The extracellular serine protease from Staphylococcus epidermidis elicits a type 2-biased immune response in atopic dermatitis patients.

Background: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with skin barrier defects and a misdirected type 2 immune response against harmless antigens. The skin microbiome in AD is characterized by a reduction in microbial diversity with a dominance of staphylococci, including Staphylococcus epidermidis (S. epidermidis). Objective: To assess whether S. epidermidis antigens play a role in AD, we screened for candidate allergens and studied the T cell and humoral immune response against the extracellular serine protease (Esp). Methods: To identify candidate allergens, we analyzed the binding of human serum IgG4, as a surrogate of IgE, to S. epidermidis extracellular proteins using 2-dimensional immunoblotting and mass spectrometry. We then measured serum IgE and IgG1 binding to recombinant Esp by ELISA in healthy and AD individuals. We also stimulated T cells from AD patients and control subjects with Esp and measured the secreted cytokines. Finally, we analyzed the proteolytic activity of Esp against IL-33 and determined the cleavage sites by mass spectrometry. Results: We identified Esp as the dominant candidate allergen of S. epidermidis. Esp-specific IgE was present in human serum; AD patients had higher concentrations than controls. T cells reacting to Esp were detectable in both AD patients and healthy controls. The T cell response in healthy adults was characterized by IL-17, IL-22, IFN-γ, and IL-10, whereas the AD patients' T cells lacked IL-17 production and released only low amounts of IL-22, IFN-γ, and IL-10. In contrast, Th2 cytokine release was higher in T cells from AD patients than from healthy controls. Mature Esp cleaved and activated the alarmin IL-33. Conclusion: The extracellular serine protease Esp of S. epidermidis can activate IL-33. As an antigen, Esp elicits a type 2-biased antibody and T cell response in AD patients. This suggests that S. epidermidis can aggravate AD through the allergenic properties of Esp.

A comprehensive genetic map of cytokine responses in Lyme borreliosis.

The incidence of Lyme borreliosis has risen, accompanied by persistent symptoms. The innate immune system and related cytokines are crucial in the host response and symptom development. We characterized cytokine production capacity before and after antibiotic treatment in 1,060 Lyme borreliosis patients. We observed a negative correlation between antibody production and IL-10 responses, as well as increased IL-1Ra responses in patients with disseminated disease. Genome-wide mapping the cytokine production allowed us to identify 34 cytokine quantitative trait loci (cQTLs), with 31 novel ones. We pinpointed the causal variant at the TLR1-6-10 locus and validated the regulation of IL-1Ra responses at transcritpome level using an independent cohort. We found that cQTLs contribute to Lyme borreliosis susceptibility and are relevant to other immune-mediated diseases. Our findings improve the understanding of cytokine responses in Lyme borreliosis and provide a genetic map of immune function as an expanded resource.

Inhibition of IL-17 ameliorates keratinocyte-borne cytokine responses in an in vitro model for house-dust-mite triggered atopic dermatitis.

A subgroup of patients suffering from atopic dermatitis (AD) does not respond to biologics therapy targeting the key players of type-2 inflammation, and it is an ongoing discussion whether skin-infiltrating Th17 cells may underlie this phenomenon. This study aimed to investigate the potential of allergen-induced, immune-cell derived IL-17 on the induction of inflammatory processes in keratinocytes. Peripheral blood mononuclear cells derived from respectively sensitized AD patients were stimulated with house dust mite (HDM) extract and cell culture supernatants were applied subsequently in absence or presence of secukinumab to primary human keratinocytes. Hereby we confirm that the immune response of sensitized AD patients to HDM contains aside from type-2 cytokines significant amounts of IL-17. Blocking IL-17 efficiently reduced the stimulation-induced changes in keratinocyte gene expression. IL-17-dependent transcriptional changes included increased expression of the cytokines IL-20 and IL-24 as well as Suppressor of Cytokine Siganling 3 (SOCS3), a negative feedback-regulator of the STAT3/IL-17/IL-24 immune response. We conclude that the immune response to HDM can induce pro-inflammatory cytokines from keratinocytes in AD, which in part is mediated via IL-17. Targeting IL-17 may turn out to be a reasonable alternative therapy in a subgroup of patients with moderate to severe AD and HDM sensitization.

