Lipocalin‐2 exacerbates psoriasiform skin inflammation by augmenting T‐helper 17 response

Lipocalin‐2 (LCN2) is an antimicrobial protein and adipokine associated with insulin resistance, obesity and atherosclerotic disease. Psoriasis is a T‐helper (Th)1/Th17‐mediated, chronic inflammatory dermatosis related to metabolic syndromes and serum LCN2 levels are elevated in psoriatic patients. We examined the in vivo effects of LCN2 on topical imiquimod (IMQ)‐induced psoriasiform skin in BALB/c mice and in vitro on human keratinocytes (KC). Clinically, i.p. injected LCN2 exacerbated erythema and scaling in IMQ‐treated murine skin compared with phosphate‐buffered saline injection alone, and it augmented interleukin (IL)‐17A, IL‐17F, IL‐22, IL‐23p19, IL‐12p40, CCL20, tumor necrosis factor‐α, chemokine (C‐X‐C motif) ligand (CXCL)1, CXCL2, DEFB4, DEFB14, LCN2 and S100A7 mRNA levels of IMQ‐treated murine skin while it did not increase the mRNA levels of interferon‐γ, IL‐12p35 or CXCL10. LCN2 in synergy with IL‐17 increased mRNA levels of CCL20, LCN2 and DEFB4A but not of CXCL10 in human KC in vitro. These results suggest that LCN2 enhances the expression of Th17 cytokines/chemokines and antimicrobial peptides in murine IMQ‐treated psoriatic skin and KC. LCN2 may potentiate the development of psoriasis via the enhancement of Th17‐ and antimicrobial peptide‐mediated inflammation.     

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.     

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.     

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.     

The human IL‐17A/F heterodimer regulates psoriasis‐associated genes through IκBζ

Antagonists of IL‐17A and its receptor have proven to be highly effective in the treatment of psoriasis. However, many of the underlying molecular mechanisms involved in the pathogenesis of psoriasis are still to be determined. IκBζ (encoded by the NFKBIZ gene) plays a key role in the development of psoriasis by mediating IL‐17A‐ and IL‐17F‐driven effects. Both IL‐17A and IL‐17F expression are increased in lesional psoriatic skin. IL‐17A/A and IL‐17F/F homodimers as well as the IL‐17A/F heterodimer signal through the same receptors. The aim of this study was to characterize the role of the IL‐17A/F heterodimer in the regulation of NFKBIZ expression and in the regulation of selected psoriasis‐associated genes. We demonstrated that IL‐17A/F stimulation of human keratinocytes significantly induced NFKBIZ expression. Moreover, silencing IκBζ by siRNA revealed that IκBζ is a key regulator of IL‐17A/F‐inducible psoriasis‐associated genes, including CCL20, DEFB4, IL‐8, CHI3L1 and S100A7. In addition, IL‐17A/F‐induced NFKBIZ expression was mediated by a mechanism involving the p38 MAPK and NF‐κB signalling pathways. In conclusion, we present IκBζ as a novel key regulator of IL‐17A/F‐driven effects in psoriasis. Thus, antagonists to IL‐17A/F or IκBζ may present a targeted approach for treating psoriasis.     

Interleukin (IL)-17 versus IL-27: opposite effects on tumor necrosis factor-α-mediated chemokine production in human keratinocytes

Tumor necrosis factor (TNF)-α is known to play a pivotal role in the pathogenesis of psoriasis. TNF-α has been shown to act directly on keratinocytes, thereby inducing the production of various kinds of chemokines, which contributes to the infiltration of leucocytes into the psoriatic lesions. Recent studies have shown that both interleukin (IL)-17 and IL-27 are increased in psoriatic lesional tissue. However, the interactions between TNF-α, IL-17 and IL-27 in chemokine production by keratinocytes have not been fully elucidated. Here, we examined in human keratinocytes how TNF-α, IL-17 and IL-27 affect production of chemokines that are involved in the pathogenesis of psoriasis. We found that IL-17 and IL-27 exert opposite effects on TNF-α-mediated chemokine production. This suggests that lesional balance of IL-17 and IL-27 is involved in the recruitment of T cells, natural killer cells, neutrophils, monocytes or dendritic cells, thereby affecting inflammation in skin diseases.     

