Th17 cytokines interleukin (IL)-17 and IL-22 modulate distinct inflammatory and keratinocyte-response pathways

Background Psoriasis vulgaris is an inflammatory skin disease mediated by Th1 and Th17 cytokines, yet the relative contribution of interferon (IFN)‐γ, interleukin (IL)‐17 and IL‐22 on disease pathogenesis is still unknown. Objectives In this study, we sought to identify the cytokines produced by skin‐resident T cells in normal skin, localize the receptors for these cytokines, and examine how these cytokines alter gene expression profiles of the cells bearing cognate receptors. Methods We used intracellular cytokine staining and flow cytometry to evaluate T cell cytokine production, and immunohistochemistry and double‐label immunofluorescence to localize cytokine receptors in skin. Gene array analysis of cytokine‐treated keratinocytes was performed using moderated paired t‐test controlling for false discovery rate using the Benjamini–Hochberg procedure. Results We demonstrate that T‐helper cells producing IL‐17, IL‐22 and/or IFN‐γ, as well as the cells bearing cognate cytokine receptors, are present in normal human skin. Keratinocytes stimulated with IL‐17 expressed chemokines that were different from those induced by IFN‐γ, probably contributing to the influx of neutrophils, dendritic cells and memory T cells into the psoriatic lesion. In contrast, IL‐22 downregulated genes associated with keratinocyte differentiation and caused epidermal alterations in an organotypic skin model. Conclusions Our results suggest that the Th17 cytokines IL‐17 and IL‐22 mediate distinct downstream pathways that contribute to the psoriatic phenotype: IL‐17 is more proinflammatory, while IL‐22 retards keratinocyte differentiation.     

Increased expression of the histamine H4 receptor following differentiation and mediation of the H4 receptor on interleukin‐8 mRNA expression in HaCaT keratinocytes

Recent in vivo studies have demonstrated involvement of the histamine H4 receptor in pruritus and skin inflammation. We previously reported that an H4 receptor antagonist attenuated scratching behaviour and improved skin lesions in an experimental model of atopic dermatitis. We also reported the expression of the H4 receptor in human epidermal tissues. In this study, we investigated the expression of H4 receptor mRNA and the function of the receptor in a culture system that mimics in vivo inflammation on the HaCaT human keratinocyte cell line. Increased expression of the H4 receptor was observed in HaCaT cells following differentiation. Treatment of HaCaT cells with histamine and TNFα enhanced the mRNA expression of interleukin (IL)‐8. These increases in expression were significantly inhibited by the H4 receptor antagonist JNJ7777120. Our results indicate that IL‐8 mRNA expression might be enhanced by histamine and TNFα via H4 receptor stimulation in keratinocytes.     

Interleukin‐21 receptor signalling is not critically required for imiquimod‐induced psoriasiform dermatitis in mice

Psoriasis is largely mediated by interleukin (IL)‐23/T helper (Th) 17 axis, and IL‐21 is a pleiotropic cytokine expressed by Th17 cells. Despite previously reported possible pathogenic roles of IL‐21 in human psoriasis, we found that IL‐21 receptor (IL‐21R) signalling was not crucial for imiquimod‐induced psoriatic inflammation, using IL‐21R^?/? mice. The severity of imiquimod‐induced psoriatic manifestation and pro‐inflammatory Th17 cytokine levels, IL‐17A‐producing γδ T cells and CD4+ T cells, and in vitro IL‐17A production by γδ T cells after IL‐23 stimulation was comparable between wild‐type and IL‐21R^?/? mice. Collectively, IL‐21R signalling was not critically involved in IMQ‐induced psoriatic inflammation despite an increased IL‐21 expression in the IMQ‐treated mouse skin. Our data may represent the significant differences between human psoriasis and murine psoriasis model, and further studies using other models will be required to elucidate the role of IL‐21 in psoriasis pathogenesis.     

Heparinoid suppresses Der p‐induced IL‐1β production by inhibiting ERK and p38 MAPK pathways in keratinocytes

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro‐inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen‐induced interleukin (IL)‐1β release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase‐1 release, suggesting that heparinoid did not affect HDM allergen‐induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro‐IL‐1β, but also suppressed IL‐1β messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal‐regulated kinase and p38 pathways, which are required for IL‐1β expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL‐1β mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte‐mediated skin inflammation.     

