Requirements and expectations of high-quality biomarkers for atopic dermatitis and psoriasis in 2021-a two-round Delphi survey among international experts.

BACKGROUND: Chronic inflammatory skin diseases such as atopic dermatitis (AD) and psoriasis (PSO) present major challenges in health care. Thus, biomarkers to identify disease trajectories and response to treatments to improve the lives of affected individuals warrant great research consideration. The requirements that these biomarkers must fulfil for use as practical clinical tools have not yet been adequately investigated. AIM: To identify the core elements of high-quality AD and PSO biomarkers to prepare recommendations for current biomarker research. METHOD: A cross-sectional two-round Delphi survey was conducted from August to October 2019 and October to November 2020. All participants were members of the BIOMAP project, an EU-funded consortium of clinicians, researchers, patient organizations and pharmaceutical industry partners. The first round consisted of three open-ended questions. Responses were qualitatively analysed, and 26 closed statements were developed. For the second round, 'agreement' was assumed when the responses of ≥70% of the participants were ≥5 points on a 7-point Likert scale for each statement. Priority classification was based on mean scores (<20th percentile = low, 20th to 60th percentile = medium, >60th percentile = high). RESULTS: Twenty-one and twenty-six individuals participated in rounds one and two, respectively. From 26 statements that were included in round 2, 18 achieved agreement (8 concerning the performance, 8 for the purpose and 2 on current obstacles). Seven statements were classified as high priority, e.g. those concerning reliability, clinical validity, a high positive predictive value, prediction of the therapeutic response and disease progression. Another seven statements were assigned medium priority, e.g. those about analytical validity, prediction of comorbidities and therapeutic algorithm. Low priority included four statements, like those concerning cost effectiveness and prediction of disease flares. CONCLUSION: The core requirements that experts agreed on being essential for high-quality AD and PSO biomarkers require rapid validation. Biomarkers can therefore be assessed based on these prioritized requirements.

IL-17C amplifies epithelial inflammation in human psoriasis and atopic eczema.

BACKGROUND: Key pathogenic events of psoriasis and atopic eczema (AE) are misguided immune reactions of the skin. IL-17C is an epithelial-derived cytokine, whose impact on skin inflammation is unclear. OBJECTIVE: We sought to characterize the role of IL-17C in human ISD. METHODS: IL-17C gene and protein expression was assessed by immunohistochemistry and transcriptome analysis. Primary human keratinocytes were stimulated and expression of cytokines chemokines was determined by qRT-PCR and luminex assay. Neutrophil migration towards supernatant of stimulated keratinocytes was assessed. IL-17C was depleted using a new IL-17C-specific antibody (MOR106) in murine models of psoriasis (IL-23 injection model) and AE (MC903 model) as well as in human skin biopsies of psoriasis and AE. Effects on cell influx (mouse models) and gene expression (human explant cultures) were determined. RESULTS: Expression of IL-17C mRNA and protein was elevated in various ISD. We demonstrate that IL-17C potentiates the expression of innate cytokines, antimicrobial peptides (IL-36G, S100A7 and HBD2) and chemokines (CXCL8, CXCL10, CCL5 and VEGF) and the autocrine induction of IL-17C in keratinocytes. Cell-free supernatant of keratinocytes stimulated with IL-17C was strongly chemotactic for neutrophils, thus demonstrating a critical role for IL-17C in immune cell recruitment. IL-17C depletion significantly reduced cell numbers of T cells, neutrophils and eosinophils in murine models of psoriasis and AE and led to a significant downregulation of inflammatory mediators in human skin biopsies of psoriasis and AE ex vivo. CONCLUSION: IL-17C amplifies epithelial inflammation in Th2 and Th17 dominated skin inflammation and represents a promising target for the treatment of ISD.

Is the humoral immunity dispensable for the pathogenesis of psoriasis?

BACKGROUND: Imbalances of T-cell subsets are hallmarks of disease-specific inflammation in psoriasis. However, the relevance of B cells for psoriasis remains poorly investigated. OBJECTIVE: To analyse the role of B cells and immunoglobulins for the disease-specific immunology of psoriasis. METHODS: We characterized B-cell subsets and immunoglobulin levels in untreated psoriasis patients (n = 37) and compared them to healthy controls (n = 20) as well as to psoriasis patients under disease-controlling systemic treatment (n = 28). B-cell subsets were analysed following the flow cytometric gating strategy based on the surface markers CD24, CD38 and CD138. Moreover, immunofluorescence stainings were used to detect IgA in psoriatic skin. RESULTS: We found significantly increased levels of IgA in the serum of treatment-naïve psoriasis patients correlating with disease score. However, IgA was only observed in dermal vessels of skin sections. Concerning B-cell subsets, we only found a moderately positive correlation of CD138+ plasma cells with IgA levels and disease score in treatment-naïve psoriasis patients. Confirming our hypothesis that psoriasis can develop in the absence of functional humoral immunity, we investigated a patient who suffered concomitantly from both psoriasis and a hereditary common variable immune defect (CVID) characterized by a lack of B cells and immunoglobulins. We detected variants in three of the 13 described genes of CVID and a so far undescribed variant in the ligand of the TNFRSF13B receptor leading to disturbed B-cell maturation and antibody production. However, this patient showed typical psoriasis regarding clinical presentation, histology or T-cell infiltrate. Finally, in a group of psoriasis patients under systemic treatment, neither did IgA levels drop nor did plasma cells correlate with IgA levels and disease score. CONCLUSION: B-cell alterations might rather be an epiphenomenal finding in psoriasis with a clear dominance of T cells over shifts in B-cell subsets.

