Enhanced type I interferon signaling and recruitment of chemokine receptor CXCR3-expressing lymphocytes into the skin following treatment with the TLR7-agonist imiquimod

INTRODUCTION: Imiquimod (Aldara) is an immune response modifier approved for the topical treatment of external genital and perianal warts which can mediate regression of several cutaneous malignancies [basal cell carcinoma (BCC), Bowen's disease, actinic keratosis, and metastasis of malignant melanoma]. Recently, it was discovered that imiquimod acts through the toll-like receptor (TLR) 7. We hypothesize that TLR7-signaling strongly induces the production of interferon (IFN) alpha, which is able to enhance Th1-mediated cellular antiviral and antitumor immunity. PATIENTS AND METHODS: In the present study we analyzed the expression of MxA, a protein specifically induced by type I IFNs during topical imiquimod treatment in several patients suffering from different cutaneous malignancies (BCC, cutaneous metastasis of melanoma, and breast cancer), and characterized the inflammatory infiltrate, along with the expression of chemokine receptor CXCR3, by immunohistochemistry. RESULTS: Treatment with the TLR7-agonist imiquimod induced a significant lesional lymphocytic inflammation, associated with strong expression of MxA, indicating the induction of type I IFN signaling. The extent of lesional MxA staining closely correlated with the number of infiltrating T lymphocytes and the expression of the chemokine receptor CXCR3, characteristic for Th1-biased immune responses. DISCUSSION: Our in vivo results suggest an important role for TLR7-induced production of type I IFN, which links innate and adaptive immunity and promotes specific Th1-biased cellular immune response capable of eliminating cutaneous malignancies. MxA appears to be a valuable parameter to demonstrate IFN-type I expression in imiquimod therapy.     

银屑病患者皮损中类浆细胞树突状细胞和IRF-7,IFN-α mRNA表达研究

目的探讨银屑病患者皮损中类浆细胞树突状细胞(plasmacytoid dendritic cells,PDCs)和干扰素调节因子7(IRF-7)、干扰素α(IFN-α)mRNA的表达及其意义。方法采用免疫组织化学和逆转录-聚合酶链反应(RT-PCR)方法检测了22例银屑病患者皮损中PDCs和IRF-7,IFN-α mRNA的表达。15例整形外科患者的皮肤作为正常人对照。结果免疫组化结果显示银屑病组皮损中CD123阳性的PDCs表达明显增多,而在正常对照组没有表达;RT-PCR检测显示,与正常人皮肤相比,寻常性银屑病患者皮损中IFN-α mRNA的表达无实质性上调,差异无显著性,但是IRF-7 mRNA的表达水平明显增高,差异有显著性(P<0.001)。结论银屑病患者皮损中PDCs浸润,IRF-7 mRNA的表达水平上调可能是银屑病发生和发展的原因之一。     

Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis

BACKGROUND: Toll-like receptors (TLRs) are part of the innate immune system involved in the response to microbial pathogens. TLR2 recognizes various ligands expressed by Gram-positive bacteria, while TLR3, TLR4 and TLR5 are specific for double-stranded RNA, Gram-negative lipopolysaccharides and bacterial flagellin, respectively. OBJECTIVES: To determine, firstly, whether epidermal keratinocytes of normal skin express TLRs and, secondly, whether modulation of TLR expression occurs in psoriasis, an inflammatory skin disease associated with certain microorganisms such as streptococci, staphylococci and yeasts. METHODS: Eight samples of normal, and 15 samples of lesional and nonlesional psoriatic skin were stained with polyclonal antibodies specific for TLR1-5 using an avidin-biotin-peroxidase technique. RESULTS: Epidermal keratinocytes in normal skin constitutively expressed TLR1, TLR2 and TLR5, while TLR3 and TLR4 were, in most cases, barely detectable. Cytoplasmic TLR1 and TLR2 were expressed throughout the epidermis, with higher staining of the latter on basal keratinocytes, while TLR5 expression was concentrated in the basal layer. In contrast, in lesional epidermis from patients with psoriasis, TLR2 was more highly expressed on the keratinocytes of the upper epidermis than on the basal layer, while TLR5 was downregulated in basal keratinocytes compared with corresponding nonlesional psoriatic epidermis. In addition, nuclear TLR1 staining was observed in the upper layers of both nonlesional and lesional psoriatic epidermis, but not in that of normal skin. CONCLUSIONS: These findings suggest that TLRs expressed by epidermal keratinocytes constitute part of the innate immune system of the skin. The relevance of altered keratinocyte TLR expression in psoriasis remains to be determined.     

