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.     

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.     

E6 and E7 of human papillomavirus type 18 and UVB irradiation corporately regulate interleukin‐6 and interleukin‐8 expressions in basal cell carcinoma

The lack of a human papillomavirus (HPV)‐infected skin cancer cell line has hampered the investigation of the interaction of UV and HPV in skin carcinogenesis. We identified a human basal cell carcinoma (BCC‐1/KMC) cell line integrated with E6 and E7 genes of high‐risk HPV type 18 and demonstrated that repression of E6 and E7 results in proliferation inhibition. Sublethal ultraviolet‐B (UVB) irradiation induced the expressions of interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8), as well as viral E6 and E7 genes, in BCC‐1/KMC cells. When E6 and E7 expressions were inhibited, IL‐6/IL‐8 expressions were repressed. Furthermore, IL‐6/IL‐8 remained inducible by UVB irradiation when E6 and E7 were inhibited. These results indicated that IL‐6 and IL‐8 can be upregulated by viral E6 and E7 proteins without UVB irradiation. Moreover, chronic exposure to UVB upregulates IL‐6 and IL‐8 when E6/E7 is induced by UVB.     

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.     

Evidence for a regulatory loop between IFN‐γ and IL‐33 in skin inflammation

Interleukin‐33 has recently gained much attention due to its role in allergic responses. It has been shown to amplify Th2 responses and to act as a damage‐associated molecular pattern. IL‐33 acts on a broad range of cells and has been proposed to link innate and adaptive features of allergic responses. It was the aim of this study to investigate this property of IL‐33 in the inflammatory response characterising atopic dermatitis (AD). We have analysed the response of skin‐resident cells derived from patients with AD and healthy donors with regard to the expression of IL‐33 and its receptor ST2. The functional impact of IL‐33 on CD4+ T cells was investigated. Keratinocytes and dermal fibroblasts clearly differ in their regulation of IL‐33. In fibroblasts, the concerted action of TNF‐α and IL‐1β was the strongest inducer, whereas IFN‐γ is clearly the key molecule that upregulates IL‐33 in keratinocytes with a more pronounced response of cells derived from patients with AD. Keratinocytes from patients with AD showed a markedly higher constitutive expression level of surface ST2. CD4+ T cells respond to IL‐33. Unexpectedly, IL‐33 failed to induce a significant secretion of IL‐5 or IL‐13. By contrast, high amounts of IFN‐γ were detectable if IL‐33 was added to the T‐cell receptor‐stimulated cells or in combination with IL‐12. These results suggest that IL‐33 and IFN‐γ are closely interlinked in epidermal AD inflammation. IFN‐γ induces IL‐33 in keratinocytes and IL‐33 acts on activated T cells to further increase the release of IFN‐γ, therefore contributing to drive skin inflammation towards chronic responses.     

UVA‐1 exposure in vivo leads to an IL‐6 surge within the skin

UVA‐1 is a known promotor of skin ageing. Cytokines like IL‐1α, Il‐1β or TNF‐α, VEGF and IL‐6 orchestrate UV effects, and IL‐6 is furthermore an effector of UVA‐induced photoageing. We investigated how fractionated UVA‐1 doses influence the cytokine milieu and especially the IL‐6 levels in the skin in vivo. In a study with 35 participants, we exposed previously unirradiated human skin to three UVA‐1 irradiation regimes. Cytokine levels in interstitial skin fluid were measured up to 48 hours postexposure and compared to unirradiated control skin fluid. Our results show that IL‐6 levels increased significantly after UVA‐1 exposure at selected time points. The other candidates IL‐1α, Il‐1β or TNF‐α and VEGF show no significant response after UVA‐1 exposure in vivo. UVA‐1 thus raises selectively IL‐6 levels in vivo, a fact that underlines its role in photoageing and has potential implications for its modulatory effect on photoageing pathology.     

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.     

The balance between pro‐ and anti‐inflammatory cytokines is crucial in human allergic contact dermatitis pathogenesis: the role of IL‐1 family members