Dupilumab strengthens herpes simplex virus type 1-specific immune responses in atopic dermatitis.

BACKGROUND: Impaired virus clearance in a subgroup of atopic dermatitis (AD) patients can lead to severe herpes simplex virus (HSV) infections called eczema herpeticum (EH). We recently identified a type 2 skewed viral immune response in EH patients. Clinical data suggest a reduced incidence of EH in AD patients treated with dupilumab, although immunologic investigations of this phenomenon are still lacking. OBJECTIVE: We examined the impact of dupilumab on the HSV type 1 (HSV-1) specific immune response in AD, focusing on patients with (ADEH+) and without (ADEH-) a history of EH. METHODS: Sera and peripheral blood mononuclear cells were collected from ADEH+ and ADEH- patients, a subgroup of whom was receiving dupilumab treatment, and healthy controls. Serum samples were tested for IgE against HSV-1 glycoprotein D (n = 85). Peripheral blood mononuclear cells were stimulated with HSV peptides, and activated CD4+ and CD8+ cells were characterized by flow cytometry after magnetic enrichment via CD154 or CD137 (n = 60). Cytokine production of HSV-1-reactive T-cell lines (n = 33) and MHC-I tetramer+ (HSV-1-UL25) CD8+ T cells was investigated by bead assay and intracellular cytokine staining (n = 21). RESULTS: We confirmed that HSV-1-specific IgE is elevated in ADEH+ patients. During dupilumab treatment, the IgE levels were significantly decreased, reaching levels of healthy controls. HSV-1-specific TC1 frequencies were elevated in ADEH- patients treated with dupilumab compared to dupilumab-negative patients. There were no changes in the frequencies of HSV-1-specific TH cells while receiving dupilumab therapy. AD patients receiving dupilumab exhibited elevated IFN-γ and reduced IL-4 production in HSV-1-UL25-epitope-specific T cells compared to dupilumab-negative patients. CONCLUSION: Dupilumab may improve the HSV-1-specific immune response in AD as a result of an increased type I immune response and a reduction of HSV-1-specific IgE.

Differentiation of IL-26+ TH17 intermediates into IL-17A producers via epithelial crosstalk in psoriasis.

Interleukin (IL)-26 is a TH17 cytokine with known antimicrobial and pro-inflammatory functions. However, the precise role of IL-26 in the context of pathogenic TH17 responses is unknown. Here we identify a population of blood TH17 intermediates that produce high levels of IL-26 and differentiate into IL-17A-producing TH17 cells upon TGF-ß1 exposure. By combining single cell RNA sequencing, TCR sequencing and spatial transcriptomics we show that this process occurs in psoriatic skin. In fact, IL-26+ TH17 intermediates infiltrating psoriatic skin induce TGF-ß1 expression in basal keratinocytes and thereby promote their own differentiation into IL-17A-producing cells. Thus, our study identifies IL-26-producing cells as an early differentiation stage of TH17 cells that infiltrates psoriatic skin and controls its own maturation into IL17A-producing TH17 cells, via epithelial crosstalk involving paracrine production of TGF-ß1.

Specific IgE against the house dust mite allergens Der p 5, 20 and 21 influences the phenotype and severity of atopic diseases.