TNFα‐ and IL‐17A‐mediated S100A8 expression is regulated by p38 MAPK

The antimicrobial peptide S100A8 is known to be upregulated in lesional psoriatic skin compared with non‐lesional psoriatic skin and is believed to play a role in the pathogenesis of psoriasis. However, little is known about the signalling pathways involved in the regulation of S100A8 expression. Using quantitative real‐time RT‐PCR analysis, we demonstrated that stimulation with TNFα and IL‐17A in combination resulted in a significant and synergistic induction of S100A8 mRNA in human keratinocytes. TNFα and IL‐17A also induced the S100A8 promoter activity synergistically. This was demonstrated by a gene reporter assay in cells transfected with a luciferase plasmid construct, consisting of 3502 base pairs of the human S100A8 promoter. The TNFα‐ and IL‐17A‐mediated induction of S100A8 mRNA and protein was mediated by a p38 MAPK‐dependent mechanism, as demonstrated by the use of a p38 MAPK inhibitor. Finally, adalimumab treatment for patients with psoriasis significantly decreased S100A8 mRNA at day fourteen after start of treatment, but not at day four. Taken together, this study demonstrates that the p38 MAPK signalling pathway plays a key role in the TNFα‐ and IL‐17A‐induced expression of S100A8 in cultured human keratinocytes.     

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.     

Increased mast cell expression of PAR‐2 in skin inflammatory diseases and release of IL‐8 upon PAR‐2 activation

Please cite this paper as: Increased mast cell expression of PAR‐2 in skin inflammatory diseases and release of IL‐8 upon PAR‐2 activation. Experimental Dermatology 2010; 19: 117–122. Abstract: Mast cells are increasingly present in the lesional skin of chronic skin inflammatory diseases including psoriasis and basal cell carcinoma (BCC). It has previously been shown that proteinase‐activated receptor (PAR)‐2 is expressed by mast cells, and tryptase is a potent activator of this receptor. In this study, skin biopsies from both healthy‐looking and lesional skin of patients with psoriasis and superficial spreading BCC were collected and the expression of PAR‐2 immunoreactivity in tryptase‐positive mast cells was analysed. PAR‐2 expression was confirmed in vitro in different mast cell populations. Cord‐blood derived mast cells (CBMC) were stimulated with a PAR‐2 activating peptide, 2‐furoyl‐LIGRLO‐NH₂. Consequently, IL‐8 and histamine production was analysed in the supernatants. We observed a significant increase in the percentage of mast cells expressing PAR‐2 in the lesional skin of psoriasis and BCC patients compared with the healthy‐looking skin. HMC‐1.2, LAD‐2 and CBMC mast cells all expressed PAR‐2 both intracellularly and on the cell surface. CBMC activation with the PAR‐2 activating peptide resulted in an increased secretion of IL‐8, but no histamine release was observed. Furthermore, both PAR‐2 and IL‐8 were co‐localized to the same tryptase‐positive mast cells in the lesional BCC skin. These results show that mast cells express increased levels of PAR‐2 in chronic skin inflammation. Also, mast cells can be activated by a PAR‐2 agonist to secrete IL‐8, a chemokine which can contribute to the progress of inflammation.     

Expression of CCL1 and CCL18 in atopic dermatitis and psoriasis

Background.  Recent studies have shown that chemoattractive proteins play an important role in the organization of the innate and adaptive immune responses. There are some reports that chemokine (C‐C motif) ligand (CCL)1 and CCL18, members of a family of chemoattractive proteins, have increased expression in atopic dermatitis (AD). Aim.  To evaluate the quantity and pattern of CCL1 and CCL18 expression in lesions and blood of patients with AD, and compare them with those of patients with psoriasis. Methods.  Biopsy specimens were taken from atopic skin and normal‐appearing skin of patients with AD and from the psoriatic skin only of patients with psoriasis. Quantitative real‐time PCR analysis and immunohistochemistry of CCL1 and CCL18 expression were performed, and the quantities of expressed CCL1 and CCL18 in acute AD were compared with those of normal‐appearing atopic skin and psoriatic skin. The serum level of CCL1 and CCL18 was assessed by ELISA. Results.  Expression of CCL1 mRNA and protein was significantly higher in the acute lesional skin of patients with AD than in their nonlesional skin or in the lesional skin of patients with psoriasis. Both CCL18 mRNA and protein were abundant in acute AD lesions and in psoriatic lesions, but were lower in the nonlesional skin of patients with AD. The serum levels of CCL1 and CCL18 were not different in patients with AD and patients with psoriasis. Conclusions.  CCL1 is a chemokine that is associated with AD. Both CCL1 and CCL18 may play important roles in the initiation and progression of atopic skin inflammation.     

