New insight into prurigo nodularis: Proadrenomedullin N-terminal 20 peptide mediates mouse mast cell activation via Mrgprb2.

BACKGROUND: Prurigo nodularis (PN) is a chronic inflammatory skin disorder that is characterized by extremely itchy nodules. Proadrenomedullin N-terminal 20 (PAMP) activates mast cell degranulation via Mas-related G protein-coupled receptor X2 (MRGPRX2), which is associated with pruritus in allergic contact dermatitis. However, the mechanisms underlying the action of PAMP and MRGPRX2 in PN remain unclear. OBJECTIVE: To determine the role of PAMP-induced mast cell activation via MRGPRX2 (mouse homologous Mrgprb2) in PN. METHODS: The expression of PAMP and the number of MRGPRX2-expressing mast cells in the skin biopsies of patients with PN, atopic dermatitis (AD), and healthy participants were analyzed using immunohistochemistry and immunofluorescence, respectively. The biphasic response of PAMP9-20 mediated by Mrgprb2 in mouse peritoneal mast cells (PMC) was validated in vitro using qRT-PCR, ELISA, flow cytometry, and siRNA techniques. RESULTS: PAMP expression and the number of MRGPRX2+ mast cells in lesional PN skin, but not in AD, were elevated compared to healthy skin. PAMP9-20 mediates the immediate and delayed phase responses of PMC, such as degranulation, histamine and ß-hexosaminidase release, and secretion of inflammatory factors such as CCL2, TNF-α, and GM-CSF. These effects were inhibited when Mrgprb2 expression was silenced. Silencing Mrgprb2 did not affect the biphasic response of PMC that was induced by IgE-FcεRI activation. CONCLUSIONS: The results show that PAMP mediates mouse mast cell activation via Mrgprb2, which may be involved in the pathogenesis of PN. The PAMP/ Mrgprb2 pathway, independent of classical IgE signaling, could be developed as a candidate drug target for treating PN.

Itch intensity in prurigo nodularis is closely related to dermal interleukin-31, oncostatin M, IL-31 receptor alpha and oncostatin M receptor beta.

Prurigo nodularis (PN) is a chronic skin dermatosis with hyperkeratotic and intensely pruritic nodules. Managing PN-associated itch is difficult because its aetiology is still unknown. This study aimed to investigate the correlation between itch intensity in PN and the expression of a pruritogenic cytokine interleukin (IL)-31, its receptor complex components IL-31 receptor α (IL-31RA) and oncostatin M receptor ß (OSMRß), and oncostatin M (OSM), which is a ligand of OSMR ß, through immunofluorescence staining examination. Itch intensity in PN was closely correlated with the number of dermal IL-31(+) cells (Spearman's r = 0.551, p < 0.05), dermal IL-31RA(+) cells (r = 0.475, p < 0.05) and dermal OSM(+) cells (r = 0.505, p < 0.05). In addition, the number of dermal OSMRß (+) cells was increased in PN (t test, p < 0.05), despite not being correlated with itch intensity (Spearman's r = 0.375, p > 0.05). Major cellular sources of dermal IL-31 were T cells (27.0% of total IL-31-expressing cells) and macrophages (35.0%), while those of OSM were mainly T cells (49.8%) and mast cells (26.8%). IL-31RA-expressing dermal cells were mostly mast cells (49.3%) and macrophages (36.6%), and OSMRß was mainly expressed by macrophages (51.8%) in the dermis. These findings indicate that IL-31 (mainly from macrophages and T cells) and OSM (principally from T cells and mast cells) stimulate dermal cells expressing IL-31RA and OSMRß (e.g. macrophages), which may further promote itch and inflammation in PN. This complex dermal milieu of cell/cytokine/receptor network can be a therapeutic target for PN-associated itch.

Aberrant Expression of Histamine-independent Pruritogenic Mediators in Keratinocytes may be Involved in the Pathogenesis of Prurigo Nodularis.

