IL‐17A synergistically enhances TLR3‐mediated IL‐36γ production by keratinocytes: A potential role in injury‐amplified psoriatic inflammation

Skin injury can trigger formation of new lesions in psoriasis (Koebner phenomenon). The mechanisms through which injury exacerbates psoriasis are unclear. During wound repair, epidermal keratinocytes are activated and produce abundant IL‐36γ, further promoting the skin inflammation. IL‐17A is the cornerstone cytokine in the pathogenesis of psoriasis. We sought to investigate the effects of IL‐17A on injury‐induced keratinocyte activation and IL‐36γ production. Here, we demonstrated that dsRNA released from necrotic keratinocytes induced the expression of IL‐36γ. Silencing of TLR3 by siRNA decreased the IL‐36γ induction by necrotic keratinocyte supernatant. Co‐stimulation with dsRNA and IL‐17A synergistically increased the expression of IL‐36γ and other proinflammatory mediators (CCL20, CXCL8, DEFB4 and LCN2) in keratinocytes. The synergistic effects were not dependent on TLR3 upregulation, TNF receptor signalling and mRNA stabilization. Co‐stimulation with dsRNA and IL‐17A resulted in an accumulation of IκBζ. The synergistic upregulation of IL‐36γ and proinflammatory mediators were inhibited by IκBζ siRNA. Co‐stimulation with IL‐17A and poly(I:C) markedly activated the p38 MAPK and NF‐κB pathway, compared with poly(I:C). Blockade of p38 MAPK and NF‐κB suppressed dsRNA/IL‐17A–mediated IκBζ and IL‐36γ induction. These findings demonstrated that IL‐17A synergistically enhanced the dsRNA‐mediated IL‐36γ production through a p38 MAPK‐, NF‐κB–, and IκBζ‐dependent mechanism.     

IL‐1 signalling determines the fate of skin grafts expressing non‐self protein in keratinocytes

Please cite this paper as: IL‐1 signalling determines the fate of skin grafts expressing non‐self protein in keratinocytes. Experimental Dermatology 2010; 19 : 723–729. Abstract: Although IL‐1 is a known inflammatory cytokine during pathogen infection, the role of IL‐1 in skin graft rejection, particularly where foreign antigen is expressed exclusively in keratinocytes, is less understood. Here, we use a syngeneic skin graft system, where antigens are expressed in epithelial cells via either a keratin 14 or keratin 5 promoter, to explore the role of IL‐1 in graft rejection and induction of epithelial antigen‐specific effector CD8⁺ T‐cell function. Keratin 5 ovalbumin (K5mOVA) transgenic skin grafts destined for rejection demonstrated increased expression of IL‐1β and its receptors compared to K14 HPV16 E7 transgenic grafts that do not reject spontaneously. Rejection of OVA grafts lacking the IL‐1 receptor (IL‐1R1) was delayed and associated with decreased numbers of antigen‐specific CD8 T cells. In contrast, K14E7 grafts survived on immunocompetent, syngeneic recipients with decreased graft levels of IL‐1β and IL‐1R1 and 2. However, in the absence of the IL‐1 receptor antagonist, IL‐1Ra, skin grafts were spontaneously rejected and an E7‐specific CD8 T‐cell response was primed. Thus, expression of the HPV16E7 oncoprotein in epithelial cells prevents IL‐1β‐associated skin graft rejection and induction of antigen‐specific CD8 T‐cell responses. Enhancing IL‐1β signalling, via blocking of the IL‐1 receptor antagonist, may represent an alternative strategy for treatment of HPV16E7‐associated cancers.     

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.     

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.     

Oral vitamin D increases the frequencies of CD38+ human B cells and ameliorates IL‐17‐producing T cells

