Neurokinin 1 receptor antagonists exhibit peripheral effects in prurigo nodularis including reduced ERK1/2 activation.

BACKGROUND: Aprepitant is a neurokinin 1 receptor (NK1R) antagonist used for its antipruritic properties in dermatoses and systemic diseases. The mode of action is still unclear. A peripheral effect is assumed as aprepitant shows efficacy in inflammatory skin diseases including prurigo nodularis (PN). OBJECTIVES: To investigate the peripheral effects of NK1R antagonism in PN and cell culture models. METHODS: Subjects with PN received an aprepitant treatment. Clinical, morphological and immunohistochemical changes were investigated in skin biopsies before and after treatment. Expression of NK1R was analysed by immunohistochemistry and for downstream pathways ((p)ERK1/2) by Western blotting in PN patients and matched healthy volunteers. Effects of NK1R blocking were analysed in cell cultures of primary keratinocytes by Western blotting for (p)ERK1/2 and by qPCR for NK1R, interleukin (IL)-1beta, IL-6, IL-8 and TNFalpha. RESULTS: Aprepitant treatment showed significant reduction in pruritus intensity (P < 0.05) in PN and relevant immunohistochemical changes (down: CD5, CD25, up: CD79a, IL4). NK1R expression was higher in keratinocytes of PN patients compared to healthy controls. After treatment, epidermal NK1R expression increased while expression and activation of ERK1/2 decreased. In vitro, receptor up-regulation and reduced expression and activation of ERK1/2 were confirmed and reduced IL-expression shown when blocking NK1R. CONCLUSION: Our data confirm that NK1R antagonists such as aprepitant exhibit effects in the skin. Epidermal receptor expression, epidermal inflammatory ILs, ERK1/2 MAPK signalling and cutaneous inflammatory infiltrate were targets of NK1R antagonism. This may explain partly the antipruritic effect of NK1R antagonists next to its role in the central nervous system.

LEKTI domains D6, D7 and D8+9 serve as substrates for transglutaminase 1: implications for targeted therapy of Netherton syndrome.

BACKGROUND: Transglutaminase (TG)1 plays a key role in the formation of the cornified envelope and thus in the maintenance of the epidermal barrier. Patients with Netherton syndrome (LEKTI deficiency) have increased activity of both TG1 and serin proteases. OBJECTIVES: To determine whether there is a functional biochemical link between TG1 and LEKTI and whether LEKTI domains could possibly serve as substrates for TG1. METHODS: We analysed the protein sequence of LEKTI for possible TG1 recognition sites using bioinformatics. Synthetic peptides and recombinant LEKTI domains D6, D7 and D8+9 were examined in vitro and in situ for possible substrate specificity. The recombinant LEKTI domains were studied for inhibitory activity in a kallikrein (KLK)5 activity test. RESULTS: We identified possible TG1 consensus sequences in LEKTI domains D6, D7 and D8+9, pointing to a novel biological link between these two proteins. Indeed, synthesized short peptides from these consensus sequences were incorporated into the TG1 activity zone of the epidermis. In vitro the entire recombinant domains of LEKTI showed substrate specificity for TG1, which was again confirmed in situ. The inhibitory activity of the recombinant LEKTI domains was confirmed by a KLK5 inhibition test. The strongest inhibition was observed for domains D8+9. CONCLUSIONS: There are specific domains of LEKTI that are recognized and processed by TG1. LEKTI domains D6, D7 and D8+9 contribute to the formation and protection of the cornified envelope. These results impact the development of protein replacement therapy approaches for Netherton syndrome. What's already known about this topic? LEKTI and transglutaminase (TG)1 are key proteins involved in the terminal differentiation of the epidermis. Lack of LEKTI causes Netherton syndrome; TG1 deficiency causes lamellar ichthyosis. The serine protease inhibitor LEKTI is processed into different functional units. Among different target proteases, kallikrein (KLK)5 appears to be a key player in disease pathology. It has been demonstrated that LEKTI domain 6 inhibits KLK5 and KLK7; LEKTI domains 8-11 also inhibit KLK14. What does this study add? The single LEKTI domains 6, 7 and the functional unit of domains 8 and 9 contain recognition motifs for TG1. We show that these domains and unit are crosslinked into the epidermis by TG1. Functional analyses of the recombinant LEKTI domains revealed that LEKTI D8+9 has the strongest inhibitory effect on KLK5. What is the translational message? The novel functional link between LEKTI and TG1 should be taken into account when considering the development of a targeted topical protein therapy for Netherton syndrome. The unit of domains D8+9 may be sufficient for this purpose.