浆细胞样树突状细胞和TLR9及其mRNA在银屑病患者皮损中的表达

目的：探讨浆细胞样树突状细胞（Plasmacytoid dendritic cells,PDCs）和toll样受体9（TLR9）在银屑病发病中的表达状态。方法：采用免疫组织化学SP法和逆转录-聚合酶链反应（RT-PCR）方法检测了22例银屑病患者皮肤损害中PDCs和TLR9及其mRNA的表达。15例整形外科患者的皮肤作为正常对照。结果：免疫组化结果显示银屑病组皮损中PDCs、TLR9的表达明显增多,而在正常对照组没有表达;RT-PCR检测显示银屑病皮损中TLR9mRNA的表达明显高于正常对照组（P〈0.01）。结论：银屑病患者皮损中有PDCs浸润,TLR9及其mRNA的表达水平上调,可能与银屑病的发病相关。     

Psoriasis and pustular dermatitis triggered by TNF-{alpha} inhibitors in patients with rheumatologic conditions

BACKGROUND: New onset or worsening of psoriasis has been reported in patients treated with tumor necrosis factor alpha (TNF-alpha) inhibitors for a variety of rheumatologic conditions. There is mounting evidence that a key innate immune pathway for triggering common human autoimmune disease, including psoriasis, involves plasmacytoid dendritic cell precursors (PDCs) and type 1 interferon (IFN) production. We present herein a case series with clinical and histopathologic evidence of psoriasis in patients with rheumatologic disease treated with TNF-alpha inhibitors. We propose that the cross regulation between TNF-alpha and IFN may have a role in the pathogenesis of this reaction. OBSERVATIONS: We observed new-onset psoriasis (n = 13) or severe exacerbation of psoriasis (n = 2) in 15 patients with a variety of rheumatologic conditions-rheumatoid arthritis (n = 13), psoriatic arthritis (n = 1), and seronegative arthritis (n = 1)-during treatment with etanercept (n = 6), infliximab (n = 5), and adalimumab (n = 4). Immunohistochemical staining of skin biopsy specimens for myxovirus-resistance protein A (MxA, a surrogate marker for lesional type 1 IFN activity) showed increased staining in TNF-alpha inhibitor-induced psoriasis compared with psoriasis vulgaris. CONCLUSIONS: New onset or severe exacerbation of psoriasis is a rare complication of TNF-alpha inhibitor therapy. The finding of increased production of IFN-alpha in TNF-alpha inhibitor-induced psoriasis is a possible pathophysiologic explanation for this reaction.     

In psoriasis lesional skin the type I interferon signaling pathway is activated, whereas interferon-alpha sensitivity is unaltered