The interleukin (IL)‐1 family includes 11 members that are important in inflammatory processes. It includes various agonists and two antagonists, IL‐1Ra and IL‐36Ra. Our aim was to investigate whether the IL‐1 family is involved in allergic contact dermatitis (ACD). The expression of IL‐1 family members was evaluated by PCR and immunohistochemistry in the positive patch test reaction site (involved skin) and in the uninvolved skin of ACD patients. We also examined these cytokines in an ex vivo model of ACD. The antagonistic activity of IL‐36Ra was evaluated by injecting recombinant IL‐36Ra in uninvolved skin biopsies of ACD patients. IL‐1Ra and IL‐36Ra expression was quantified in mononuclear cells of nickel‐sensitized patients challenged in vitro with nickel. IL‐33 involvement in ACD was investigated by intra‐dermal injection of anti‐IL‐33 in the uninvolved skin of patients ex vivo. Results showed that IL‐1β, IL‐1Ra, IL‐36α, IL‐36β, IL‐36γ and IL‐33 expression, but not IL‐36Ra expression, was enhanced in ACD‐involved skin. Immunohistochemical analysis and ex vivo skin cultures confirmed these results. Injection of anti‐IL‐33 in ACD‐uninvolved skin inhibited IL‐8 expression, whereas IL‐36Ra inhibited IL‐36α, IL‐36β, IL‐36γ and IL‐8 expression. Nickel induced IL‐1Ra expression in lymphocytes of nickel‐sensitized patients. Hence, various IL‐1 agonists and antagonists may be involved in ACD pathogenesis.     

High‐fat diet exacerbates imiquimod‐induced psoriasis‐like dermatitis in mice

Psoriasis, a chronic inflammatory skin disease, is closely related to systemic metabolism. An elevated body mass index (BMI) is a risk factor for psoriasis; inflammasomes are activated by adipose tissue macrophages in obese subjects. We hypothesized that hyperlipidaemia is involved in the pathogenesis of psoriasis and examined the role of a high‐fat diet (HFD) in the development of psoriasis in imiquimod (IMQ)‐treated mice. The body weight and serum level of cholesterol were significantly higher in mice fed an HFD than in a regular diet (RD). HFD mice had higher psoriasis skin scores, and the number of neutrophils infiltrating into the lesional skin was elevated. IL‐17A mRNA expression was significantly increased in the skin of IMQ‐treated HFD mice; the expression of IL‐22, IL‐23 and TNF‐α mRNA was not enhanced. Caspase‐1 and IL‐1β were activated in the skin of IMQ‐treated HFD mice, and their serum level of IL‐17A, TNF‐α and IL‐1β was significantly upregulated. Our findings strongly suggest that hyperlipidaemia is involved in the development and progression of psoriasis via systemic inflammation and inflammasome activation.     

CD1d‐dependent,iNKT‐cell cytotoxicity against keratinocytes in allergic contact dermatitis

Conventional CD8+ T‐lymphocytes are thought to be major effector cells in allergic contact dermatitis (ACD). However, previous work has demonstrated a significant population of invariant natural killer T‐cells (iNKT‐cells) in the elicitation phase of ACD. In this study, we investigate whether iNKT‐cells have the capacity to serve as effector lymphocytes in ACD. Using in situ staining of skin biopsy specimens from ACD lesions, we observed intra‐epidermal iNKT‐cells. Presence of these cells provides the possibility of interactions with keratinocytes (KC), Langerhans cells (LC) and CD1d‐bearing antigen‐presenting cells (APC). Investigation into gene expression profiles of cytotoxic effector molecules in seven different cases of ACD found that the expression of perforin and granzymes A, B and K were significantly elevated in ACD relative to paired clinically normal skin. Immunostaining of ACD skin biopsy specimens revealed that these cytotoxic granules indeed localized to iNKT‐cells. Studies of antigen presentation of KC to iNKT‐cells show that these epithelial cells do not activate the expression of cytotoxicity effector genes in resting iNKT‐cells, but had the capacity to serve as targets for activated iNKT‐cells, which was dependent on CD1d expression. Mature LC were not able to present glycolipids to iNKT‐cells and did not up‐regulate CD1d in vitro to a variety of maturational stimuli or in vivo during ACD. These data suggest that iNKT‐cells can serve as effector cells during human ACD and provide the rationale for developing inhibitory glycolipids as therapeutic agents for ACD.     

Evidence on the direct correlation between miR‐31 and IL‐22 axis in IMQ‐induced psoriasis

Psoriatic skin is characterized by a deregulated profile of miRNAs that are contributing in disease development. In this study, we focus on miR‐31, one of the upregulated miRNAs known to promote keratinocytes proliferation and migration. Moreover, miR‐31 expression induction was dependent on a large panel of cytokines including IL‐22. Here, we aimed at investigating the relationship between miR‐31‐5p and IL‐22 axis; and by searching novel molecular target for miR‐31‐5p in keratinocytes. Our data identify a direct correlation between miR‐31‐5p and IL‐22 in psoriasis with Pwp1 as new potential target. These findings confirm the important role of miR‐31 in psoriasis onset and provide a basis for further investigations in miRNAs field in context of skin diseases.     