Sensitive skin.

Sensitive skin is a clinical condition defined by the self-reported facial presence of different sensory perceptions, including tightness, stinging, burning, tingling, pain and pruritus. Sensitive skin may occur in individuals with normal skin, with skin barrier disturbance, or as a part of the symptoms associated with facial dermatoses such as rosacea, atopic dermatitis and psoriasis. Although experimental studies are still pending, the symptoms of sensitive skin suggest the involvement of cutaneous nerve fibres and neuronal, as well as epidermal, thermochannels. Many individuals with sensitive skin report worsening symptoms due to environmental factors. It is thought that this might be attributed to the thermochannel TRPV1, as it typically responds to exogenous, endogenous, physical and chemical stimuli. Barrier disruptions and immune mechanisms may also be involved. This review summarizes current knowledge on the epidemiology, potential mechanisms, clinics and therapy of sensitive skin.

In vitro induced CD8+ regulatory T cells inhibit skin inflammation.

CD8(+) regulatory T cells appear impaired in number and/or function in some autoimmune diseases. However, the role of CD8(+) regulatory T cells in the pathogenesis of skin inflammation and psoriasis remains unknown. In this study, we set out to analyze the capability of CD8(+) regulatory T cells to inhibit skin inflammation in a murine model and to determine the frequency of CD8(+) regulatory T cells in patients with psoriasis. We demonstrate that murine fully competent CD8(+) regulatory T cells can be induced by stimulating naïve CD8(+) T cells in the presence of TGF-ß and retinoic acid (RA). Importantly, in vitro induced CD8(+) regulatory T cells significantly suppressed skin inflammation in vivo. Furthermore, we found that the frequency of regulatory CD8(+)CD25(+)Foxp3(+) T cells is decreased in peripheral blood but increased in lesional psoriatic skin of patients with psoriasis. Thus, our study suggests a previously unappreciated role of CD8(+)CD25(+)Foxp3(+) T cells in skin disorders, and induction of these cells in vitro may be an effective immunotherapy for skin inflammation.

Ichthyosis vulgaris: novel FLG mutations in the German population and high presence of CD1a+ cells in the epidermis of the atopic subgroup.

BACKGROUND: Ichthyosis vulgaris (IV) is a genetic disorder with a prevalence of 1:250-1000 caused by filaggrin (FLG) mutations, which also predispose to atopic diseases. OBJECTIVES: To study the genotype/phenotype relationship in IV and to analyse whether the suggested skin barrier defect is associated with differences of epidermal dendritic cells. PATIENTS/METHODS: We evaluated a cohort of 26 German patients with IV, established an IV severity score and analysed epidermal ultrastructure, histology, filaggrin and CD1a antigens. Mutations were screened by restriction enzyme analysis. Particular sequencing techniques allowed the complete FLG analysis to reveal novel mutations. RESULTS: The combined null allele frequency of R501X and 2282del4 was 67.3%. Patients also showed the mutations S3247X and R2447X as well as five novel FLG mutations: 424del17 and 621del4 (profilaggrin S100 domain), 2974delGA (repeat 2), R3766X (repeat 10(1)) and E4265X (repeat 10(2)). Their combined allele frequency in controls was <0.7%. No mutation was found in one IV patient, all in all approximately 27% were heterozygous, and the majority (approximately 69%) showed two null alleles. The IV severity score and ultrastructure showed a significant correlation with genotypes. Interestingly, CD1a cell counts showed a significant difference between nonatopic and atopic IV patients both with eczema and without eczema. CONCLUSIONS: We confirm that the mutations R501X and 2282del4 represent the most frequent genetic cause in German IV patients. The novel mutations are probably population and family specific. The observed differences of CD1a cells support the hypothesis that there is a barrier defect that predisposes to atopic manifestations, possibly independent of atopic eczema.