Prurigo nodularis is a highly pruritic and hyperplastic chronic dermatosis with unknown pathogenesis. Many pruritogenic mediators, including nerve growth factor, interleukin (IL)-31, thymic stromal lymphopoietin, and endothelin-1, are implicated in chronic itch and inflammation. This study investigated the mRNA levels and immunoreactivity of the nerve growth factor, IL-31, thymic stromal lymphopoietin, and endothelin axes in both lesional and perilesional skin in prurigo nodularis by using quantitative real-time PCR and immunohistochemistry studies. The nerve growth factor high-affinity receptor tyrosine kinase receptor A was upregulated while the low affinity receptor p75 neurotrophin receptor was downregulated in prurigo nodularis lesions. Downregulated expression of IL-31/IL-31 receptor A and endothelin-3/endothelin receptor B and upregulation of thymic stromal lymphopoietin receptor were found in prurigo nodularis lesions. Aberrant expression of nerve growth factor, IL-31, thymic stromal lymphopoietin and endothelin axes was found in prurigo nodularis lesions, especially in the epidermis, indicating the importance of keratinocytes in prurigo nodularis pathogenesis.

The use of biodegradable microneedle patches to increase penetration of topical steroid for prurigo nodularis.

BACKGROUND: The stratum corneum is an almost impermeable barrier. Recently, microneedles have been used to increase drug delivery passing the stratum corneum by incorporating the drug within the microneedle or by coating the surface of the microneedle with the drug. OBJECTIVE: This study was performed to investigate whether applying a biodegradable microneedle patch after topical steroid application increases penetration of the steroid in vitro, as well as treatment efficacy in patients with prurigo nodularis. MATERIALS & METHODS: In vitro penetration of topical steroids after biodegradable microneedle patch application was measured using a 3D skin model. To evaluate the treatment efficacy of the combination of biodegradable microneedle and topical steroids, a split-body clinical study was performed. RESULTS: Penetration of topical steroid in the in vitro skin model was significantly greater in the microneedle-applied skin. In a split-body clinical study with prurigo nodularis patients, the area and height of skin lesions decreased after four weeks of treatment on both sides, however, the microneedle patch side exhibited a significantly greater decrease in both area and height, compared to the control side. The pruritus visual analogue scale was also significantly lower on the microneedle side. CONCLUSION: We suggest that simply applying a microneedle patch after topical steroid application could be a useful strategy for treating refractory skin diseases such as prurigo nodularis.

Role of Substance P and Its Receptor Neurokinin 1 in Chronic Prurigo: A Randomized, Proof-of-Concept, Controlled Trial with Topical Aprepitant.

Substance P (SP) and its receptor neurokinin 1 (NK1R) are thought to be involved in the pathogenesis of chronic prurigo. Here, we assessed SP serum levels, cutaneous NK1R expression, and the effects of topical aprepitant, an NK1R antagonist, in patients with chronic prurigo. SP and NK1R were increased, compared with controls, in the serum and in lesional vs. non-lesional skin of the patients, respectively. Aprepitant, in a randomized, placebo-controlled, split-sided, doubleblind trial, reduced the intensity of pruritus as assessed by visual analogue scale by >50% from baseline to day 28 (-35.2), but so did placebo vehicle (-38.1, p= 0.76). Overall clinical scores improved significantly by day 28 in both treatment groups, with no significant difference between the 2 groups (p=0.32). Our findings imply that both SP and NK1R are involved in the pathogenesis of chronic prurigo. Parallel groupdesigned trials are needed to assess the efficacy of topical aprepitant treatment in this condition.

Possible Involvement of F1F0-ATP synthase and Intracellular ATP in Keratinocyte Differentiation in normal skin and skin lesions.

The F1F0-ATP synthase, an enzyme complex, is mainly located on the mitochondrial inner membrane or sometimes cytomembrane to generate or hydrolyze ATP, play a role in cell proliferation. This study focused on the role of F1F0-ATP synthase in keratinocyte differentiation, and its relationship with intracellular and extracellular ATP (InATP and ExATP). The F1F0-ATP synthase ß subunit (ATP5B) expression in various skin tissues and confluence-dependent HaCaT differentiation models was detected. ATP5B expression increased with keratinocyte and HaCaT cell differentiation in normal skin, some epidermis hyper-proliferative diseases, squamous cell carcinoma, and the HaCaT cell differentiation model. The impact of InATP and ExATP content on HaCaT differentiation was reflected by the expression of the differentiation marker involucrin. Inhibition of F1F0-ATP synthase blocked HaCaT cell differentiation, which was associated with a decrease of InATP content, but not with changes of ExATP. Our results revealed that F1F0-ATP synthase expression is associated with the process of keratinocyte differentiation which may possibly be related to InATP synthesis.

The pathogenesis of Prurigo nodularis--'Super-Itch' in exploration.