Immunostimulatory activity of murine keratinocyte‐derived exosomes

It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble factors. Exosomes, small membrane vesicles of endocytotic origin, have been implicated in intercellular communication processes such as the transfer of tumor cell antigens and the activation of recipient dendritic cells (DC). However, little is known about immunomodulatory functions of keratinocyte‐derived exosomes. To address this question, we analysed exosome secretion of the murine keratinocyte cell line MPEK under steady state as well as inflammatory conditions (+/? IFNγ). These exosomes were readily taken up by bone marrow‐derived DC (BMDC) in vitro resulting in a matured phenotype, as evidenced by increased CD40 expression as well as by the production of large amounts of IL‐6, IL‐10 and IL‐12. When the transfer of antigen‐specific information through exosomes was investigated, it was found that keratinocytes took up antigen (ovalbumin) and transferred it to their exosomes. However, these antigen‐harbouring exosomes failed to induce antigen‐specific T cell responses via BMDC. Together, this novel biological function suggests that keratinocytes are able to direct unspecific immune processes but do not elicit specific immune responses.     

HPV8 early genes modulate differentiation and cell cycle of primary human adult keratinocytes

Human papillomaviruses (HPV) have been associated with the development of non-melanoma skin cancer (NMSC) but the molecular mechanisms of the role of the virus in NMSC development are not clearly understood. Abnormal epithelial differentiation seen in malignant transformation of keratinocytes is associated with changes in keratin expression. The purpose of this study was to investigate the phenotype of primary human adult keratinocytes expressing early genes of HPV8 with specific reference to their differentiation and cell cycle profile to determine whether early genes of HPV8 lead to changes that are consistent with transformation. The expression of HPV8 early genes either individually or simultaneously caused distinct changes in the keratinocyte morphology and induced an abnormal keratin expression pattern, that included simple epithelial (K8, K18, K19), hyperproliferation-specific (K6, K16), basal-specific (K14, K15) and differentiation-specific (K1, K10) keratins. Our results indicate that expression of HPV8 early genes disrupts the normal keratin expression pattern in vitro. Expression of HPV8-E7 alone caused polyploidy that was associated with decreased expression of p21 and pRb. Expression of individual genes or in combination differentially influenced cell morphology and cell cycle distribution which might be important in HPV8-induced keratinocyte transformation.     

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.     

Continuous high‐dose antigen exposure preferentially induces IL‐10, but intermittent antigen exposure induces IL‐4

IL‐10 plays a critical role in the induction of specific T‐cell tolerance. To date, whether IL‐10 induction by antigen application is dose‐ or time‐dependent remains unclear. In this study, IL‐10 induction by allergen exposure was investigated in the several schedules. Oxazolone was repeatedly applied to mouse ear, and mRNA of inflammatory cytokines in lesional skins was measured. The results indicated that continuous high‐dose antigen exposure induces IL‐4 as well as abundant IL‐10 production. Monocytes/dendritic cells and T cells are major source of IL‐10. Allergen‐specific immunotherapy is resumed before antigen scattering: preseason. We evaluated safe‐loading dose of allergens in preseasonal therapy focusing Tr1 induction. Restarting immunotherapy with high dose effectively augmented IL‐10 expression accompanied with further induction of IL‐4 and inflammatory cytokines. Therefore, the protocol restarting with low‐dose antigen is preferential to obviate the risk of exacerbation or anaphylaxis.     

Decreased interleukin‐21 expression in skin and blood in advanced mycosis fungoides

Interleukin (IL)‐21 is regarded as a potent antitumor agent, which increases the cytotoxicity of both natural killer (NK) and CD8⁺ T cells. In this study, we investigated the role of IL‐21 in mycosis fungoides (MF). IL‐21 mRNA expression levels in patch and plaque MF were significantly higher than those in normal skin. IL‐21 mRNA expression levels in tumor MF were significantly decreased compared with those in patch and plaque MF. Interestingly, mRNA expression levels of IL‐21 in MF lesional skin significantly correlated with those of T‐helper type‐1 cytokines/chemokines such as CXCL10, CXCL11 and γ‐interferon. Immunohistochemistry showed that IL‐21 was expressed by keratinocytes in patch and plaque MF. Furthermore, serum IL‐21 levels in patients with tumor MF were significantly lower than those of healthy controls and plaque MF. Thus, IL‐21 expression was significantly downregulated in skin and blood of patients with tumor MF, which may contribute to progression of MF. Our study suggests that recombinant IL‐21 would be a promising therapy for MF.     

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.