Who is really in control of skin immunity under physiological circumstances - lymphocytes, dendritic cells or keratinocytes?

Our views of the skin immunity theatre are undergoing constant change. These not only reflect paradigm shifts in general immunology and skin biology, but also have profound clinical implications, which call for strategic changes in dermatological therapy. Nowhere can this be witnessed at a greater level of instructiveness and fascination than when addressing the question posed by this new Controversies feature. Thus, after a very long period of dominance by T cells and Langerhans cells as 'lead actors' on the skin immunity stage, the lowly keratinocyte has recently made an astounding theatrical appearance as a key protagonist of the innate skin immunity system, which may control even acquired skin immune responses. Further enhancing dramatic complexity and tension, the mast cell has entered as an additional actor claiming centre stage, and the epidermal Langerhans cell has slipped in a surprise appearance as the chief agent of immunotolerance. May you, esteemed reader, enjoy the spectacle offered here by selected immunodermatology authorities who double as 'stage managers' pushing their respective favourite actors into the limelight. You get everything you may expect from a good performance - complete with the impresario's overture that lures you into the theatre and sets the stage, competing divas, recently discovered new talents and even the critic's digest while the performance is still ongoing. By the time the curtain drops, you will have reached your own, independent conclusions on how to answer the title question of this play - at least for the time being...

Therapeutic modulation of cutaneous autoimmunity by regulatory T cells.

Cutaneous autoimmunity is characterized by the presence of autoantibodies and/or autoreactive T cells. Several mechanisms have been developed to avoid loss of immunotolerance to self such as activation-induced cell death, deletion, ignorance and active suppression of autoreactivity. Regulatory ('suppressor') T cells play a pivotal role in inhibiting the activation and function of effector T cells. CD4(+)CD25(+) T cells constitute a subset of regulatory T cells, which have been shown to suppress the development of organ-specific autoimmunity in mice. Recent understanding in the generation and function of human regulatory T cells indicates that these cells are involved in the appearance of inflammatory as well as bullous autoimmune dermatosis. These findings suggest that modulation of regulatory T cell numbers or function might be a promising therapeutic alternative for the treatment of such disorders. In the following, recent strategies aimed at inducing antigen-specific or non-specific regulatory T cells for the immunotherapy of ongoing cutaneous autoimmunity are presented and discussed. Hopefully, pursuing these strategies in the future will result in the initiation of randomized clinical studies analysing the usefulness of regulatory T cells for human skin diseases in great detail.

In vitro-generated regulatory T cells induced by Foxp3-retrovirus infection control murine contact allergy and systemic autoimmunity.

Regulatory T cells are promising candidates for the modulation of inflammation and autoimmunity. To generate regulatory T cells in vitro, we have infected naïve CD4+CD25- T cells with a retrovirus encoding the transcription factor Foxp3. Foxp3-infected T cells are similar to naturally occurring regulatory T cells as evidenced by surface marker expression and function. To investigate the effects of Foxp3-infected T cells on contact hypersensitivity (CHS) responses, sensitized mice were injected with Foxp3- or control virus-infected T cells. Only injection of Foxp3-infected T cells into sensitized mice significantly inhibited CHS compared to controls, indicating that Foxp3-infected T cells are suppressive in vivo. These findings prompted treatment of autoimmune-prone CD40L transgenic (tg) mice, which develop a severe systemic autoimmune disease including autoreactive T cells and autoantibodies, with Foxp3-infected T cells. Interestingly, injections of Foxp3-infected T cells into CD40L tg mice inhibited the ongoing development of autoimmune dermatitis and activation of cytotoxic CD8+ T cells. Strikingly, treatment with Foxp3-infected T cells reduced serum concentrations of antinuclear antibodies in CD40L tg mice, which was paralleled with reduced renal immunoglobulin depositions and increased kidney function. Together, these findings indicate that newly in vitro-generated regulatory T cells can be successfully used to treat inflammatory and ongoing autoimmune disorders.