The epidermal phenotype as observed in psoriatic skin results from inflammation and abnormal proliferation and terminal differentiation of keratinocytes. Mice deficient for interferon regulatory factor-2, a repressor of interferon signaling, display psoriasis-like skin inflammation. The development of this phenotype is strictly dependent on type I interferon (interferon-alpha/beta) signaling. The aim of this study was to assess the involvement of interferon-alpha/beta in the pathogenesis of human psoriasis. In psoriatic skin, we measured an increased expression of components that play central and crucial roles in interferon-alpha/beta signal transduction. Culturing keratinocytes or healthy skin biopsies with recombinant interferon-alpha stimulated this signaling pathway; however, this did not induce the expression of markers that are generally used to define the psoriasis phenotype. Furthermore, skin from psoriasis patients responded identically to interferon-alpha stimulation, demonstrating that psoriatic skin does not have an aberrant sensitivity to type I interferon. We conclude that in psoriatic lesional skin the type I interferon signaling pathway is activated, despite an unaltered interferon-alpha sensitivity. Our data furthermore show that type I interferon, in contrast to interferon-gamma, does not act directly on keratinocytes to induce a psoriatic phenotype. Thus, if the observed activated type I interferon signaling is indeed functionally involved in the pathogenesis of psoriasis, its contribution might be indirect, putatively involving other cell types besides keratinocytes.     

Cathelicidin antimicrobial peptide LL-37 in psoriasis enables keratinocyte reactivity against TLR9 ligands

Here we show that keratinocytes in psoriatic lesional skin express increased Toll-like receptor (TLR) 9 that similarly localizes with elevated expression of the cathelicidin antimicrobial peptide LL-37. In culture, normal human keratinocytes exposed to LL-37 increased TLR9 expression. Furthermore, when keratinocytes were exposed to LL-37 and subsequently treated with TLR9 ligands, such as CpG or genomic DNA, they greatly increased production of type I IFNs. This response mimicked observations in the epidermis of psoriatic lesional skin as keratinocytes in psoriatic lesions produce greater amounts of IFN-β than normal skin lacking LL-37. The mechanism for induction of type I IFNs in keratinocytes was dependent on TLR9 expression but not on a DNA-LL-37 complex. These findings suggest that keratinocytes recognize and respond to DNA and can actively participate in contributing to the immunological environment that characterizes psoriasis.     

Etanercept decreases the innate immune wounding response in psoriasis

Cathelicidin is increased when normal skin is injured and in psoriasis lesions where it has been suggested to play a pivotal role in inflammation through interactions with self‐DNA and toll‐like receptor 9 (TLR‐9) in keratinocytes and plasmacytoid dendritic cells. Because of etanercept's success in treating psoriasis, we hypothesized that etanercept may suppress TLR‐9 and cathelicidin induction. Examination of experimentally induced wounds of psoriatic lesional and non‐lesional skin, and comparison with wounded normal skin, shows that the induction of cathelicidin and TLR‐9 is greatly enhanced in lesional psoriatic skin. Six weeks of etanercept appears not to affect the baseline expression of cathelicidin or TLR‐9, but does blunt the induction of cathelicidin in psoriasis with wounding. These findings support the role of cathelicidin in the enhancement of local inflammation in psoriasis and may partially explain one of the mechanisms enabling TNF‐α inhibitors to successfully treat this disorder.     

Reduction of different inflammatory cell types of the innate immune system in psoriatic skin during etanercept treatment

Please cite this paper as: Reduction of different inflammatory cell types of the innate immune system in psoriatic skin during etanercept treatment. Experimental Dermatology 2010; 19 : 754–756. Abstract: To investigate whether specific markers for innate immunity would diminish with successful treatment in psoriasis, we analyzed lesional and non‐lesional skin biopsies taken from patients with moderate to severe psoriasis during 12 weeks of treatment with etanercept in correlation with the clinical response. In the clinical responders (PASI reduction >50%), all markers (CD3, CD68, CD161, elastase, BDCA‐2, TNF‐α) showed a decline during treatment, indicating a pivotal role for innate immunity in the pathogenesis of psoriasis.     