BACKGROUND: House dust mites (HDM) are among the most important sources for airborne allergens with high relevance for atopic diseases. Routine tests contain only 4 of 32 registered allergens of Dermatophagoides pteronyssinus. Clinical relevance and pathomechanistic properties of many allergens are not well understood. OBJECTIVE: The association of several HDM allergens with allergic rhinitis, allergic asthma, and atopic dermatitis was investigated to identify allergens with biomarker potential and to transfer them into diagnostics. METHODS: Eight out of nine D. pteronyssinus allergens (nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 10, rDer p 13, rDer p 20, rDer p 21, rDer p 23) were recombinantly expressed and purified. Sensitization patterns of 384 HDM-allergic individuals exhibiting different clinical phenotypes were analyzed with a serum-saving multiplex array. RESULTS: Sensitization to more than three mite allergens (sensitization count) was associated with allergic asthma and/or atopic dermatitis. Reactions to Der p 5 and Der p 21 were more frequent in allergic asthma compared to allergic rhinitis. Atopic dermatitis patients were more often sensitized to Der p 5, Der p 20, and Der p 21 among others. Der p 20-IgE > 80 kU/L was associated with severe atopic dermatitis in 75% of patients. CONCLUSION: This study demonstrates the clinical importance of the sensitization count and of certain allergens (Der p 5, Der p 20, and Der p 21) not available for routine diagnostics yet. Implementing them as well as the sensitization count in diagnostic measures will improve diagnosis and risk assessment of HDM-allergic patients.

Single-cell profiles reveal distinctive immune response in atopic dermatitis in contrast to psoriasis.

BACKGROUND: Understanding the complex orchestrated inflammation in atopic dermatitis (AD), one of the most common chronic inflammatory diseases worldwide, is essential for therapeutic approaches. However, a comparative analysis on the single-cell level of the inflammation signatures correlated with the severity is missing so far. METHODS: We applied single-cell RNA and T-cell receptor (TCR) sequencing on immune cells enriched from skin biopsies and matched blood samples of AD in comparison with psoriasis (PS) patients. RESULTS: Clonally propagated skin-derived T cells showed disease-specific TCR motifs shared between patients which was more pronounced in PS compared to AD. The disease-specific T-cell clusters were mostly of a Th2/Th22 sub-population in AD and Th17/Tc17 in PS, and their numbers were associated with severity scores in both diseases. Herein, we provide for the first time a list that associates cell type-specific gene expression with the severity of the two most common chronic inflammatory skin diseases. Investigating the cell signatures in the patients´ PBMCs and skin stromal cells, a systemic involvement of type-3 inflammation was clearly detectable in PS circulating cells, while in AD inflammatory signatures were most pronounced in fibroblasts, pericytes, and keratinocytes. Compositional and functional analyses of myeloid cells revealed the activation of antiviral responses in macrophages in association with disease severity in both diseases. CONCLUSION: Different disease-driving cell types and subtypes which contribute to the hallmarks of type-2 and type-3 inflammatory signatures and are associated with disease activities could be identified by single-cell RNA-seq and TCR-seq in AD and PS.

T-cell receptor sequencing specifies psoriasis as a systemic and atopic dermatitis as a skin-focused, allergen-driven disease.

BACKGROUND: Atopic dermatitis (AD) and psoriasis represent two of the most common inflammatory skin diseases in developed countries. A hallmark of both diseases is T-cell infiltration into the skin. However, it is still not clarified to what extent these infiltrating T cells are antigen-specific skin-homing T cells or unspecific heterogeneous bystander cells. METHODS: To elucidate this, T cells from lesional skin and from blood of 9 AD and 10 psoriasis patients were compared by receptor (TCR) sequencing. Therefore, peripheral blood mononuclear cells (PBMC) were cell-sorted according to expression of the cutaneous leukocyte antigen (CLA) into skin-homing (CLA+ ) and non-skin-homing (CLA- ) subfractions. Aeroallergen-specific T-cell lines were grown from AD patients' PBMC in parallel. RESULTS: Intra-individual comparison of TCRB CDR3 regions revealed that clonally expanded T cells in skin lesions of both AD and psoriasis patients corresponded to skin-homing circulating T cells. However, in psoriasis patients, these T-cell clones were also detectable to a larger extent among CLA- circulating T cells. Up to 28% of infiltrating cells in AD skin were identified as allergen-specific by overlapping TCR sequences. CONCLUSIONS: Our data show that in line with the systemic nature of psoriasis, T-cell clones that infiltrate psoriatic skin lesions do not exclusively possess skin-homing ability and are therefore most probably specific to antigens that are not exclusively expressed or located in the skin. T cells driving AD skin inflammation appear to home nearly exclusively to the skin and are, to a certain extent, specific to aeroallergens.