Vitamin D deficiency (serum 25‐hydroxyvitamin D < 50 nm ) has been associated with the onset of immunological diseases including atopic dermatitis (AD), cutaneous or systemic lupus erythematosus and allergic asthma. In this study, we assessed whether oral vitamin D (cholecalciferol) supplementation leads to a systemic modulation of the phenotype of circulating lymphocyte populations and whether a defined serum 25‐hydroxyvitamin D (25(OH)D) concentration can be related to the effects on lymphocytes. Cholecalciferol was administered in a dose‐escalation setting to vitamin D–deficient individuals from 2000 up to 8000 IU daily for 12 weeks. Individuals without cholecalciferol intake served as controls. Peripheral B cells and T cells were examined by multicolour flow cytometric analysis. The mean serum 25(OH)D concentrations increased upon cholecalciferol intake up to 159 ± 28.7 nm , and remained low in the control group 30.0 ± 12.5 nm . Following cholecalciferol intake, the frequencies of circulating CD38 expressing B cells were significantly increased and IFN‐γ⁺, and/or IL‐17⁺ CD4⁺ T helper cells were decreased. These data were identified to correlate with the serum 25(OH)D levels by applying two different analysis approaches (ROC and a nonlinear regression analysis). Our data indicate that increasing 25(OH)D serum concentrations are associated with an increased expression of CD38 on B cells and a decreased T‐cell‐dependent proinflammatory cytokine production. The therapeutical role of our findings in systemic immunological diseases should be explored in the future by further controlled clinical studies.     

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.     

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.     

Brodalumab: A new way to inhibit IL‐17 in psoriasis

Psoriasis is a chronic inflammatory disease that affects 2% to 4% of the population; about 20% of the patients present a moderate‐to‐severe form. The IL‐23/Th17/IL‐17 molecular axis is considered crucial in the pathogenesis of psoriasis and IL‐17 is fundamental in the maintenance of the immune and inflammatory alterations causing psoriasis. Expression of IL‐17A, IL‐17F, and IL‐17C is strongly increased in psoriatic plaques. Effective therapy leads to restoration of the expression of a wide range of genes (including effector cytokines and chemokines downstream of IL‐17) to near normal levels. Brodalumab is the first biologic drug targeting specifically the subunit A of the IL‐17 receptor (IL‐17RA) and thus inhibiting not only IL‐17A but also other members of the IL‐17 family. Brodalumab is very effective and safe in treating moderate‐to severe psoriasis.     

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.     

TNF stimulates IL‐6, CXCL8 and VEGF secretion from human keratinocytes via activation of mTOR,inhibited by tetramethoxyluteolin

Psoriasis is an autoimmune skin disease characterized by keratinocyte hyperproliferation and chronic inflammation. The pathogenesis of psoriasis involves proinflammatory cytokines, such as tumor necrosis factor (TNF), but the mechanism of keratinocyte activation is not well understood. Here, we show that TNF (10 or 50 ng/mL) stimulates a significant (P < .0001) gene expression and secretion of proinflammatory IL‐6, CXCL8 and VEGF from both cultured human HaCaT and normal epidermal human keratinocytes (NHEKs). This effect occurs via activation of the mammalian target of rapamycin (mTOR) signalling complex as shown by Western blot analysis and phospho‐ELISAs. Pretreatment with the novel natural flavonoid tetramethoxyluteolin (10‐100 μmol L^?1) significantly (P < .0001) inhibits gene expression and secretion (P < .0001) of all 3 mediators in a concentration‐dependent manner. Moreover, tetramethoxyluteolin (50 μmol L^?1) appears to be a potent inhibitor of the phosphorylated mTOR substrates (pmTOR^Ser2448, pp70S6K^Thr389 and p4EBP1^Thr37/46) as compared to known mTOR inhibitors in keratinocytes. The present findings indicate that TNF stimulates skin inflammation via mTOR signalling. Inhibition by tetramethoxyluteolin may be used in the treatment for psoriasis.     

Epidermal growth factor receptor tyrosine kinase inhibitors induce CCL2 and CCL5 via reduction in IL‐1R2 in keratinocytes

