoriasis and eczema.

Novel specific therapies for psoriasis and eczema have been developed, and they mark a new era in the treatment of these complex inflammatory skin diseases. However, within their broad clinical spectrum, psoriasis and eczema phenotypes overlap making an accurate diagnosis impossible in special cases, not to speak about predicting the clinical outcome of an individual patient. Here, we present a novel robust molecular classifier (MC) consisting of NOS2 and CCL27 gene that diagnosed psoriasis and eczema with a sensitivity and specificity of >95% in a cohort of 129 patients suffering from (i) classical forms; (ii) subtypes; and (iii) clinically and histologically indistinct variants of psoriasis and eczema. NOS2 and CCL27 correlated with clinical and histological hallmarks of psoriasis and eczema in a mutually antagonistic way, thus highlighting their biological relevance. In line with this, the MC could be transferred to the level of immunofluorescence stainings for iNOS and CCL27 protein on paraffin-embedded sections, where patients were diagnosed with sensitivity and specificity >88%. Our MC proved superiority over current gold standard methods to distinguish psoriasis and eczema and may therefore build the basis for molecular diagnosis of chronic inflammatory skin diseases required to establish personalized medicine in the field.

Proposing the use of dental pulp stem cells as a suitable biological model of neurofibromatosis type 1.

PURPOSE: This study aims to propose the dental pulp stem cells (DPSCs) as a model for studying two features related to neurofibromatosis type 1 (NF1), i.e. augmented proliferative capacity and altered osteogenic differentiation. METHODS: We isolated a DPSC from the pulp of deciduous teeth of a 6-year-old NF1 patient and two other healthy children of similar age. Cell proliferation was assayed by counting with a haemocytometer after successive cell re-plating. In order to compare osteogenic differentiation, we used osteoblast-differentiating medium and quantified alizarin stain, which relates to degree of calcification, and evaluated the expression of osteoblastic markers by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The DPSCs isolated from the NF1 patient displayed a greater rate of proliferation when compared to the control cells. Osteogenic differentiation occurred as expected for both NF1 and control, which concerned cell morphology and expression of osteoblast marker genes ALP, BMP2, BMP4, OCN and SPP1. However, alizarin staining denoted a markedly lower calcification level in the cells from the NF1-diagnosed child, considering that less calcium deposits were visualized under light microscopy and a smaller amount of alizarin could be quantified by spectrophotometry after extraction from the stained cells. CONCLUSION: DPSCs seem to be useful as a model for studying NF1 and predicting prognosis of patients, since their in vitro behaviour seems to mimic at least two features of this disorder: higher tendency to develop bone abnormalities and neoplastic cell proliferation.

CCR10 regulates balanced maintenance and function of resident regulatory and effector T cells to promote immune homeostasis in the skin.

BACKGROUND: CCR10 and CCL27 make up the most skin-specific chemokine receptor/ligand pair implicated in skin allergy and inflammatory diseases, including atopic dermatitis and psoriasis. This pair is thought to regulate the migration, maintenance, or both of skin T cells and is suggested to be therapeutic targets for treatment of skin diseases. However, the functional importance of CCR10/CCL27 in vivo remains elusive. OBJECTIVE: We sought to determine the expression and function of CCR10 in different subsets of skin T cells under both homeostatic and inflammatory conditions to gain a mechanistic insight into the potential roles of CCR10 during skin inflammation. METHODS: Using heterozygous and homozygous CCR10 knockout/enhanced green fluorescent protein knockin mice, we assessed the expression of CCR10 on regulatory and effector T cells of healthy and inflamed skin induced by chemicals, pathogens, and autoreactive T cells. In addition, we assessed the effect of CCR10 knockout on the maintenance and functions of different T cells and inflammatory status in the skin during different phases of the immune response. RESULTS: CCR10 expression is preferentially induced on memory-like skin-resident T cells and their progenitors for their maintenance in homeostatic skin but not expressed on most skin-infiltrating effector T cells during inflammation. In CCR10 knockout mice the imbalanced presence and dysregulated function of resident regulatory and effector T cells result in over-reactive and prolonged innate and memory responses in the skin, leading to increased clearance of Leishmania species infection in the skin. CONCLUSION: CCR10 is a critical regulator of skin immune homeostasis.

Presence of CTAK/CCL27, MCP-3/CCL7 and LIF in human colostrum and breast milk.

