Development and validation of serological immunoassays in laminin γ-1 pemphigoid and epidermolysis bullosa acquisita

Pemphigoid diseases encompass a heterogeneous group of subepidermal autoimmune bullous diseases of the skin and mucosa. These disorders are characterized by autoantibodies that target distinct structural proteins of the basement membrane zone which are crucial for the integrity of the skin. Targeting of these proteins leads to skin fragility, detachment of the epidermis and eventually formation of blisters and skin inflammation. The clinical presentation of the pemphigoids is heterogeneous, the subtypes are divided by clinical symptoms, target antigens and response to treatment. A crucial step for the proper classification is the serological detection of the autoantigens directed against distinct components of the dermal epidermal junction. Thus, it is crucial to develop reliable and reproducible diagnostic methods which help clinicians to differentiate between different entities and therefore to select the best treatment. This thesis is focused on Laminin γ-1 pemphigoid and epidermolysis bullosa acquisita, the study of their clinical appearance and the development and validation of serological assays to correctly diagnose these two entities. Both autoimmune blistering diseases are rare and often hard to diagnose with standard serological assays. In the first study presented in this thesis, I discuss the development of an immunoblot assay for the detection of autoantibodies against Laminin γ-1. Laminin γ-1 pemphigoid is a novel subepidermal blistering disease which has been first described in 1996. Since then, different immune serological methods for the detection of circulating autoantibodies have been described, however these are often not reproducible for most of the centers worldwide, since often advanced lab skills are required. In our study, we present a novel immunoblot assay based on three commercially available Laminin γ-1 recombinants, which can be reproduced in almost every laboratory with basic skills. In the second study, we thought to serologically identify for the best immunoassay for the detection of serum IgG autoantibodies against anti-human collagen VII in patients with epidermolysis bullosa acquisita. Epidermolysis bullosa acquisita is a severe blistering diseases characterized by circulating IgG autoantibodies targeting human collagen VII. In this study, four different diagnostic assays (immunoblot, indirect immunofluorescence with saline split human skin and 2 enzyme linked immunosorbent assays) have been tested and compared with regard to their sensitivity and specificity in a large cohort of epidermolysis bullosa acquisita sera (n=95). In summary, our study showed that enzyme linked immunosorbent assays based on recombinant human collagen VII NC1-NC2 or collagen VII-NC1 proteins provide the highest sensitivity and specificity for the detection of anticollagen VII IgG and should be preferred to immunoblot analysis or indirect immunofluorescence on saline split human skin in making the diagnosis of epidermolysis bullosa acquisita

Emerging Topical and Systemic JAK Inhibitors in Dermatology

Accumulating data on cellular and molecular pathways help to develop novel therapeutic strategies in skin inflammation and autoimmunity. Examples are psoriasis and atopic dermatitis, two clinically and immunologically well-defined disorders. Here, the elucidation of key pathogenic factors such as IL-17A/IL-23 on the one hand and IL-4/IL-13 on the other hand profoundly changed our therapeutic practice. The knowledge on intracellular pathways and governing factors is shifting our attention to new druggable molecules. Multiple cytokine receptors signal through Janus kinases (JAKs) and associated signal transducer and activators of transcription (STATs). Inhibition of JAKs can simultaneously block the function of multiple cytokines. Therefore, JAK inhibitors (JAKi) are emerging as a new class of drugs, which in dermatology can either be used systemically as oral drugs or locally in topical formulations. Inhibition of JAKs has been shown to be effective in various skin disorders. The first oral JAKi have been recently approved for the treatment of rheumatoid arthritis and psoriatic arthritis. Currently, multiple inhibitors of the JAK/STAT pathway are being investigated for skin diseases like alopecia areata, atopic dermatitis, dermatomyositis, graft-versus-host-disease, hidradenitis suppurativa, lichen planus, lupus erythematosus, psoriasis, and vitiligo. Here, we aim to discuss the immunological basis and current stage of development of JAKi in dermatology

Janus kinase signaling as risk factor and therapeutic target for severe SARS‐CoV‐2 infection

Cytokine signaling, especially interferon (IFN) signaling is closely linked to several aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. During initial SARS-CoV-2 infection, symptomatic patients present with impaired type I/III IFN-mediated antiviral responses. Interestingly, IFNs regulate the cellular entry receptor for SARS-CoV-2 on epithelial and endothelial cells. As reported recently, critically ill COVID-19 patients show genetic polymorphisms in one IFN receptor gene (IFNRA2) and in a gene locus near the Janus kinase (JAK) TYK2, which is key for IFN, interleukin (IL)-12 and IL-23 signaling, and T helper (Th) 1/Th17 cell-mediated antiviral immune responses. In the advanced stage of the disease, critically ill COVID-19 patients develop a cytokine storm where many inflammatory mediators using the JAK/STAT signaling pathway such as IL-6, IFN-gamma, the granulocyte colony-stimulating factor (G-CSF) or IL-2, and chemokines result in an influx of macrophages and neutrophils damaging the lung tissue. The knowledge on the cytokine and JAK/STAT signaling pathways in severe COVID-19 disease explains the promising first results with JAK inhibitors like baricitinib, which not only dampen the inflammation but in the case of baricitinib also affect virus replication and endocytosis in target cells. Here, we summarize the current immunological associations of SARS-CoV-2 infection with cytokine signaling, the JAK/STAT pathway, and the current clinical stage of JAK inhibitors for improving severe COVID-19 disease