IL‐24 is expressed during wound repair and inhibits TGFα‐induced migration and proliferation of keratinocytes

Please cite this paper as: IL‐24 is expressed during wound repair and inhibits TGFα ‐induced migration and proliferation of keratinocytes. Experimental Dermatology 2010; 19 : 714–722. Abstract: Interleukin (IL)‐24 is the protein product of melanoma differentiation‐associated gene 7 (MDA‐7). Originally identified as a tumor suppressor molecule, MDA‐7 was renamed IL‐24 and classified as a cytokine because of its chromosomal location in the IL‐10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL‐24 is expressed by cytokine‐activated monocytes and T lymphocytes. Here, we show that IL‐24 is expressed in keratinocytes during wound repair. Paraffin‐embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL‐24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming growth factor α (TGFα), TGFβ, IFNγ, and IFNβ, upregulated IL‐24 protein expression in normal human epidermal keratinocytes (NHEKs). Examination of the function of IL‐24 in both in vitro wound repair and migration assays demonstrated that IL‐24 inhibits TGFα‐induced proliferation and migration of NHEKs. These data support the hypothesis that IL‐24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes.     

CD4+ T cells producing interleukin (IL)‐17, IL‐22 and interferon‐γ are major effector T cells in nickel allergy

Background It has been suggested that interleukin (IL)‐17 and IL‐22 play important roles in the elicitation of human allergic contact dermatitis; however, the frequencies of T cell subtypes producing IL‐17 and IL‐22 in human allergic contact dermatitis are unknown. Objectives To determine the frequencies of CD4⁺, CD8⁺ and γδ T cells producing IL‐17, IL‐22 and interferon (IFN)‐γ in the blood and skin from nickel‐allergic patients. Patients/materials/methods Blood samples were collected from 14 patients and 17 controls, and analysed by flow cytometry. Biopsies were taken from 5 patients and 6 controls, and analysed by immunohistochemistry and flow cytometry of skin lymphocytes. Results We found an increased frequency of γδ T cells in the blood, but no differences in the distribution of cytokine‐producing CLA⁺ T cell subtypes in nickel‐allergic patients as compared with controls. In nickel‐allergic patients, there was massive cellular infiltration dominated by CD4⁺ T cells producing IL‐17, IL‐22 and IFN‐γ in nickel‐challenged skin but not in vehicle‐challenged skin. Conclusion CD4⁺ T cells producing IL‐17, IL‐22 and IFN‐γ are important effector cells in the eczematous reactions of nickel‐induced allergic contact dermatitis in humans.     

UV‐B‐activated B16 melanoma cells or HaCaT keratinocytes accelerate signaling pathways associated with melanogenesis via ANGPTL 2 induction,an activity antagonized by Chrysanthemum extract

Sunburn causes inflammation, which increases melanin production in skin and causes hyperpigmentation. Angiopoietin‐like protein (ANGPTL) 2 is an inflammatory mediator induced in sun‐exposed skin areas. However, whether ANGPTL2 functions in melanin production remains unclear. To assess this possibility, we overexpressed Angptl2 in the melanoma line B16 and in the keratinocyte line HaCaT. Relative to controls, Angptl2‐expressing B16 cells produced higher melanin levels via tyrosinase induction. Accordingly, Angptl2‐expressing HaCaT cells secreted relatively high levels of both endothelin‐1 (ET‐1) and α‐melanocyte‐stimulating hormone (α‐MSH). Moreover, treatment with an extract from Chrysanthemum indicum × Erigeron annuus (CE) suppressed ANGPTL2 expression and repressed tyrosinase induction in melanocytes and of α‐MSH and ET‐1 in keratinocytes. Our data suggest that ANGPTL2 expression in keratinocytes and melanin‐producing cells accelerates pigment production and that treatment of skin with a CE extract could prevent melanin accumulation.     