The single‐chain anti‐TNF‐α antibody DLX105 induces clinical and biomarker responses upon local administration in patients with chronic plaque‐type psoriasis

It is not clear whether TNF‐α antagonists used in the treatment of psoriasis need to act systemically, or whether local inhibition of skin‐produced TNF‐α would be sufficient to silence skin inflammation. To answer this question, we conducted two multicentre, double‐blinded, randomized, placebo‐controlled clinical trials with the novel single‐chain anti‐TNF‐α‐PENTRA^®‐antibody DLX105. Upon intra‐dermal injection, DLX105 induced a mean local PASI decrease of 33% over baseline after 2 weeks of treatment, while the placebo response was only 12% (P = 0.001). The clinical response was accompanied by changes in biomarkers such as reductions in K16, Ki67 and epidermal thickness as well as decreased mRNA levels of IL‐17, TNF‐α, IL‐23p19, IL‐12p40 and IFN‐γ. Next, we applied the drug topically twice daily in a 0.5% hydrogel formulation. While the local PASI did not change, topical DLX105 mediated significant reductions of mRNA levels of key proinflammatory cytokines when compared to placebo, and this effect was further enhanced after weekly tape stripping of plaques to increase drug penetration. These results suggest that longer treatment periods and/or increased local drug concentrations might result in better therapeutic efficacy of topically applied DLX105. In sum, we can show for the first time that local inhibition of TNF‐α is sufficient to mediate a biological response in psoriasis that translates into clinical efficacy.     

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.     

IL6Rα function in myeloid cells modulates the inflammatory response during irritant contact dermatitis

Irritant contact dermatitis (ICD) is characterized by epidermal hyperplasia, infiltration of leucocytes into lesional skin and inflammatory cytokine release. The cellular infiltrate during ICD comprises primarily cells of the myeloid lineage. Our group has previously shown that the cytokine IL‐6 confers a protective effect to lesional skin during ICD. How IL‐6Rα function in myeloid cells is involved in the inflammatory response during ICD is, however, unknown. In the present study, utilizing a chemical model of ICD, it is shown that mice with a myeloid‐specific knockout of the IL‐6Rα (IL‐6Rα^Δmyeloid) display an exaggerated inflammatory response to benzalkonium chloride (BKC) and Jet propellant‐8 (JP8) fuel, two well‐characterized irritants relative to littermate control. Results from immunohistochemical and flow cytometric analyses revealed that IL‐6Rα^Δmyeloid mouse skin displayed increased epidermal hyperplasia and inflammatory monocyte influx into lesional skin but lower numbers of resident macrophages relative to littermate controls after irritant exposure. Multiplex immunoassay revealed significantly higher levels of pro‐inflammatory cytokines IL‐1α and TNF‐α, but reduced expression of chemokine proteins including CCL2‐5, CCL7, CCL11, CXCL1 and CXCL10 in IL‐6Rα^Δmyeloid mouse skin relative to littermate control following irritant exposure. These results highlight a previously unknown role of IL‐6Rα function in myeloid cells in modulating the inflammatory response and myeloid population dynamics during ICD.     

Increased CCL18 expression in patients with cutaneous T‐cell lymphoma: association with disease severity and prognosis

Background CC chemokine ligand (CCL) 18 is expressed by monocytes and dendritic cells (DCs), and has potent chemotactic activity for T cells, B cells and DCs. CCL18 expression is up‐regulated in lesional skin of atopic dermatitis and bullous pemphigoid, suggesting its important roles in the development of these skin diseases. Objective To investigate roles of CCL18 in cutaneous T‐cell lymphoma (CTCL). Methods The CCL18 messenger RNA (mRNA) expression in CTCL skin (n = 21) and in normal skin (n = 7) was examined by quantitative RT‐PCR. CCL18 expression was also examined by immunohistochemistry. Serum CCL18 levels were measured in 38 patients with CTCL and 20 healthy controls by enzyme‐linked immunosorbent assay. We also analysed correlation between serum CCL18 levels and other clinical and laboratory data. Results The CTCL lesional skin contained higher levels of CCL18 mRNA than normal skin. CCL18 was expressed by dermal macrophages and DCs in CTCL skin. Serum CCL18 levels in patients with CTCL were significantly higher than those of healthy controls and correlated with types of skin lesions. They also significantly correlated with modified severity‐weighted assessment scores, serum sIL‐2R, LDH, IL‐4, IL‐10, IL‐31, CCL17 and CCL26 levels. Patients with high serum levels of CCL18 showed significantly poor prognosis compared with those with low CCL18 levels. Conclusion CCL18 mRNA is up‐regulated in CTCL lesional skin, and serum CCL18 levels are significantly increased and correlated with the severity of CTCL. These results suggest that CCL18 may be associated with the development of CTCL.