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.     

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.     

Primary human keratinocytes efficiently induce IL‐1‐dependent IL‐17 in CCR6+ T cells

Abstract: Keratinocytes and activated T cells interact in skin inflammation by virtue of chemokines and cytokines. T cell‐derived IL‐17 has been described to play an important role in the course of psoriatic inflammation. In this study, we addressed how keratinocytes influence the secretion of IL‐17 in autologous T cell subsets. We found that a co‐culture of autologous keratinocytes and T cell‐receptor‐stimulated T cells markedly enhanced the production of IL‐17. Besides the importance of direct cell contact, this effect was mainly mediated by IL‐1 and could be blocked by the IL‐1 antagonist anakinra. An additional increase in IL‐17 production by IL‐23 was only seen in the presence of IL‐1, which thus plays a permissive role for the action of IL‐23. Importantly, co‐culture of keratinocytes with CCR6+ CD4+ T cells that are enriched for Th17 cells resulted in significantly higher IL‐17 production compared to co‐culture with CD4+ T cells.     

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.     

The crucial role of IL‐22 and its receptor in thymus and activation regulated chemokine production and T‐cell migration by house dust mite extract

House dust mite (HDM) is known as one of the factors that causes atopic dermatitis (AD). Interleukin (IL)‐22 and thymus and activation regulated chemokine (TARC) are related to skin inflammatory disease and highly expressed in AD lesions. However, the effects of HDM on IL‐22 production in T cells and on TARC production and IL‐22Rα receptor expression in keratinocytes are unknown. To identify the role of HDM in keratinocytes and T cells, we investigated IL‐22Rα expression and TARC production in the human keratinocyte cell line HaCaT and IL‐22 production in T cells treated with HDM extract as well as their roles in HDM‐induced skin inflammation. HDM extract not only increased IL‐22Rα expression and TARC production in HaCaT but also enhanced IL‐22, tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ production in T cells. The HDM extract‐induced IL‐22 from T cells significantly increased the production of IL‐1α, IL‐6 and TARC in HaCaT cells. In addition, we found that TARC produced in HDM extract‐treated HaCaT induced T‐cell recruitment. These results suggest that there is a direct involvement of HDM extract‐induced IL‐22 in TARC production and T‐cell migration. Taken together, TARC production in HaCaT through the interaction between IL‐22 and IL‐22Rα facilitates T‐cell migration. These data show one of the reasons for inflammation in the skin lesions of AD patients.     

Therapeutic potential of topically administered γ‐AlOOH on 2,4‐dinitrochlorobenzene‐induced atopic dermatitis‐like lesions in Balb/c mice

Boehmite (γ‐AlOOH) has a wide range of applications in a variety of industrial and biological fields. However, little is known about its potential roles in skin diseases. The current study investigated its effect on atopic dermatitis (AD). Following characterization, cytotoxicity, pro‐inflammatory response and oxidative stress associated with boehmite were assessed, using TNF‐α‐induced keratinocytes and mast cells. In addition, therapeutic effects of boehmite, topically administered to Balb/c mice induced by 2,4‐dinitrochlorobenzene (DNCB), were evaluated. Expression of cytokines (TLSP, IL‐25 and IL‐33) and the generation of ROS from keratinocytes induced by TNF‐α were significantly inhibited by boehmite without affecting cell viability. MAPKs (ERK, JNK and p38) required for cytokine expression were suppressed by boehmite treatment. Up‐regulation of cytokines (TSLP, IL‐4, IL‐5, IL‐13, RANTES) in human mast cells treated with phorbol 12‐myristate 13‐acetate and calcium ionophore was also suppressed by boehmite. Boehmite improved the AD severity score, epidermal hyperplasia and transepidermal water loss in DNCB‐induced AD‐like lesions. Moreover, Th2‐mediated cytokine expression, mast cell hyperplasia and destruction of the skin barrier were improved by boehmite treatment. Overall, we demonstrated that boehmite may potentially protect against AD.     

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‐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.     

Effects of β‐carotene on oxazolone‐induced atopic dermatitis in hairless mice

Skin acts as a barrier, which protects internal tissues and promotes moisture retention. Atopic dermatitis (AD) is an inflammatory skin disease associated with a variety of genetic and environmental factors that involve helper T cells. β‐Carotene (provitamin A) exhibits antioxidant activity and activates the immune system. However, it is not clear whether inflammation in AD skin is improved by posttreatment with β‐carotene. In the current study, we investigated the effects of β‐carotene on the skin of hairless mice with oxazolone‐induced inflammation/oedema (Ox‐AD mice). We found that skin inflammation was significantly reduced by oral administration of β‐carotene. In addition, treatment with β‐carotene suppressed protein levels of TNF‐α, IL‐1β and MCP‐1, as well as mRNA expression associated with IL‐1β, IL‐6, IL‐4 and Par‐2 in skin tissues. Furthermore, the mRNA and protein levels of filaggrin, a structural protein in the epidermal stratum corneum, were elevated by β‐carotene administration as compared with Ox‐AD mice. β‐Carotene significantly reduced the activity of proMMP‐9, but not proMMP‐2. These results suggest that in Ox‐AD mice, β‐carotene improves skin inflammation by suppressing the expression of inflammatory factors, promoting filaggrin expression and reducing MMP‐9 activity. β‐Carotene is a potent anti‐inflammatory agent that improves the barrier functions of AD skin.     