Prurigo nodularis (PN) is characterized by multiple hyperkeratotic nodules, papules and the presence of intensive pruritus. This leads to an impaired quality of life and high burden due not only to the severe itch but also the chronic, skin lesions and lack of treatment options. The pathogenesis of PN is not completely clarified. Previous studies have demonstrated just how important the interaction between cutaneous nerve fibres and immune cells is. Besides a reduced intraepidermal nerve fibre density, there are increased dermal levels of neuropeptides such as substance P, calcitonin gene-related peptide and nerve growth factor, as well as a predominant presence of eosinophils and mast cells. An interaction of these factors results in a complex relationship which will be discussed in this article.

METEORIN-LIKE is a cytokine associated with barrier tissues and alternatively activated macrophages.

Cytokines are involved in many functions of the immune system including initiating, amplifying and resolving immune responses. Through bioinformatics analyses of a comprehensive database of gene expression (BIGE: Body Index of Gene Expression) we observed that a small secreted protein encoded by a poorly characterized gene called meteorin-like (METRNL), is highly expressed in mucosal tissues, skin and activated macrophages. Further studies indicate that Metrnl is produced by Alternatively Activated Macrophages (AAM) and M-CSF cultured bone marrow macrophages (M2-like macrophages). In the skin, METRNL is expressed by resting fibroblasts and IFNγ-treated keratinocytes. A screen of human skin-associated diseases showed significant over-expression of METRNL in psoriasis, prurigo nodularis, actinic keratosis and atopic dermatitis. METRNL is also up-regulated in synovial membranes of human rheumatoid arthritis. Taken together, these results indicate that Metrnl represents a novel cytokine, which is likely involved in both innate and acquired immune responses.

Evaluation of the antipruritic effects of topical pimecrolimus in non-atopic prurigo nodularis: results of a randomized, hydrocortisone-controlled, double-blind phase II trial.

BACKGROUND: In the treatment of atopic dermatitis, pimecrolimus has high antipruritic effects. OBJECTIVE: To investigate the efficacy of 1% pimecrolimus cream in comparison to 1% hydrocortisone cream in non-atopic prurigo nodularis (PN). METHODS: A randomized, controlled, double-blind study with intraindividual randomization was done in 30 patients (17 females, 13 males; mean age 58.5 years) with PN. RESULTS: Pruritus intensity decreased significantly (p < 0.001) on both treated sides as early as after 10 days of treatment; scratch lesions improved (p < 0.001). Quality of life as assessed by the Dermatology Life Quality Index improved significantly. However, a significant advantage of pimecrolimus over hydrocortisone was not found. CONCLUSION: The results suggest that the non-steroid pimecrolimus is an effective alternative for PN treatment.

The utility of ATF3 in distinguishing cutaneous squamous cell carcinoma among other cutaneous epithelial neoplasms.

The histopathologic distinction between benign and malignant cutaneous keratinocytic proliferations can pose a difficult diagnostic challenge - often with important clinical implications. Activating transcription factor 3 (ATF3) has emerged as a potential biomarker which may aid in the segregation of these lesions, and we hypothesize that ATF3 expression may be a specific marker of cutaneous squamous cell carcinoma (SCC). Using immunohistochemistry, we characterized ATF3 expression in a series of 126 cutaneous epithelial proliferations, including SCC (n = 27), basal cell carcinomas (BCC, n = 59), seborrheic keratoses with atypia (SK, n = 16), hyperplastic actinic keratoses (AK, n = 12) and prurigo nodularis cases (PN, n = 12). We showed strong, nuclear and/or nucleolar expression of ATF3 in a statistically significant number of cases of SCC compared to BCC, SK and PN. We conclude that ATF3 expression is a surrogate of malignancy (or pre-malignancy) in keratinocytic epithelial proliferations and thus helps distinguish SCC from other cutaneous epithelial neoplasms.

IL-31 expression by inflammatory cells is preferentially elevated in atopic dermatitis.

Interleukin-31 (IL-31) is a recently discovered cytokine expressed in many human tissues, and predominantly by activated CD4(+) T cells. IL-31 signals through a heterodimeric receptor consisting of IL-31 receptor alpha (IL-31RA) and oncostatin M receptor beta (OSMR). Earlier studies have shown involvement of IL-31 and its receptor components IL-31RA and OSMR in atopic dermatitis, pruritus and Th2-weighted inflammation at the mRNA level. The aim of this study was to investigate IL-31 protein expression in skin of such conditions. Immunohistochemical staining for IL-31, IL-31RA and OSMR was performed in formalin-fixed paraffin-embedded biopsy specimens. IL-31 expression was increased in the inflammatory infiltrates from skin biopsies taken from subjects with atopic dermatitis, compared with controls (p ≤ 0.05). IL-31, IL-31RA and OSMR protein immunoreactivity was not increased in biopsies from subjects with other Th2-weighted and pruritic skin diseases. Our results confirm, at the protein level, the relationship between IL-31 expression and atopic dermatitis. Our results do not support a general relationship between expression of IL-31/IL-31R and pruritic or Th2-mediated diseases.