Sebocytes contribute to skin inflammation by promoting the differentiation of T helper 17 cells.

BACKGROUND: The main function of sebocytes is considered to be the production of lipids to moisturize the skin. However, it recently became apparent that sebocytes release chemokines and cytokines and respond to proinflammatory stimuli as well as the presence of bacteria. OBJECTIVES: To analyse the functional communication between human sebocytes and T cells. METHODS: Immunofluorescence stainings for CD4 and interleukin (IL)-17 were performed on acne sections and healthy skin. Migration assays and T-cell-stimulation cultures were performed with supernatants derived from unstimulated or prestimulated SZ95 sebocytes. Dendritic cells were generated in the presence of SZ95 supernatant and subsequently used in mixed leucocyte reactions. RESULTS: We showed that CD4+ IL-17+ T cells accumulate around the pilosebaceous unit and are in close contact with sebocytes in acne lesions. By using SZ95 sebocyte supernatant, we demonstrate a chemotactic effect of sebocytes on neutrophils, monocytes and T cells in a CXCL8-dependent manner. Furthermore, sebocyte supernatant induces the differentiation of CD4+ CD45RA+ naive T cells into T helper (Th)17 cells via the secretion of IL-6, transforming growth factor-ß and, most importantly, IL-1ß. No direct effects of sebocytes on the function of CD4+ CD45RO+ memory T cells were detected. Moreover, sebocytes functionally interact with Propionibacterium acnes in the maturation of dendritic cells, leading to antigen-presenting cells that preferentially prime Th17 cells. CONCLUSIONS: Our study provides evidence that human sebocytes actively participate in inflammatory processes in the skin by recruiting and communicating with immune cells. This interaction leads to the generation of Th17 cells, which might contribute to the pathogenesis not only of acne vulgaris, but also of several inflammatory skin diseases.

Immune response patterns in non-communicable inflammatory skin diseases.

Non-communicable inflammatory skin diseases (ncISD) such as psoriasis or atopic eczema are a major cause of global disease burden. Due to their impact and complexity, ncISD represent a major challenge of modern medicine. Dermatology textbooks describe more than 100 different ncISD based on clinical phenotype and histological architecture. In the last decades, this historical description was complemented by increasing molecular knowledge - and this knowledge is now being translated into specific therapeutics. Combining the enormous advances made in lymphocyte immunology and molecular genetics with clinical and histological phenotyping reveals six immune response patterns of the skin - type I immune cells cause the lichenoid pattern characterized by immune-mediated cell death of keratinocytes; type II immune cells underlie the eczematous pattern with impaired epidermal barrier, infection and eosinophils as well as the bullous pattern with loss of epithelial integrity; Th17 cells and ILC3 mediate the psoriatic pattern characterized by acanthosis, high metabolic activity and neutrophils; dysbalance of regulatory T cells causes either the fibrogenic pattern with rarefication of cells and dermal thickening or the granulomatous pattern defined by formation of granulomas. With more and more specific therapeutic agents approved, classifying ncISD also according to their immune response pattern will become highly relevant. This review defines the six immune response patterns of ncISD and highlights therapeutic strategies targeting key lymphocyte mediators.

Alpha-gal is a possible target of IgE-mediated reactivity to antivenom.