Obesity exacerbates imiquimod‐induced psoriasis‐like epidermal hyperplasia and interleukin‐17 and interleukin‐22 production in mice

Psoriasis is a chronic inflammatory skin disorder that is accompanied by an imbalance between the proliferation and differentiation of keratinocytes. A number of studies have suggested an association between obesity and severe psoriasis; however, it remains to be clarified whether obesity exacerbates psoriasis. To address this unsolved question, we induced psoriasiform dermatitis in mouse models for obesity. We found that obesity exaggerated the severity of psoriasiform dermatitis induced by topical application of the Toll‐like receptor (TLR) 7 agonist, imiquimod. Ear swelling and epidermal hyperplasia were more prominent in the obese mice than in the control mice. When compared to imiquimod‐treated control mice, imiquimod‐treated obese mice expressed higher levels of psoriasis mediators, interleukin‐17A (IL‐17A) and IL‐22 in the skin. Food intake restriction partially abrogated enhanced ear swelling and cytokine overproduction in obese mice. Furthermore, the obesity environment and imiquimod treatment synergistically induced an IL‐17A downstream molecule, regenerating islet‐derived 3γ (Reg3γ), which is a critical molecule for psoriatic epidermal hyperplasia. Palmitic acid, one of the fatty acids released by subcutaneous adipocytes, increased the expression of REG3A (a human homologue of mouse Reg3γ) in both the HaCaT keratinocyte cell line and normal human keratinocytes. Taken together, these results strongly suggest that obesity exacerbates psoriasiform dermatitis in mice by upregulating IL‐17A, IL‐22 and Reg3γ.     

Mild electrical stimulation with heat shock reduces inflammatory symptoms in the imiquimod‐induced psoriasis mouse model

Psoriasis is a chronic skin disease caused by immune disorder. The chronic skin inflammation involves inflammatory molecules that are released from T lymphocytes and keratinocytes. Therefore, developing an anti‐inflammatory therapy that is suitable for long‐term treatment is needed. Electrical stimulation induces biological responses by modulating intracellular signaling pathways. Our previous studies showed that the optimized combination treatment of mild electrical stimulation (MES, 0.1‐millisecond; ms, 55‐pulses per second; pps) and heat shock (HS, 42°C) modulates inflammatory symptoms of metabolic disorders and chronic kidney disease in mice models and clinical trials. Here, we investigated the effect of MES+HS treatment on imiquimod‐induced psoriasis mouse model. Topical application of imiquimod cream (15 mg) to mice ear induced keratinocyte hyperproliferation and psoriasis‐like inflammation. In MES+HS‐treated mice, imiquimod‐induced skin hyperplasia was significantly decreased. MES+HS treatment reduced the protein expression of IL‐17A and the infiltration of CD3‐positive cells in lesioned skin. In addition, MES+HS‐treated mice had decreased mRNA expression level of antimicrobial molecules (S100A8 and Reg3γ) which aggravate psoriasis. In IL‐17A‐stimulated HaCaT cells, MES+HS treatment significantly lowered the mRNA expression of aggravation markers (S100A8, S100A9 and β‐defensin2). Taken together, our study suggested that MES+HS treatment improves the pathology of psoriasis via decreasing the expression of inflammatory molecules.     

Modulation of HMGB1 translocation and RAGE/NFκB cascade by quercetin treatment mitigates atopic dermatitis in NC/Nga transgenic mice

Quercetin, glycosylated form of flavonoid compound, has potent antioxidant and anti‐inflammatory properties. In this study, we have investigated the effects of quercetin on skin lesion, high‐mobility group box (HMGB)1 cascade signalling and inflammation in atopic dermatitis (AD) mouse model. AD‐like lesion was induced by the application of house dust mite extract to the dorsal skin of NC/Nga transgenic mouse. After AD induction, quercetin (50 mg/kg, p.o) was administered daily for 2 weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for HMGB1, receptor for advanced glycation end products (RAGE), toll‐like receptor (TLR)4, nuclear factor (NF)κB, nuclear factor erythroid‐2‐related factor (Nrf)2, kelch‐like ECH‐associated protein (Keap)1, extracellular signal‐regulated kinase (ERK)1/2, cyclooxygenase (COX)2, tumor necrosis factor (TNF)α, interleukin (IL)‐1β, IL‐2Rα and other inflammatory markers in the skin of AD mice. In addition, serum levels of T helper (Th) cytokines (interferon (IFN)γ, IL‐4) were measured by enzyme‐linked immunosorbent assay. Quercetin treatment attenuated the development of AD‐like skin lesions. Histological analysis showed that quercetin inhibited hyperkeratosis, parakeratosis, acanthosis, mast cells and infiltration of inflammatory cells. Furthermore, quercetin treatment downregulated cytoplasmic HMGB1, RAGE, nuclear p‐NFκB, p‐ERK1/2, COX2, TNFα, IL‐1β, IL‐2Rα, IFNγ and IL‐4 and upregulated nuclear Nrf2. Our data demonstrated that the HMGB1/RAGE/NFκB signalling might play an important role in skin inflammation, and quercetin treatment could be a promising agent for AD by modulating the HMGB1/RAGE/NFκB signalling and induction of Nrf2 protein.     