CCL7 contributes to the TNF‐alpha‐dependent inflammation of lesional psoriatic skin

Chemokines are small chemotactic proteins that have a crucial role in leukocyte recruitment into tissue. Targeting these mediators has been suggested as a potential therapeutic option in inflammatory skin diseases such as psoriasis. Using quantitative RT‐PCR, we found CCL7, a chemokine ligand known to interact with multiple C‐C chemokine receptors, to be markedly increased in lesional psoriasis as opposed to atopic dermatitis, lichen planus, non‐lesional psoriatic and normal control skin. Surprisingly, this increase in CCL7 mRNA expression exceeded that of all other chemokines investigated, and keratinocytes and dermal blood endothelial cells were identified as its likely cellular sources. In an imiquimod‐induced psoriasis‐like mouse model, CCL7 had a profound impact on myeloid cell inflammation as well as on the upregulation of key pro‐psoriatic cytokines such as CCL20, IL‐12p40 and IL‐17C, while its blockade led to an increase in the antipsoriatic cytokine IL‐4. In humans receiving the TNF‐α‐blocker infliximab, CCL7 was downregulated in lesional psoriatic skin already within 16 hours after a single intravenous infusion. These data suggest that CCL7 acts as a driver of TNF‐α‐dependent Th1/Th17‐mediated inflammation in lesional psoriatic skin.     

Fc gamma-receptors and HLA-DR antigens on endothelial cells in psoriatic skin lesions

Receptors for the Fc-part of IgG (FcR) and HLA-DR antigens on endothelial cells in normal and lesional skin from patients with psoriasis were studied in cryostat sections, using soluble immune complexes and monoclonal antibodies. FcR and HLA-DR antigens were detected on endothelial cells of dermal vessels both in sections of normal and lesional skin. The expression of FcR varied from one vessel to another and on endothelial cells within one and the same vessel. The expression of FcR and HLA-DR antigens was enhanced in sections of lesional skin compared with normal skin and most pronounced in lesional skin from active psoriasis. The enhanced expression may be mediated by interferon produced in psoriatic lesions. The presence of FcR and HLA-DR antigens on endothelial cells adds further evidence of he involvement of these cells in immune processes in the skin.     

银屑病患者皮损中Toll样受体2及相关因子的检测

目的探讨Toll样受体2(TLR2)、信号途径下游分子NF-κBp65以及可能的效应分子TGF-α在银屑病患者皮损中的表达及其与银屑病严重程度的关系。方法采用EliVision免疫组化法对40例银屑病患者进行期皮损、11例非皮损区及15例正常人皮肤中TLR2,NF-κBp65,TGF-α的表达进行检测,对其在皮损中的表达进行相关性分析,并将结果与PASI评分进行相关性分析。结果与正常人皮肤及银屑病患者非皮损区相比,银屑病患者皮损表皮中TLR2,NF-κBp65,TGF-α的表达明显上调,而非皮损区TLR2的表达也高于正常人皮肤,差异均有统计学意义(P<0.05)。银屑病患者皮损表皮中TLR2,NF-κBp65的表达水平与PASI评分之间存在正相关(P<0.05)。银屑病患者皮损表皮中TLR2与NF-κBp65,TLR2与TGF-α,NF-κBp65与TGF-α表达水平之间均存在正相关(P<0.05)。结论TLR2,NF-κBp65,TGF-α在银屑病中表达异常,可能共同参与了银屑病的发病过程。     

Toll样受体2和4在银屑病皮损中的表达

目的研究Toll样受体（TLR）2和4在银屑病皮损中的表达，探讨其与银屑病发病的关系。方法选用16例滴状银屑病、13例斑块状银屑病患者及10例正常人皮肤的石蜡切片．用免疫组化的方法研究TLR2和TLR4的表达。结果10例正常人皮肤的基底层均有较弱的TLR2表达而无TLR4表达．真皮血管内皮细胞未见TLR2及TLR4表达。所有16例滴状银屑病、13例斑块状银屑病皮损的基底细胞层均可见明显的TLR2表达，棘层也有弱表达；TLR4则呈现表皮全层的弥漫性强表达。银屑病真皮浅层血管内皮细胞可见明显的TLR2及TLR4表达。结论TLR2、TLR4在银屑病皮损均有表达，TLR4的表达更高：提示感染相关免疫与银屑病发病关系密切。     

Early migration of cutaneous lymphocyte-associated antigen (CLA) positive T cells into evolving psoriatic plaques