Please cite this paper as: Epidermal growth factor receptor tyrosine kinase inhibitors induce CCL2 and CCL5 via reduction in IL‐1R2 in keratinocytes. Experimental Dermatology 2010; 19 : 730–735. Abstract: Epidermal growth factor receptor tyrosine kinase (EGFR‐TK) is a transducer of mitogenic signals, and is involved in the pathogenesis and progression of a number of cancers, including non‐small cell lung cancer (NSCLC). Gefitinib is an EGFR‐TK inhibitor that is clinically used to treat NSCLC; however, this drug frequently causes adverse effects, including skin eruptions. The mechanism underlying these skin reactions is elusive, although it is assumed that they are caused by the inhibition of EGFR‐TK signalling in epidermal and adnexal cells. In this article, we demonstrate by immunocytochemistry that the skin lesions of patients treated with oral gefitinib had higher expression of CCL2 and CCL5 compared to normal human epidermis. Further, PD153035, a gefitinib prototype, induced CCL2 and CCL5 mRNA and protein expression in HaCaT and HSC‐1 keratinocyte cell lines with or without interleukin‐1 (IL‐1) treatment in vitro. PD153035 also reduced the levels of interleukin‐1 receptor 2 (IL‐1R2), an IL‐1 decoy receptor. Moreover, we demonstrate that reduction in IL‐1R2 by RNA interference increased IL‐1‐mediated CCL2 and CCL5 mRNA and protein expression. Taken together, our data strongly suggest that IL‐1‐mediated signalling is activated to induce the high expression of CCL2 and CCL5 via reduction in IL‐1R2 in the skin lesions caused by gefitinib.     

Interleukin‐17A affects extracellular vesicles release and cargo in human keratinocytes

Psoriasis is a chronic inflammatory systemic disease caused by deregulation of the interleukin‐23/‐17 axis that allows the activation of Th17 lymphocytes and the reprogramming of keratinocytes proliferative response, thereby inducing the secretion of cyto‐/chemokines and antimicrobial peptides. Beside cell‐to‐cell contacts and release of cytokines, hormones and second messengers, cells communicate each other through the release of extracellular vesicles containing DNA, RNA, microRNAs and proteins. It has been reported the alteration of extracellular vesicles trafficking in several diseases, but there is scarce evidence of the involvement of extracellular vesicles trafficking in the pathogenesis of psoriasis. The main goal of the study was to characterize the release, the cargo content and the capacity to transfer bioactive molecules of extracellular vesicles produced by keratinocytes following recombinant IL‐17A treatment if compared to untreated keratinocytes. A combined approach of standard ultracentrifugation, RNA isolation and real‐time RT‐PCR techniques was used to characterize extracellular vesicles cargo. Flow cytometry was used to quantitatively and qualitatively analyse extracellular vesicles and to evaluate cell‐to‐cell extracellular vesicles transfer. We report that the treatment of human keratinocytes with IL‐17A significantly modifies the extracellular vesicles cargo and release. Vesicles from IL‐17A‐treated cells display a specific pattern of mRNA which is undid by IL‐17A neutralization. Extracellular vesicles are taken up by acceptor cells irrespective of their content but only those derived from IL‐17A‐treated cells enable recipient cells to express psoriasis‐associated mRNA. The results imply a role of extracellular vesicles in amplifying the pro‐inflammatory cascade induced in keratinocyte by pro‐psoriatic cytokines.     

Imbalance of T‐helper 17 and regulatory T cells in patients with alopecia areata

There is mounting evidence that T helper (Th)17 cells and regulatory T cells (Treg) play parts in the pathogenesis of autoimmune disease. Hence, levels of these T‐cell subsets in patients with alopecia areata (AA) merit investigation. Our goal was to assess Th17 and Treg levels in peripheral blood mononuclear cells (PBMC) and scalp lesions of patients with AA, correlating the findings with clinical characteristics. PBMC of 177 patients with AA (test group) and 42 healthy controls and scalp tissues of 33 patients and 15 healthy controls were collected. Levels of Th17 and Treg subsets were then determined via flow cytometry and immunohistochemical staining, correlating results in test subjects with clinical features of AA. Th17 levels were significantly higher in patients, whereas Treg levels were lower by comparison. Furthermore, Th17 levels in patients with disease of short duration or in the active phase were significantly higher, relative to their respective counterparts. Th17 levels also negatively correlated with disease duration. While Treg levels were higher in severe AA than in mild AA. Results of lesions were parallel to findings of PBMC. Our data indicates an imbalance in the immune state of patients with AA.     

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.     

E‐FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro

Epidermal fatty acid‐binding protein (E‐FABP) is a lipid carrier, originally discovered in human epidermis. We show that E‐FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E‐FABP induces overexpression of K10 and involucrin. On the other hand, E‐FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF‐κB and JNK signalling pathways. E‐FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E‐FABP as compared to the same population in normal epidermis. E‐FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high‐calcium conditions, E‐FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E‐FABP plays an important role in keratinocyte differentiation. Moreover, E‐FABP modulates differentiation in psoriatic keratinocytes.