Interleukin‐1 from epithelial cells fosters T cell‐dependent skin inflammation

Background Chronic inflammatory skin diseases such as atopic dermatitis and psoriasis are characterized by the infiltration of lymphocytes into the epidermal compartment. Several studies point to an active role of skin epithelial cells in the pathophysiology of such diseases. Objectives In this study we addressed the regulatory function of primary human keratinocytes in the interaction with autologous T cells and monocytes. Methods We used a human coculture model with keratinocytes grown from epidermal stem cells of the outer root sheath of human hair follicles and autologous T cells. Results In our coculture system we observed a high production of interferon (IFN)‐γ, but not Th2 cytokines, in the presence of superantigen or antigen‐pulsed autologous monocytes. Critical parameters for this effect were: (i) T‐cell receptor activation, (ii) an intercellular adhesion molecule‐1/lymphocyte function‐associated antigen (LFA)‐1‐dependent interaction between keratinocytes and T cells, and (iii) secretion of interleukin (IL)‐1β. Remarkably, in the presence of activated T cells, epithelial cells seemed to be a more significant source of IL‐1β than monocytes. Application of the LFA‐1 blocker efalizumab or IL‐1 receptor antagonist anakinra enabled us to suppress completely the production of IFN‐γ by T cells in the coculture. Conclusions IL‐1 secretion and the physical contact between keratinocytes and activated, infiltrating T cells may be central for the development of chronic inflammatory skin conditions.     

Role of IL‐10 and TGF‐β in melanoma

IL‐10 and TGF‐β are immunosuppressive cytokines expressed in tumors including melanoma and, therefore, deemed major cause for failing antitumor immune responses. Re‐evaluating their role, we compared their expression by quantitative RT‐PCR in melanoma and skin of healthy individuals, tested their induction in dendritic cells and T cells co‐cultured with tumor cells, and their effects on the immune cells. Both cytokines as well as their receptors were expressed in melanoma at significantly lower levels than in healthy skin. Consequently, the expressions of IL‐10‐responsive SOCS‐3 and TGF‐β‐responsive Smad‐7 were low in tumors but high in healthy skin. T cells co‐cultured with tumor cells developed an anergic state without increased IL‐10 or TGF‐β expression. In vitro tumor‐induced immature dendritic cells produced high IL‐10 levels and less efficiently induced T‐cell proliferation. Nonetheless, they could be induced to mature, and blocking IL‐10 did not alter the capacity of the resulting mature dendritic cells to stimulate T cells. Mature dendritic cells co‐cultured with tumor cells produced increased IL‐10 but decreased TGF‐β and more efficiently induced T‐cell proliferation. The lack of correlation of IL‐10 and TGF‐β with immune deficits in situ and in vitro suggests re‐evaluating their roles in cancer.     

Particulate matter induces pro‐inflammatory cytokines via phosphorylation of p38 MAPK possibly leading to dermal inflammaging

Particulate matter (PM) is known to have harmful effects on human health. Epidemiological studies have suggested that PM exposure is related to skin diseases and extrinsic skin ageing. However, the mechanisms by which PM affects skin are unclear. The aim of this study was to investigate the mechanism of action of PMs on epidermal inflammation and skin ageing using a co‐culture of human keratinocytes (HaCaT) and fibroblasts (HDF). SRM 1648a (pmA) and 1649b (pmB), which mainly comprise heavy metals and polycyclic aromatic hydrocarbons, respectively, were used as reference PMs. Cytotoxic effects, activation of AhR, phosphorylation of p38 kinase and ROS generation were examined in PM‐treated HaCaT cells. The phosphorylation of p38 MAPK induced by PMs was shown to be critically important for the increases in IL‐1α and IL‐1β expression. Moreover, the mRNA and protein expression levels of MMP1 and COX2 were markedly increased in HDF cells co‐cultured with PM‐treated HaCaT cells. In conclusion, PMs induce the expression of pro‐inflammatory cytokines in keratinocytes via the p38 MAPK pathway, and these interleukins increase the expression of MMP1 and COX2 in HDF cells. These results suggest that PMs trigger skin ageing via p38 MAPK activation and interleukin secretion in epidermal keratinocytes.     