The majority of T cells in lesional psoriatic skin express the skin homing receptor, cutaneous lymphocyte-associated antigen (CLA). We investigated whether this reflects the selective migration of CLA positive cells into evolving psoriatic plaques, consistent with an important role in disease onset, or whether this occurs in the context of an established cutaneous inflammatory response. We identified the advancing edge of plaques in 16 patients with chronic plaque psoriasis using scanning laser Doppler fluxmetry, and performed immunohistochemical analysis of i) lesional psoriatic skin, ii) clinically normal skin immediately in front of the advancing plaque edge, and iii) uninvolved skin distant from the plaque edge. The T-cell infiltrate was characterized using monoclonal antibodies to CD3, CLA and the integrin alphaEbeta7, which is associated with the retention of lymphocytes at mucosal sites. Epithelial proliferation was assessed using a monoclonal antibody to the nuclear proliferation marker Ki67. There was enrichment of CLA positive T cells in evolving psoriatic skin compared to distant, uninvolved skin (mean CLA positive 75.9% vs 47.8%; P<0.003). This accumulation of CLA positive cells occurred before epidermal hyperproliferation was evident, suggesting that this population of cells plays an important, early role in disease pathogenesis. Established lesional psoriatic skin contained a mixed infiltrate of CLA positive (mean 53.2%) and alphaEbeta7 positive (mean 18.2%) cells, suggesting less tissue-specific T-cell infiltration, although an additional, specific role for alphaEbeta7 in cutaneous inflammation cannot be excluded. Furthermore, this study has highlighted scanning laser Doppler fluxmetry as a useful investigative tool, permitting analysis of the earliest and therefore potentially most important changes in psoriatic plaque formation.     

IFN-alpha enhances poly-IC responses in human keratinocytes by inducing expression of cytosolic innate RNA receptors: relevance for psoriasis

Keratinocytes play a key role in innate immune responses of the skin to bacterial and viral pathogens. Viral double-stranded RNA and its synthetic analogue polyriboinosinic-polyribocytidylic acid (poly-IC) are recognized via multiple pathways involving the receptors Toll-like receptor 3 (TLR3), protein kinase R (PKR), and the recently described cytosolic RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We show that preincubation of human keratinocytes with IFN-alpha enhances the proinflammatory responses to poly-IC. Kinetic studies suggest that this is mediated via upregulation of the receptors TLR3, PKR, RIG-I, and MDA5. Interestingly, expression of RIG-I, MDA5, and PKR was significantly increased in lesional skin from patients with psoriasis, a chronic inflammatory skin disease that is characterized by high IFN-alpha levels. These results suggest that psoriatic keratinocytes show increased sensitivity to viral RNA intermediates, thereby leading to excessive proinflammatory responses and maintenance of the inflammatory skin phenotype. Here, we provide early evidence that point toward a role for the recently described cytosolic innate RNA receptors in non-viral chronic inflammatory diseases.     

Immune response modifiers--mode of action

The innate immune system governs the interconnecting pathways of microbial recognition, inflammation, microbial clearance, and cell death. A family of evolutionarily conserved receptors, known as the Toll-like receptors (TLRs), is crucial in early host defense against invading pathogens. Upon TLR stimulation, nuclear factor-kappaB activation and the interferon (IFN)-regulatory factor 3 pathway initiate production of pro-inflammatory cytokines, such as interleukin-1 and tumor necrosis factor-alpha, and production of type I IFNs (IFN-alpha and IFN-beta), respectively. The innate immunity thereby offers diverse targets for highly selective therapeutics, such as small molecular synthetic compounds that modify innate immune responses. The notion that activation of the innate immune system is a prerequisite for the induction of acquired immunity raised interest in these immune response modifiers as potential therapeutics for viral infections and various tumors. A scenario of dermal events following skin cancer treatment with imiquimod presumably comprises (i) an initial low amount of pro-inflammatory cytokine secretion by macrophages and dermal dendritic cells (DCs), thereby (ii) attracting an increasing number type I IFN-producing plasmacytoid DCs (pDCs) from the blood; (iii) Langerhans cells migrate into draining lymph nodes, leading to an increased presentation of tumor antigen in the draining lymph node, and (iv) consequently an increased generation of tumor-specific T cells and finally (v) an accumulation of tumoricidal effector cells in the treated skin area. The induction of predominately T helper (Th)1-type cytokine profiles by TLR agonists such as imiquimod might have further benefits by shifting the dominant Th2-type response in atopic diseases such as asthma and atopic dermatitis to a more potent Th1 response.     