Human colostrum and breast milk are known to contain high levels of cytokines and chemokines, which are thought to contribute to the development of the newborn. The aim of this study was to investigate the difference in the presence and levels of 21 soluble cytokines and chemokines in paired samples of human colostrum (day 2 after delivery) and breast milk (day 4-5 after delivery) by using the multiplex technology. Of the 21 cytokine investigated in 10 pairs of samples, only ß-NGF was absent in both colostrum and milk, while INF-α2, SCF and TNF-ß were present in colostrum but not in human milk. As a general rule, colostrum contained higher concentrations of cytokines and chemokines with respect to breast milk. The majority of cytokines, detected in colostrum alone or in colostrum and human milk (IL-1α, IL-2Rα, IL-3, IL-16, IL-18, GRO-α, HGF, IFN-α2, M-CSF, MIF, MIG, TNF-ß, SDF-1α, TRAIL) have been described in previous studies, while for the first time we describe the presence of additional cytokines either in colostrum alone (SCF) or in both colostrum and breast milk (CTAK/CCL27, MCP-3/CCL7, LIF). Our data confirm and expand previous studies showing that some cytokines/chemokines, which might contribute to the development of the gastro-intestinal and nervous systems, are overexpressed in human colostrum and breast milk, and might contribute to the development of these systems.

Analysis of chemotactic molecules in bone marrow-derived mesenchymal stem cells and the skin: Ccl27-Ccr10 axis as a basis for targeting to cutaneous tissues.

BACKGROUND AIMS: Adult stem cells produce a plethora of extracellular matrix molecules and have a high potential as cell-based therapeutics for connective tissue disorders of the skin. However, the primary challenge of the stem cell-based approach is associated with the inefficient homing of systemically infused stem cells to the skin. METHODS: We examined chemotactic mechanisms that govern directional migration of mesenchymal stem cells (MSCs) into the skin by conducting a comprehensive expression analysis of chemotactic molecules in MSCs and defined cutaneous tissues from normal and hereditary epidermolysis bullosa (EB)-affected skin. RESULTS: Analysis of chemokine receptors in short-term and long-term MSC cultures showed tissue culture-dependent expression of several receptors. Assessment of epidermis-derived and dermis-derived chemokines showed that most chemotactic signals that originate from the skin preferentially recruit different sets of leukocytes rather than MSCs. Analysis of the chemotactic molecules derived from EB-affected non-blistered skin showed only minor changes in expression of selected chemokines and receptors. Nevertheless, the data allowed us to define the Ccl27-Ccr10 chemotactic axis as the most potent for the recruitment of MSCs to the skin. Our in vivo analysis demonstrated that uniform expression of Ccr10 on MSCs and alteration of Ccl27 level in the skin enhance extravasation of stem cells from circulation and facilitate their migration within cutaneous tissue. CONCLUSIONS: Collectively, our study provides a comprehensive analysis of chemotactic signals in normal and EB-affected skin and proof-of-concept data demonstrating that alteration of the chemotactic pathways can enhance skin homing of the therapeutic stem cells.

Connexin26 Modulates Radiation-Induced Skin Damage by Regulating Chemokine CCL27 through MAPK Signaling.

Connexin26 (Cx26) plays an important role in ionizing radiation-induced damage, and CC chemokine ligand 27 (CCL27) regulates the skin immune response. However, the relationship between Cx26 and CCL27 in radiation-induced skin damage is unclear. After X-ray irradiation, clonogenic survival and micronucleus formation were assessed in immortalized human keratinocytes (HaCaT). Proteins in the mitogen activated protein kinase (MAPK) signaling pathway and CCL27-related proteins were detected by immunoblotting. HaCaTCx26-/- cells were constructed to verify the effects of Cx26 on CCL27 secretion. A mouse model was established to examine the expression of CCL27 and skin inflammation in vivo. The degree of skin injury induced by 6 MV of X rays was closely related to CCL27. The phosphorylation of ERK, p38 and NF-κB was significantly increased in irradiated cells. The secretion of CCL27 was significantly decreased in HaCaT wild-type cells relative to HaCaTCx26-/- cells. Whereas cell survival fractions decreased, and the micronuclei formation rate increased as a function of increasing X-ray dose in HaCaT cells, the opposite trend occurred in HaCaTCx26-/- cells. Our findings show that Cx26 likely plays a role in the activation of the MAPK and NF-κB/COX-2 signaling pathways and regulates the secretion of CCL27 in keratinocytes after X-ray radiation-induced skin damage.

The CCL27-CCR10 axis contributes to promoting proliferation, migration, and invasion of lung squamous cell carcinoma.