Interleukin‐33 modulates the expression of human β‐defensin 2 in human primary keratinocytes and may influence the susceptibility to bacterial superinfection in acute atopic dermatitis

Background Interleukin (IL)‐33 is a member of the IL‐1 family and has been implicated in Th2‐driven allergic diseases such as atopic dermatitis (AD) and asthma. The principal Th2 cytokine IL‐4, found highly expressed in acute allergic eczema, is known to downregulate human β‐defensin 2 (hBD2) expression in human keratinocytes and this is associated with superinfection in patients with AD. Objectives To investigate the effect of IL‐33 on the expression of hBD2 in human keratinocytes. Methods hBD2 production by stimulated keratinocytes was measured by enzyme‐linked immunosorbent assay. Results Our results showed that serum is a very potent inducer of hBD2 and 2·5% human serum was much more potent in inducing hBD2 than 20 ng mL^?1 of tumour necrosis factor‐α. Interestingly, serum from patients with AD showed an impaired ability to induce hBD2 in normal keratinocytes. IL‐33 significantly downregulated serum‐induced hBD2. The downregulatory capacity of IL‐33 was found to be 1·5‐ to 2‐fold weaker compared with IL‐4. Conclusions Our data suggest that IL‐33 can significantly contribute to the decreased expression of hBD2 in acute eczematous reaction clinically characterized by spongiosis and oozing – thus indicative for contact of the epidermis with serum components.     

Oral administration of poly‐γ‐glutamic acid prevents the development of atopic dermatitis in NC/Nga mice

Bacillus subtilis‐derived poly‐γ‐glutamic acid (γPGA) has demonstrated adjuvant activity in promoting Th1/Th17 cell differentiation. Here, the NC/Nga (NC) mouse model was used to determine whether γPGA modulates the outcome of atopic dermatitis (AD), which is known to be a Th2‐biased immune disease. We found that oral administration of γPGA dramatically reduced the development of AD in NC mice. Antigen‐presenting cells activated with γPGA produced pro‐inflammatory cytokines, such as IL12/23 and IFNγ, which, in turn, induced the differentiation of Th1 and Th17 cells. Concomitantly, Th2 responses, such as high levels of serum IgE, were dramatically decreased. Furthermore, in vivo γPGA treatment altered several cellular components of allergic reactions, such as mast cells and eosinophils. Taken together, our results strongly demonstrate that in vivo treatment with γPGA at early time points can prevent the development of AD in NC mice and suggest that γPGA may have therapeutic applications for human AD.     

Increased interleukin‐36γ expression in skin and sera of patients with atopic dermatitis and mycosis fungoides/Sézary syndrome

Interleukin (IL)‐36γ is expressed by keratinocytes and functions as a key initiator of inflammation in the skin. IL‐36γ expression is enhanced by tumor necrosis factor‐α and IL‐17A, having a strong association with psoriasis. In this study, we examined the role of IL‐36γ in atopic dermatitis (AD) and mycosis fungoides (MF)/Sézary syndrome (SS). Serum levels of IL‐36γ in AD patients and MF/SS patients were elevated compared with those of healthy controls. Importantly, serum IL‐36γ levels in AD patients positively correlated with Eczema Area and Severity Index and those of MF/SS patients positively correlated with serum soluble IL‐2 receptor levels. IL‐36γ mRNA levels in AD skin and MF/SS skin were significantly higher than those of normal skin. IL‐36γ mRNA levels in MF/SS skin positively correlated with IL‐17A mRNA levels. Immunohistochemical staining revealed that IL‐36γ was highly expressed in keratinocytes in lesional skin of AD and MF/SS. Taken together, our study demonstrated that IL‐36γ expression was increased in sera and skin of patients with AD and MF/SS as was reported in psoriatic patients.     

Immune evasion during varicella zoster virus infection of keratinocytes

T cells are sensitized during varicella‐zoster virus (VZV) infection and are important for control of viral spread and reactivation. In this report, we show that human keratinocytes infected with VZV inhibited upregulation of major histocompatibility complex (MHC) class I, MHC class II and intercellular adhesion molecule‐1 after interferon (IFN)‐γ treatment. The ability of keratinocytes to upregulate MHC class I in response to IFN‐α, tumour necrosis factor (TNF)‐α and Toll‐like receptor (TLR)‐3 ligand was also diminished upon VZV infection. VZV‐infected keratinocytes treated with IFN‐γ had significantly reduced capacity to stimulate antigen‐specific T cells compared with uninfected cells. Interference with IFN‐α, TNF‐α, IFN‐γ and TLR‐3 signalling in keratinocytes by VZV may contribute to immune evasion of the adaptive immune response.     