Increased expression of TRAIL and its death receptors DR4 and DR5 in plaque psoriasis

TNF-related apoptosis-inducing ligand (TRAIL) is recognized as an important regulator of immune responses during infections and various autoimmune-mediated pathologies. Its role in inflammatory dermatoses is largely unknown. We aimed to investigate the expression of TRAIL and its receptors DR4 and DR5 in psoriasis vulgaris. Immunohistochemistry for TRAIL, DR4 and DR5 was performed on samples of lesional (n = 10) and non-lesional (n = 10) skin of patients with plaque psoriasis and skin of healthy volunteers (n = 10). Expression of TRAIL and its receptors was further examined by means of double immunofluorescence staining and co-localization with CD4, CD8, CD11c, CD68, CD16 and CD56 markers. Immunohistochemical staining for TRAIL was significantly enhanced in psoriatic lesional as well as non-lesional epidermis compared to the epidermis of healthy skin. Lesional epidermis also showed increased immunoreactivity for DR5. In addition, expression of TRAIL and both of its receptors was significantly increased in the dermis of lesional skin. As evidenced by double immunofluorescence, TRAIL was readily expressed by most of the examined cells of the inflammatory infiltrate in psoriatic lesions. In contrast, the expression of DR4 was found mostly among CD4+ and CD8+ cells but was only nuclear, while DR5 showed cytoplasmic staining in rare CD16+, CD56+ and CD68+ cells. According to abundant in situ presence of TRAIL and its receptors in lesional psoriatic skin, it seems that this cytokine participates in the complex interplay between keratinocytes and cells of the dermal infiltrate and thus contributes to the inflammatory cycle in psoriasis vulgaris.     

Flow-cytometric characterization of normal versus psoriatic epidermis using improved cell separation methodology

Recently a new approach for epidermal cell characterization was developed: three-parameter flow cytometrical analysis of pure and complete epidermal cell suspensions prepared from punch biopsies followed by dermoepidermal separation by thermolysin. The aim of the present communication is the comparison between psoriatic lesional skin and normal skin using this new approach with respect to the percentage of suprabasal keratinocytes (keratin 10+ cells), mesenchymal cells, including the infiltrate cells (vimentin+ cells) and the percentage of basal cells in SG2 M phase, in order to validate this methodology in studies on psoriatic skin. Punch biopsies were taken from 7 healthy volunteers and in 7 psoriatic patients 4 biopsies were taken in each of them from comparable lesions. The present study reconfirmed that the percentage of basal keratinocytes in psoriasis was increased and the percentage of keratin 10+ cells was substantially decreased as compared to normal skin. The new methodology revealed data with a narrow range. In psoriatic lesional skin the intra individual variation was less compared to the inter individual variation.     

Current concepts in the immunopathogenesis of psoriasis

There is much evidence to support the concept that psoriasis is a type 1 autoimmune disease, primarily mediated by interferon gamma and other inflammatory cytokines. There has been renewed interest in the role of components of the innate immune system, however,and it may be that overlap between the innate and acquired arms of the immune system can better explain immunopathogenesis in psoriasis. Relevant cell types, receptors, and immune mediators within these traditional boundaries of the immune system are discussed.Finally, pathogenic contributions from important psoriatic mouse models and recent genomic data using the new gene chip technology are elaborated.