IL‐33 is secreted by psoriatic keratinocytes and induces pro‐inflammatory cytokines via keratinocyte and mast cell activation

IL‐33 is a novel pro‐inflammatory cytokine and ligand for the orphan receptor ST2. Although originally defined as an inducer of Th2‐mediated responses, IL‐33 was recently found to be involved in arthritis, a Th1/Th17‐mediated disease. Here, we assessed the ability of IL‐33 to promote inflammation via mast cells (MCs) and keratinocytes (KCs) activation in psoriasis. IL‐33 resulted elevated in the skin but not in the serum of psoriasis patients. IL‐33 was secreted by psoriasis KCs and HaCaT cells after TNF‐α stimulation. In HMC‐1, TNF‐α, but not IL‐17, could induce a robust increase in IL‐33 expression. In HaCaT cells, TNF‐α was able to induce IL‐6, MCP‐1 and VEGF, and the addition of IL‐33 reinforced these increases. TNF‐α + IL‐33 combination showed similar results in primary KCs and ex vivo skin organ culture. In conclusion, our study suggests that IL‐33 may be involved in psoriasis biology via MCs and KCs.     

Mechanism of pathogenesis of imiquimod‐induced skin inflammation in the mouse: A role for interferon‐alpha in dendritic cell activation by imiquimod

Topical application of imiquimod (IMQ), a Toll‐like receptor (TLR)7 ligand, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. In a mouse model of IMQ‐induced psoriasis‐like skin inflammation, T‐helper (Th)17 cells and interleukin (IL)‐17/IL‐22‐producing γδ‐T cells have been shown to play a pivotal role. However, the mechanisms of induction of the Th17 pathway and development of psoriasis‐like skin inflammation by IMQ treatment remain unclear. In this study, we investigated pathogenic mechanisms of IMQ‐induced psoriasis‐like skin inflammation in mice. We first confirmed that, together with an increase in IL‐17 and IL‐22 production, application of IMQ to mouse skin induced the expression of cytokines required for activation of the Th17 pathway, and pro‐inflammatory mediators involved in the pathology of psoriasis. Analysis of Tlr7^?/? mice demonstrated that most of the in vivo effects of IMQ were mediated via TLR7. In an in vitro study using plasmacytoid dendritic cells (DCs), IMQ induced production of interferon (IFN)‐α, IL‐23, IL‐6 and tumor necrosis factor (TNF)‐α. Furthermore, when we analyzed in vitro‐generated bone marrow‐derived DCs with features similar to TNF‐α and inducible nitric oxide synthase (iNOS)‐producing DCs, IL‐23, IL‐6, IL‐1β, TNF‐α and iNOS/NO production was weakly induced by IMQ alone and further enhanced after co‐stimulation with IMQ and IFN‐α. These in vitro effects of IMQ were also mediated via TLR7 and the synergistic effect of IMQ, and IFN‐α was suggested to be caused by upregulation of TLR7 expression by IFN‐α. These results demonstrate part of the mechanism by which the Th17 pathway and psoriasis‐like skin inflammation are induced by IMQ and IFN‐α in a mouse model.     

T‐lymphocyte‐induced,fas‐mediated apoptosis is associated with early keratinocyte differentiation

Please cite this paper as: T‐lymphocyte‐induced, fas‐mediated apoptosis is associated with early keratinocyte differentiation. Experimental Dermatology 2009. Abstract: The development of eczematous lesions is thought to be due in part to a breakdown in skin barrier function as a result of T lymphocytes (T cells) invading the skin causing epidermal keratinocyte apoptosis. In this study, we investigated the interaction of T cells and keratinocytes on apoptosis and terminal differentiation using an in vitro co‐culture system. Experiments were performed using the HaCaT keratinocyte cell line or normal human epidermal keratinocytes. Activated human peripheral blood‐derived T cells were found to induce Fas‐dependent keratinocyte apoptosis by up to sixfold. Increased Fas was associated with increased IFN‐γ. The T‐cell apoptotic signal was found to target preferentially keratinocytes in the very early stages of terminal differentiation, such as those with low levels of α6‐integrin expression, and result in subsequent increased caspase 3 activity. This observation was accompanied by a marked increase in keratinocyte ICAM‐1 expression and its ligand LFA‐1 on T cells. Our data suggest that T cells may initiate the onset of keratinocyte terminal differentiation making them more susceptible to Fas‐dependent cell death signals delivered by the T cells.