IL‐36γ has proinflammatory effects on human endothelial cells

Interleukin‐36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL‐36 is now a recognised characteristic of psoriatic skin inflammation. IL‐36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL‐36 on endothelial cells are unexplored. We here show that endothelial cells including dermal microvascular cells express a functionally active IL‐36 receptor. Adhesion molecules VCAM‐1 and ICAM‐1 are upregulated by IL‐36γ stimulation, and this is reversed by the presence of the endogenous IL‐36 receptor antagonist. IL‐36γ‐stimulated endothelial cells secrete the proinflammatory chemokines IL‐8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T‐cells following IL‐36γ stimulation of endothelial cells. These results suggest a role for IL‐36γ in the dermal vascular compartment, and it is likely to enhance psoriatic skin inflammation by activating endothelial cells and promoting leucocyte recruitment.     

Expression analysis of PD‐1 and Tim‐3 immune checkpoint receptors in patients with vitiligo; positive association with disease activity

The contribution of immune checkpoint receptors in the immunopathogenesis of various autoimmune diseases has been addressed in previous reports. In this study,  the expression profile of T‐cell immunoglobulin and mucin‐domain containing‐3 (Tim‐3) and programmed cell death‐1 (PD‐1) checkpoint molecules was investigated in CD8⁺ T cells of Vitiligo patients. The association of Tim‐3 and PD‐1 expression with disease activity was also explored. The frequency of Tim‐3⁺/PD‐1⁺/CD8⁺ T cells in 30 patients with vitiligo and 30 sex‐ and age‐matched controls was determined by flow cytometry. CD8⁺ T cells were then positively isolated by magnetic beads, and the mRNA expression of PD‐1 and Tim‐3 was determined by TaqMan‐based real‐time PCR. To measure the cytokines production, PBMCs were stimulated with PMA/ionomycin and concentrations of IL‐4, IFN‐γ and TNF‐α were measured in culture supernatants by ELISA. Disease activity of patients with vitiligo was determined using the Vitiligo Area Severity Index. Patients with vitiligo have significantly shown more expression of Tim‐3 and PD‐1 on their CD8⁺ T cells compared with controls. Expression analysis of Tim‐3 mRNA, but not PD‐1, confirmed the results obtained from flow cytometry. While the production levels of TNF‐α and IFN‐γ were found higher by patients with vitiligo, IL‐4 production was lower in patients compared with controls. A direct association was observed between the Tim‐3 and PD‐1 expression and also the production of pro‐inflammatory cytokines with disease activity of patients with vitiligo. Our results indicate that Tim‐3 and PD‐1 are involved in immune dysregulation mechanisms of CD8⁺ T cells in vitiligo and may introduce as potential biomarkers for disease progression and targeted immunotherapy.     

Disseminated cutaneous Mycobacterium kansasii infection presenting with Rosai–Dorfman disease‐like histological features in a patient carrying anti‐interferon‐γ autoantibodies

Typical cutaneous non‐tuberculous mycobacteria (NTM) infections show a histopathology pattern of granulomas with admixed Langhans giant cells, and abscesses may be observed in acute lesions. Herein, we describe a patient carrying a high titer of autoantibodies to interferon (IFN)‐γ with disseminated Mycobacterium kansasii infection presenting with emperipolesis and Rosai–Dorfman disease (RDD)‐like histopathological features characterized by remarkable, large, pale‐staining “RD cells”, which were CD68 and S100 positive and CD1a negative. The patient was misdiagnosed with RDD initially, but exhibited a poor response to all interventions. A re‐biopsy revealed Langhans‐type multinucleated giant cells; multiple definite acid‐fast bacilli were also found. M. kansasii was isolated from cultured tissues. Anti‐NTM treatment was initiated. After treatment, all lesions resolved almost completely within the following month. High‐titer anti‐IFN‐γ autoantibodies were detected during follow up, leading to the diagnosis of adult‐onset immunodeficiency syndrome. In conclusion, patients carrying high‐titer autoantibodies to IFN‐γ who also have a disseminated cutaneous M. kansasii infection may present with RDD‐like histopathological features, which may be a pitfall in the diagnosis of disseminated cutaneous NTM infections.     

Granzyme A potentiates chemokine production in IL‐17‐stimulated keratinocytes

Plaque psoriasis presents with focal skin inflammation, partially maintained by IL‐17‐mediated interactions between infiltrating epidermal T cells and activated keratinocytes. Here we show that the majority of lesional epidermal CD8 T cells express granzyme A, alone or in combination with IL‐17. To assess proinflammatory properties of granzyme A in psoriasis, primary human keratinocytes were stimulated with granzyme A in the presence or absence of IL‐17. Out of 33 analysed keratinocyte‐derived inflammatory mediators, granzyme A potentiated IL‐17‐induced secretion of CXCL 1, CXCL 12 and CCL 4. Intriguingly, all three chemokines are implicated in psoriasis pathogenesis and are involved in recruitment of T cells, neutrophils and pDCs into inflamed tissues. Our results indicate that granzyme A produced by lesional CD8 T cells specifically increase the chemokine production from inflamed keratinocytes, thereby amplifying a chemotactic inflammatory loop that sustains psoriasis lesions.