Nuclear localization of activated STAT6 and STAT3 in epidermis of prurigo nodularis.

BACKGROUND: Prurigo nodularis (PN) is a chronic dermatitis characterized by discrete, raised, and firm papulonodules with intense pruritus. The pathogenesis still remains to be elucidated. OBJECTIVES: To clarify the role of Th1 and Th2 cytokines in the pathogenesis of PN. METHODS: We examined the cytokine signatures, such as phosphorylation of STAT1, STAT3 and STAT6, HLA-DR and hyaluronan accumulation, to reveal the Th1 and Th2 cytokine influence on the lesional epidermis of PN. RESULTS: We first optimized antigen retrieval methods to detect these signatures with antibodies for phospho-STAT1 (pSTAT1), phospho-STAT3 (pSTAT3), phospho-STAT6 (pSTAT6), HLA-DR and hyaluronic acid binding protein (HABP) on the formalin-fixed paraffin-embedded sections of psoriasis, lichen planus and atopic dermatitis biopsy samples. Activation of STAT1 and STAT6 in epidermis by Th1 and Th2 cytokines was further confirmed in a cultured skin equivalent model treated with interferon-γ or interleukin (IL)-4/IL-13. With the relevant immunostaining methods, we examined the cytokine signatures in 22 cases of PN. The results revealed that (i) the entire epidermis of 19 cases was stained with anti-pSTAT6 antibody, (ii) 21 cases demonstrated nuclear staining with anti-pSTAT3 antibody, (iii) the entire epidermis of 21 cases was stained with HABP, (iv) the epidermis of eight cases showed scattered staining with anti-pSTAT1 antibody, and (v) six cases were positive for HLA-DR membrane expression. CONCLUSIONS: These data indicated that Th2 cytokines related to STAT6 activation together with some unknown stimuli that activate STAT3 play a principal role in the pathogenesis of PN.

Actinic prurigo.

Actinic Prurigo (AP), an uncommon idiopathic photodermatosis, presents a distinct clinical picture and can be severely debilitating. The clinical features, investigation and treatment of AP are reviewed. We report the experience of an Australian photobiology unit with this condition.

IL-31: a new link between T cells and pruritus in atopic skin inflammation.

BACKGROUND: IL-31 is a novel T-cell-derived cytokine that induces severe pruritus and dermatitis in transgenic mice, and signals through a heterodimeric receptor composed of IL-31 receptor A and oncostatin M receptor. OBJECTIVE: To investigate the role of human IL-31 in pruritic and nonpruritic inflammatory skin diseases. METHODS: The expression of IL-31 was analyzed by quantitative real-time PCR in skin samples of healthy individuals and patients with chronic inflammatory skin diseases. Moreover, IL-31 expression was analyzed in nonlesional skin of atopic dermatitis patients after allergen or superantigen exposure, as well as in stimulated leukocytes. The tissue distribution of the IL-31 receptor heterodimer was investigated by DNA microarray analysis. RESULTS: IL-31 was significantly overexpressed in pruritic atopic compared with nonpruritic psoriatic skin inflammation. Highest IL-31 levels were detected in prurigo nodularis, one of the most pruritic forms of chronic skin inflammation. In vivo, staphylococcal superantigen rapidly induced IL-31 expression in atopic individuals. In vitro, staphylococcal enterotoxin B but not viruses or T(H)1 and T(H)2 cytokines induced IL-31 in leukocytes. In patients with atopic dermatitis, activated leukocytes expressed significantly higher IL-31 levels compared with control subjects. IL-31 receptor A showed most abundant expression in dorsal root ganglia representing the site where the cell bodies of cutaneous sensory neurons reside. CONCLUSION: Our findings provide a new link among staphylococcal colonization, subsequent T-cell recruitment/activation, and pruritus induction in patients with atopic dermatitis. Taken together, these findings show that IL-31 may represent a novel target for antipruritic drug development.