BACKGROUND: Antivenoms are mammalian immunoglobulins with the ability to neutralize snake venom components and to mitigate the progression of toxic effects. Immediate hypersensitivity to antivenoms often occurs during the first administration of these heterologous antibodies. A comparable clinical situation occurred after introduction of cetuximab, a chimeric mouse-human antibody, for cancer treatment. The carbohydrate epitope galactose-alpha-1,3-galactose, located on the Fab region of cetuximab, was identified as the target responsible for IgE reactivity. OBJECTIVE: To investigate whether serum IgE antibodies directed to the α-gal epitope are associated with hypersensitivity to equine antivenoms. METHODS: Antivenoms were screened for α-gal epitopes via immunoblot and in comparison with cetuximab and pork kidney by IgE reactivity assays. Basophil activation tests were used to investigate reactivity to antivenoms in samples from 20 patients with specific IgE antibodies to α-gal and 10 controls. Additional IgE detection, IgE inhibition, ImmunoCAP inhibition, and skin prick tests were performed using samples from selected patients. RESULTS: Both antivenoms and cetuximab induced positive skin prick test results in patients with sIgE to α-gal. Alpha-gal epitopes were detected by immunoblotting on antivenoms. Measurements of IgE reactivity and ImmunoCAP inhibition indicated that the antivenoms contained lower α-gal contents than cetuximab. Deglycosylation assays and IgE inhibition tests confirmed that IgE-mediated reactivity to antivenom is associated with α-gal. Antivenoms, pork kidney, and cetuximab activated basophils from patients with IgE to α-gal. CONCLUSION: Alpha-gal is a potential target of IgE-mediated reactivity to equine antivenom and a possible cause of the high incidence of hypersensitivity reactions during the first application of equine antivenom.

Sebum lipids influence macrophage polarization and activation.

BACKGROUND: As lipids are known to regulate macrophage functions, it is reasonable to suppose that a sebocyte-macrophage axis mediated by sebum lipids may exist. OBJECTIVES: To investigate if sebocytes could contribute to the differentiation, polarization and function of macrophages with their secreted lipids. METHODS: Oil Red O lipid staining and Raman spectroscopy were used to assess the dermal lipid content and penetration. Immunohistochemistry was used to analyse the macrophage subsets. Human peripheral blood monocytes were differentiated in the presence of either supernatant from human SZ95 sebocytes or major sebum lipid components and activated with Propionibacterium acnes. Macrophage surface markers and their capacity to uptake fluorescein isothiocyanate-conjugated P. acnes were detected by fluorescence-activated cell sorting measurements. Cytokine protein levels were evaluated by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS: Sebaceous gland-rich skin had an increased dermal lipid content vs. sebaceous gland-poor skin to which all the tested sebum component lipids could contribute by penetrating the dermoepidermal barrier. Of the lipids, oleic acid and linoleic acid promoted monocyte differentiation into alternatively activated macrophages. Moreover, linoleic acid also had an anti-inflammatory effect in P. acnes-activated macrophages, inhibiting the secretion of interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α. Squalene, palmitic acid, stearic acid and oleic acid augmented the secretion of IL-1ß, even in the absence of P. acnes, whereas oleic acid had a selective effect of inducing IL-1ß but downregulating IL-6 and TNF-α secretion. CONCLUSIONS: Our results suggest a role for sebaceous glands in modulating innate immune responses via their secreted lipids that are of possible pathological and therapeutic relevance.

Newly acquired kiwi fruit allergy after bone marrow transplantation from a kiwi-allergic donor.

BACKGROUND: The phenomenon of allergy transfer from an allergic donor to a non-allergic recipient via hematopoietic cell transplantation has been described by several reports. However, it could not yet been conclusively shown that allergic reaction of the recipient is elicited by the donor's cells. OBJECTIVES: In the case of a 46-year-old male patient who - for the first time in his life - had two episodes of oral allergic syndrome upon kiwi consumption after having received myeloablative hematopoietic stem cell transplantation (HCT) from his kiwi-allergic sister, we aimed to clarify the origin of allergen reactive cells in the donor. We not only intended to demonstrate if allergy was transferred by HCT but also to present an experimental workup for the analysis of allergy transfer by HCT. METHODS: Allergic sensitization to kiwi in recipient and donor was proven by ImmunoCAP. Furthermore, origin of peripheral blood mononuclear cells (PBMCs) was analyzed by chromosomal fluorescence in situ hybridization (FISH). To confirm allergic reaction and activation of hematopoietic cells by customized kiwi extract, we performed basophil activation test from whole blood as well as T cell proliferation assays from purified PBMCs of both recipient and donor. RESULTS: Basophil activation upon kiwi extract was demonstrated in both recipient and donor. Besides, we showed proliferation of CD4(+) T cells after incubation with kiwi extract. FISH analysis proved that hematopoietic cells of the male recipient completely originated from the female donor. CONCLUSION: Exemplified in this patient, we show for the first time that allergy transfer is mediated by the donor's cells. Moreover, our experimental approach using customized kiwi extract to prove contribution of kiwi-specific T and B cells in both kiwi-allergic recipient and donor could serve as a model approach for future studies.

Dissecting susceptibility from exogenous triggers: the model of alopecia areata and associated inflammatory skin diseases.