Lung cancer is characterized by its high mortality and morbidity. A deep understanding of the molecular mechanisms of lung cancer tumorigenesis helps to develop novel lung cancer diagnostic and therapeutic strategies. However, the picture of the associated molecular landscape is not yet complete. As understood, chemokine-receptor interactions contribute much to lung cancer tumorigenesis, in which CCR10 also plays an important role. This study aimed to expand the knowledge of CCR10 in lung squamous cell carcinoma (LUSC) in the manner of molecular mechanism and biological functions. Using GEPIA database, the survival analysis between LUSC patients with high and low CCR10 expressions was performed, showing that CCR10 could be regarded as a risk factor for LUSC patients. Subsequently, CCR10 protein and mRNA expressions in LUSC were examined by qRT-PCR and western blot respectively. The results indicated that CCR10 was highly expressed in LUSC cells. The results of CCK-8, colony formation, and Transwell assays presented that CCL27, the ligand of CCR10, promoted proliferative, migratory, and invasive abilities of LUSC cells by activating CCR10. Also, the PI3K/AKT signaling pathway was verified as the involved pathway by western blot. Overall, it could be concluded that the CCL27-CCR10 regulatory axis can activate the PI3K/AKT pathway fostering the malignant features of LUSC cells.

Serum and tissue CTACK/CCL27 chemokine levels in early mycosis fungoides may be correlated with disease-free survival following treatment with interferon alfa and psoralen plus ultraviolet A therapy.

BACKGROUND: Neoplastic T-cell recruitment into the skin is a critical step in the pathogenesis of mycosis fungoides (MF), and the cutaneous T-cell attracting chemokine, CTACK/CCL27, might be involved. OBJECTIVES: To investigate the clinical and prognostic significance of CTACK/CCL27 levels in patients with early-stage MF. METHODS: Serum samples and skin biopsy specimens were collected from 15 patients at the time of diagnosis and after the end of treatment with psoralen plus ultraviolet A/interferon alfa-2b combination therapy. Serum samples were also collected from 20 healthy donors as controls. CTACK/CCL27 serum levels were analysed by enzyme-linked immunosorbent assays. CTACK/CCL27 tissue expression was determined by immunohistochemistry on skin biopsy specimens taken at diagnosis and after therapy. Event-free survival was taken as the primary clinical outcome. RESULTS: In patients with MF at diagnosis, CTACK/CCL27 serum levels were not significantly different from healthy controls, whereas CTACK/CCL27 expression in the skin was increased in 87% of cases compared with normal controls. After therapy, all patients obtained a clinical complete remission, serum levels did not change significantly and tissue expression remained abnormal in 80% of patients, even if complete histological remission was recorded. Serum levels were not significantly different in cases with different intensity of cutaneous immunostaining. Eight patients experienced a relapse: the combination of high CTACK/CCL27 levels both in sera and skin increased the probability of experiencing an event at 51 months from 36% to 83%. CONCLUSIONS: Our data seem to indicate that CTACK/CCL27 levels in skin and sera after therapy might be correlated with risk of recurrence.

NMR analysis of the structure, dynamics, and unique oligomerization properties of the chemokine CCL27.

Chemokines have two essential interactions in vivo, with G protein-coupled receptors, which activate intracellular signaling pathways, and with glycosaminoglycans (GAGs), which are involved in cell surface localization and transport. Although it has been shown that chemokines bind and activate their respective G protein-coupled receptors as monomers, many chemokines oligomerize upon GAG binding, and the ability to oligomerize and bind GAGs is required for in vivo function. In this study, we investigated the structure, dynamics, and oligomerization behavior of cutaneous T-cell-attracting chemokine (CTACK, also known as CCL27) by NMR. (15)N relaxation and translational self-diffusion rates indicate that CCL27 oligomerizes, but in contrast to many other chemokines that form relatively discrete oligomers, CCL27 transitions between monomer, dimer, and tetramer species over a relatively narrow concentration range. A three-dimensional structure determination was pursued under conditions where CCL27 is primarily dimeric, revealing the standard motif for a chemokine monomer. Analysis of chemical shift perturbations of (1)H-(15)N HSQC spectra, relaxation-dispersion experiments, and filtered nuclear Overhauser effects suggest that CCL27 does not adopt a discrete CXC or CC dimer motif. Instead, CCL27 has uncommon oligomerization behavior, where several equilibria involving relatively low affinity interactions between different interfaces seem to be simultaneously at work. However, interaction with heparin avidly promotes oligomerization under conditions where CCL27 is monomeric by itself. We hypothesize that the plasticity in the oligomerization state may enable CCL27 to adopt different oligomeric structures, depending on the nature of the GAG binding partner, thereby providing a mechanism for increased diversity and specificity in GAG-binding and GAG-related functions.