Transcription factor AP-2alpha triggers apoptosis in cardiac myocytes.

Idiopathic-dilated cardiomyopathy (IDC) is a common primary myocardial disease of unknown etiology associated with apoptosis, cardiac dilatation, progressive heart failure and increased mortality. An elevation of the transcription factor activator protein 2alpha (AP-2alpha) is involved in vertebrate embryonic development and oncogenesis. Here, we show that AP-2alpha protein is expressed in the human heart and increased in human failing myocardium with IDC. Adenovirus-mediated overexpression of human AP-2alpha triggered apoptosis and increased mRNA levels of Bcl-2 family members Bax and Bcl-x in rat cardiomyocytes. Immunohistological analysis of human myocardium revealed an increased percentage of AP-2alpha-positive nuclei in IDC and, interestingly, a colocalization of AP-2alpha-positive but not -negative cells with a caspase-cleaved fragment of poly(ADP-ribose)polymerase. We suggest AP-2alpha as a novel cardiac regulator implicated in the activation of apoptosis in IDC.

Overexpression of CD40 ligand in murine epidermis results in chronic skin inflammation and systemic autoimmunity.

CD40-CD40 ligand (L) interactions play a pivotal role in immune-mediated inflammatory responses via the activation of antigen-presenting cells (APCs). To investigate the effects of continuous activation of resident tissue APCs, in this case the Langerhans cells (LCs) of the skin, CD40L expression was targeted to the basal keratinocytes of the epidermis of mice using the keratin-14 promoter. Approximately 80% of the transgenic (Tg) mice spontaneously developed dermatitis on the ears, face, tail, and/or paws. Compared with littermates, Tgs had a >90% decrease in epidermal LCs yet increased numbers within the dermis suggestive of enhanced emigration of CD40-activated LCs. Tgs also displayed massive regional lymphadenopathy with increased numbers of dendritic cells and B cells. Moreover, a decrease in IgM and an increase in IgG1/IgG2a/IgG2b/IgE serum concentrations was detectable. Screening for autoantibodies revealed the presence of antinuclear antibodies and anti-dsDNA antibodies implicative of systemic autoimmunity. Accordingly, renal Ig deposits, proteinuria, and lung fibrosis were observed. Adoptive transfer of T cells from Tgs to nonTg recipients evoked the development of skin lesions similar to those found in the Tgs. Dermatitis also developed in B cell-deficient CD40L Tg mice. These findings suggest that in situ activation of LCs by CD40L in the skin not only leads to chronic inflammatory dermatitis but also to systemic mixed-connective-tissue-like autoimmune disorders, possibly by breaking immune tolerance against the skin.

The fungal acl1 and acl2 genes encode two polypeptides with homology to the N- and C-terminal parts of the animal ATP citrate lyase polypeptide.

ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl-CoA, which is mainly used for the biosynthesis of fatty acids and sterols. In this paper, we show for the first time that in filamentous fungi two different subunits of ACL are encoded by two separate genes. This is in contrast to animals where ACL is encoded by a single gene. Data are presented on acl genes from the filamentous fungi Sordaria macrospora and Gibberella pulicaris. In S. macrospora, both genes, acl1 and acl2, are clustered within a region of 10 kb and are divergently transcribed.

Induction of a polyubiquitin gene (ubi1) by potato phytoalexins and heat shock in Gibberella pulicaris.

Gibberella pulicaris, a causal agent of potato dry rot, infects potato tubers via wounds, where it is exposed to the phytoalexins rishitin and lubimin. Incubation of mycelium on agar supplemented with phytoalexins transiently induced the transcription of a polyubiquitin gene consisting of four ubiquitin units arranged head to tail; the fourth unit contains a 54-bp intron and an additional glutamine at the C-terminus of the encoded protein. Southern analysis of the G. pulicaris genome revealed one copy of the isolated polyubiquitin gene and one or two copies of other ubiquitin genes. Increased transcription of the gene was detectable above a threshold of 100 microg/ml of rishitin and at elevated temperatures, which indicates that exposure to phytoalexins causes a stress reaction of hyphal cells similar to that after heat shock.