Congenital deficiency reveals critical role of ISG15 in skin homeostasis.

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-ß1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.

Specific T cells targeting Staphylococcus aureus fibronectin-binding protein 1 induce a type 2/type 1 inflammatory response in sensitized atopic dermatitis patients.

BACKGROUND: Atopic dermatitis (AD) is one of the most common inflammatory skin diseases worldwide and Staphylococcus aureus colonization and secondary infections occur in the majority of AD patients. Allergic sensitizations against microbial antigens have been discussed as possible trigger factors of AD. Recently, we reported IgE sensitization against fibronectin-binding protein 1 (FBP1), an essential virulence component in S. aureus, in a subgroup of patients suffering from AD. To expand these findings by investigating delayed-type immune reactions, the objective of this study was to detect and phenotypically characterize FBP1-specific T cells as possible trigger factors in AD. METHODS: Immunodominant T-cell epitopes were mapped by proliferation testing of patient-derived FBP1-specific T-cell lines after stimulation with single 15mer peptides, which were derived from different functional domains of the FBP1 sequence. Major histocompatibility complex class II tetramers carrying immunodominant epitopes successfully stained T helper cells in 8 out of 8 HLA-matched, IgE-sensitized AD patients. RESULTS: Cytokine profiling of multimer-sorted cells revealed that predominantly the type 2 cytokines IL-13 and IL-4 were secreted by these cells. In contrast, IL-17, the marker cytokine for response to extracellular pathogens, was scarcely detectable. CONCLUSIONS: We demonstrate that FBP1 contains immunodominant peptides that induce a specific pro-inflammatory T helper cell response with increased Th2 levels that can drive an allergic inflammation in sensitized AD patients.

Barrier defect in atopic dermatitis - possibilities and limits of basic skin therapy.

The increased permeability of the skin barrier towards environmental factors such as allergens is considered a key factor in the pathogenesis of atopic dermatitis (AD). Strengthening the skin barrier through basic skin therapy represents the basis of any therapy for AD. It is well known that genetic factors as well as the skin inflammation itself contribute to the weakening of the barrier; here, recent studies have led to a deeper understanding of the complex structures of the epidermis. The possibility of counteracting the disease preventively by the use of basic skin therapy from birth on has been studied intensively in recent years. This article summarizes recent findings on the effects of basic skin therapy as a primary and secondary preventive measure.

Dupilumab: An Opportunity to Unravel In Vivo Actions of IL-4 and IL-13 in Humans.

The application of biologics in clinical practice allows immunological observations under real-life conditions. In a new article in the Journal of Investigative Dermatology, Bakker et al. (2021) use deep immune cell phenotyping to demonstrate how dupilumab acts in a targeted fashion on skin-homing T cells, the driver cells of atopic dermatitis.

Eczema herpeticum in atopic dermatitis.

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases leading to pruritic skin lesions. A subset of AD patients exhibits a disseminated severe HSV infection called eczema herpeticum (EH) that can cause life-threatening complications. This review gives an overview of the clinical picture, and characteristics of the patients as well as the diagnosis and therapy of EH. A special focus lies on the pathophysiological hallmarks identified so far that predispose for EH. This aspect covers genetic aberrations, immunological changes, and environmental influences displaying a complex multifactorial situation, which is not completely understood. Type 2 skewing of virus-specific T cells in ADEH+ patients has been implicated in immune profile abnormalities, along with impaired functions of dendritic cells and natural killer cells. Furthermore, aberrations in interferon pathway-related genes such as IFNG and IFNGR1 have been identified to increase the risk of EH. IL-4, IL-25, and thymic stromal lymphopoietin (TSLP) are overexpressed in EH, whereas antimicrobial peptides like human ß-defensins and LL-37 are reduced. Concerning the epidermal barrier, single nucleotide polymorphisms (SNPs) in skin barrier proteins such as filaggrin were identified in ADEH+ patients. A dysbalance of the skin microbiome also contributes to EH due to an increase of Staphylococcus aureus, which provides a supporting role to the viral infection via secreted toxins such as α-toxin. The risk of EH is reduced in AD patients treated with dupilumab. Further research is needed to identify and specifically target risk factors for EH in AD patients.