CCL27 expression is regulated by both p38 MAPK and IKKß signalling pathways.

The skin-specific chemokine CCL27 is believed to play a pivotal role in establishing the inflammatory infiltrate characteristic for common inflammatory skin diseases. Through binding to the chemokine receptor 10 (CCR10), CCL27 mediates inflammation by promoting lymphocyte migration into the skin. Little is known about the regulation of CCL27 gene expression. The purpose of our study was to investigate the regulation of the IL-1ß-induced CCL27 gene expression in normal human keratinocytes (NHEK). Preincubation of NHEK with the inhibitory κB (IκB) kinase (IKK) inhibitor, SC-514, or the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB202190, revealed a profound reduction in both CCL27 mRNA and CCL27 protein expression indicating the significance of these pathways in the regulation of CCL27 expression. Furthermore, the impact of inhibitors of mitogen- and stress-activated kinase 1 (MSK1) or the mitogen-activated protein kinase-interacting kinases (Mnk1+2), downstream kinases of p38 MAPK, on IL-1ß-induced CCL27 expression in NHEK were investigated. We identified seven NF-κB binding elements upstream from the CCL27 gene start codon using electrophoretic mobility shift assay (EMSA). Supershift analyses demonstrated the involvement of the p50/p65 NF-κB heterodimer. We conclude that IL-1ß-induced CCL27 gene expression in NHEK is regulated through the p38 MAPK/MSK1/Mnk1+2 as well as the IKKß/NF-κB signalling pathways.

The role of mitogen- and stress-activated protein kinase 1 and 2 in chronic skin inflammation in mice.

Mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2) are two kinases phosphorylated by both ERK1/2 and p38 MAPK. Recently, MSK1 and 2 have been reported to act as negative regulators of acute inflammation. In this study, we investigated the role of MSK1/2 in chronic skin inflammation using an oxazolone-induced allergic contact dermatitis model in MSK1/2 knockout mice and wild-type mice. MSK1/2 knockout mice were demonstrated to have significantly increased inflammation compared with wild-type mice. This was measured by an increased ear thickness, elevated infiltration of neutrophils in the skin and increased inflammatory histological changes. Furthermore, we found significantly elevated levels of the proinflammatory cytokines Tumor necrosis factor-α (TNF-α), IL-1ß and IL-6 at both mRNA and protein levels in MSK1/2 knockout mice compared with wild-type mice after oxazolone treatment. In addition, the mRNA expression of the chemokine Thymus and activation regulated chemokine (TARC) was demonstrated to be significantly elevated in oxazolone-treated MSK1/2 knockout mice compared with wild-type mice. The increased expression of TARC was paralleled by increased infiltration of cells positive for the TARC receptor, CCR4, in the dermis of MSK1/2 knockout mice. Our results indicate that MSK1/2 are involved in the activation of feedback mechanisms that dampen oxazolone-induced skin inflammation.

Kinetics and differential expression of the skin-related chemokines CCL27 and CCL17 in psoriasis, atopic dermatitis and allergic contact dermatitis.

CCL27 and CCL17 are chemokines believed to be involved in the process of establishing the inflammatory infiltrate, characteristic for the various inflammatory skin diseases. The skin-specific CCL27 binds the chemokine receptor-10 (CCR10), and CCL17 is a chemokine receptor-4 (CCR4) ligand. The purpose of our study was to characterize the expression of CCL27 and CCL17 in the inflammatory skin diseases: psoriasis, atopic dermatitis (AD) and acute allergic contact dermatitis (ACD) induced in nickel-sensitive individuals. Surprisingly, our studies revealed a markedly decreased CCL27 mRNA and protein expression in psoriatic lesions compared with non-lesional psoriatic skin. A minor CCL17 mRNA increase was measured in lesional psoriatic skin. No alterations were found in AD. In ACD, we found a pronounced (90-fold) raise in CCL17 mRNA and a 50-fold increase in CCL17 protein compared with normal skin. A kinetic ACD study of CCL17 expression showed the highest mean value 24 h after hapten application. Furthermore, we found the mRNA levels of CCR10 and CCR4 paralleling the results of their corresponding ligands. Overall, our principal findings were a distinct decrease in CCL27 in lesional psoriatic skin and a marked upregulation of CCL17 in ACD. These findings underscore the differential cutaneous T-cell recruitment in different inflammatory diseases.