BACKGROUND: Alopecia areata (AA) is a T-cell-driven autoimmune disease of the hair follicle and frequently reported to be associated with inflammatory skin diseases (ISD) such as atopic eczema (AE) or psoriasis. Interestingly, AA on the one hand and both AE and psoriasis on the other hand are believed to be driven by mutually antagonistic T-cell subsets. OBJECTIVE: To characterize AA-specific T-cell profiles and inflammatory pattern by intra-individual comparison of AA and coexistent ISD. METHODS: 112 patients with AA were recruited and investigated for coexisting ISD. In-depth analyses were performed in patients with AA and AE (n = 2), AA and psoriasis (n = 1), AA and psoriasis and AE (n = 1) and AA and lichen planus (n = 1), using histology, immunohistochemistry and cytokine staining of T cells isolated from lesional skin. RESULTS: Of 112 AA patients investigated, 23 suffered from an ISD. The prevalence of AE, vitiligo, psoriasis and lichen planus was higher in the investigated AA cohort than in the normal population. The clinical as well as histological phenotype of AA the coexistent ISD were unequivocal. In line with this, T-cell infiltrates were found to be disease-characteristics with AA and lichen planus dominated by CD8+ and IFN-γ+ TNF-α+ producing T cells while psoriasis lesions in the same patients were dominated by IL-17+ and AE by IL-4+ T cells. CONCLUSION: AA patients have a higher incidence of various T-cell-driven inflammatory skin diseases than the normal population, a phenomenon which might relate to over-activation of skin-homing T cells and to specific immune triggers as the primary cause of inflammation. More importantly, we showed that by using AA as a model disease, our approach of intra-individual comparison of distinct inflammatory responses in the same patient is feasible and offers the unique possibility to gain insights into disease pathogenesis independent from genetic susceptibilities.

A common atopy-associated variant in the Th2 cytokine locus control region impacts transcriptional regulation and alters SMAD3 and SP1 binding.

BACKGROUND: Type 2 immune responses directed by Th2 cells and characterized by the signature cytokines IL4, IL5, and IL13 play major pathogenic roles in atopic diseases. Single nucleotide polymorphisms in the human Th2 cytokine locus in particular in a locus control region within the DNA repair gene RAD50, containing several RAD50 DNase1-hypersensitive sites (RHS), have been robustly associated with atopic traits in genome-wide association studies (GWAS). Functional variants in IL13 have been intensely studied, whereas no causative variants for the IL13-independent RAD50 signal have been identified yet. This study aimed to characterize the functional impact of the atopy-associated polymorphism rs2240032 located in the human RHS7 on cis-regulatory activity and differential binding of transcription factors. METHODS: Differential transcription factor binding was analyzed by electrophoretic mobility shift assays (EMSAs) with Jurkat T-cell nuclear extracts. Identification of differentially binding factors was performed using mass spectrometry (LC-MS/MS). Reporter vector constructs carrying either the major or minor allele of rs2240032 were tested for regulating transcriptional activity in Jurkat and HeLa cells. RESULTS: The variant rs2240032 impacts transcriptional activity and allele-specific binding of SMAD3, SP1, and additional putative protein complex partners. We further demonstrate that rs2240032 is located in an RHS7 subunit which itself encompasses repressor activity and might be important for the fine-tuning of transcription regulation within this region. CONCLUSION: The human RHS7 critically contributes to the regulation of gene transcription, and the common atopy-associated polymorphism rs2240032 impacts transcriptional activity and transcription factor binding.

Spatial transcriptomics landscape of lesions from non-communicable inflammatory skin diseases.

Abundant heterogeneous immune cells infiltrate lesions in chronic inflammatory diseases and characterization of these cells is needed to distinguish disease-promoting from bystander immune cells. Here, we investigate the landscape of non-communicable inflammatory skin diseases (ncISD) by spatial transcriptomics resulting in a large repository of 62,000 spatially defined human cutaneous transcriptomes from 31 patients. Despite the expected immune cell infiltration, we observe rather low numbers of pathogenic disease promoting cytokine transcripts (IFNG, IL13 and IL17A), i.e. >125 times less compared to the mean expression of all other genes over lesional skin sections. Nevertheless, cytokine expression is limited to lesional skin and presented in a disease-specific pattern. Leveraging a density-based spatial clustering method, we identify specific responder gene signatures in direct proximity of cytokines, and confirm that detected cytokine transcripts initiate amplification cascades of up to thousands of specific responder transcripts forming localized epidermal clusters. Thus, within the abundant and heterogeneous infiltrates of ncISD, only a low number of cytokine transcripts and their translated proteins promote disease by initiating an inflammatory amplification cascade in their local microenvironment.

Novel key cytokines in allergy: IL-17, IL-22.

The biology of the T cell cytokines Interleukin (IL-)17 and IL-22 has been a main focus in the field of clinical immunology in the last decade. This intensive interest in both cytokines has resulted in almost 5,000 scientific publications (www.pubmed.com) dealing with the molecular structure, extra- and intracellular signaling pathways, specific transcription factors and the function of IL-17 and IL-22. This review article highlights the main findings concerning IL-17 and IL-22 in the last years.