Immunotyping of clinically divergent p.Phe508del homozygous monozygous cystic fibrosis twins.

Blood of the three clinically most concordant and most discordant p.Phe508del homozygous monozygous twin pairs of the European Cystic Fibrosis Twin and Sibling Study was examined in two postzygotic attributes that generate diversity between monozygous twins, i.e. the repertoire of the CDR3 region of the T-cell receptor ß chains and the DNA methylation at 450,000 genomic CpG sites. Methylation patterns in peripheral blood of twins changed at selected cell-type-independent positions and the immune cells of the twins showed individual profiles of the T cell receptor repertoire reflecting the plasticity of the immune system of genetically identical humans with cystic fibrosis to cope with the environment.

The clinical impact of cross-reactions between allergens on allergic skin diseases.

PURPOSE OF REVIEW: The route of allergen sensing via the skin appears to influence the immune system towards mounting a type 2 response, especially in genetically predisposed individuals. Allergens recognized this way may derive from microbial, animal, food, or other plant sources and trigger atopic dermatitis. Allergens can be grouped into families depending on their structure and function, harboring significant structural and sequence similarities. Cross-reactivity between allergens is believed to arise as a consequence, and to underlie the development of further atopic diseases. RECENT FINDINGS: Especially for the plant allergens of the families of PR10-related proteins and profilins, immune cross-reactions have been described. Actual studies support that food and pollen allergens can aggravate skin lesions in patients suffering from atopic dermatitis. Further on, allergens derived from air-borne or skin-borne fungi belong to common allergen families and bear cross-reactivity potential. Cross-reactivity to human homologous proteins, so-called autoallergens, is discussed to contribute to the chronification of atopic dermatitis. SUMMARY: Due to high evolutionary conservation, allergic reactions can be triggered by highly homologous members of allergen families on the humoral as well as on the cellular level.

Autoimmunity (or Not) in Atopic Dermatitis.

Atopic dermatitis (AD), one of the most frequent inflammatory skin diseases worldwide, is believed to result from a disturbed skin barrier as well as aberrant immune reactions against per se harmless allergens. Starting mostly during childhood with a chronic, remitting relapsing course, the disease can persist into adulthood in about one fifth of patients. Immune reactions to self-proteins have been observed in AD patients already in the beginning of the Twentieth century, when human cellular extracts were shown to provoke skin lesions. However, the term "autoimmunity" has never been claimed, since AD is first and foremost an atopic disease. In contrast, this IgE-hallmarked autoreactivity was termed "autoallergy" and is ongoing discussed regarding its impact on the disease. Since severely affected patients tend to develop IgE-hypersensitivity reactions to numerous environmental allergens, the impact of immune responses to self-proteins is difficult to determine. On the other hand: any autoreactivity, irrespective of the magnitude, implicates the potential of driving the chronification of the disease while shaping the immune response. This review article revisits the observations made on autoallergy from an actual point of view and tries to approach the question whether these still point to a contribution to the disease.

Common and different roles of IL-4 and IL-13 in skin allergy and clinical implications.

PURPOSE OF REVIEW: This review summarizes the mode of action of IL-4 and IL-13 in skin allergy, upcoming therapeutics and depicts key outcomes of the latest clinical trials. RECENT FINDINGS: Atopic dermatitis is considered to be one of the most common inflammatory skin disease in industrialized countries. Accompanied by strong pruritus, atopic dermatitis has a significant impact on quality of life in severely affected individuals. Aside from unspecific immunosuppressant medications, therapeutics targeting the key cytokines IL-4 and IL-13 and their downstream mediators are under development or have been approved just recently with outstanding potential. SUMMARY: The recent development of several biologics and small compounds has the potential to revolutionize the treatment of atopic dermatitis, and applying this set of state-of-the-art drugs will provide a unique chance to gain insights into this skin disorder, patient subgroups, and key inflammatory mediators.