Case Report: Apremilast for Therapy-Resistant Pemphigus Vulgaris

Background: In pemphigus, elucidating the disease-causing immune mechanism and developing new therapeutic strategies are needed. In this context, the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is gaining attention. cAMP is important in hematological and auto-inflammatory disorders. A class of enzymes called phosphodiesterases (PDEs) control intracellular cAMP levels. In pemphigus, cAMP levels increase following IgG binding to Dsg3. This appears to be a mechanism to preserve epithelial integrity. Objectives: To determine whether apremilast, an inhibitor of the PDE4 normally used in psoriasis, may be of benefit in the blistering skin disorder pemphigus. Methods: Here we report of a 62 years old patient with chronic debilitating and recalcitrant pemphigus not responding to several previous treatments, who received treatment with apremilast over a period of 32 weeks. Desmoglein autoantibody levels were assessed by Enzyme-linked Immunosorbent Assay (ELISA), whereas disease severity and quality of life were assessed by the Autoimmune Bullous Skin Disorder Intensity Score (ABSIS). In an attempt to explain the effects of apremilast in pemphigus, peripheral blood mononuclear cells (PBMCs) were analyzed for the duration of treatment by flow cytometry for the distribution of specialized T cell subsets. The frequencies of circulating T helper (Th) 1, Th2, Th17, Th17.1 and T follicular helper (Tfh) 1, Tfh2, Tfh17, and Tfh17.1 were analyzed by CCR6, CXCR3, and CXCR5 expression of CD4+ T cells. Further, based on the different expressions of CXCR5, CD127, and CD25, we analyzed the T regulatory (Treg) and T follicular regulatory (Tfreg) compartment. Results: In response to apremilast treatment, Dsg-specific autoantibody titers decreased, blistering ceased and lesions healed, showing a long-lasting effect. While the frequencies of most of the Th and Tfh cell subsets remained unchanged, we observed a continuous increase in Treg and Tfreg cell levels. Conclusion: Our findings are encouraging and warrant extension of the beneficial effect of PDE4 inhibition on a larger cohort of pemphigus patients

Case Report: Apremilast for Therapy-Resistant Pemphigus Vulgaris

Background: In pemphigus, elucidating the disease-causing immune mechanism and developing new therapeutic strategies are needed. In this context, the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is gaining attention. cAMP is important in hematological and auto-inflammatory disorders. A class of enzymes called phosphodiesterases (PDEs) control intracellular cAMP levels. In pemphigus, cAMP levels increase following IgG binding to Dsg3. This appears to be a mechanism to preserve epithelial integrity. Objectives: To determine whether apremilast, an inhibitor of the PDE4 normally used in psoriasis, may be of benefit in the blistering skin disorder pemphigus. Methods: Here we report of a 62 years old patient with chronic debilitating and recalcitrant pemphigus not responding to several previous treatments, who received treatment with apremilast over a period of 32 weeks. Desmoglein autoantibody levels were assessed by Enzyme-linked Immunosorbent Assay (ELISA), whereas disease severity and quality of life were assessed by the Autoimmune Bullous Skin Disorder Intensity Score (ABSIS). In an attempt to explain the effects of apremilast in pemphigus, peripheral blood mononuclear cells (PBMCs) were analyzed for the duration of treatment by flow cytometry for the distribution of specialized T cell subsets. The frequencies of circulating T helper (Th) 1, Th2, Th17, Th17.1 and T follicular helper (Tfh) 1, Tfh2, Tfh17, and Tfh17.1 were analyzed by CCR6, CXCR3, and CXCR5 expression of CD4(+) T cells. Further, based on the different expressions of CXCR5, CD127, and CD25, we analyzed the T regulatory (Treg) and T follicular regulatory (Tfreg) compartment. Results: In response to apremilast treatment, Dsg-specific autoantibody titers decreased, blistering ceased and lesions healed, showing a long-lasting effect. While the frequencies of most of the Th and Tfh cell subsets remained unchanged, we observed a continuous increase in Treg and Tfreg cell levels. Conclusion: Our findings are encouraging and warrant extension of the beneficial effect of PDE4 inhibition on a larger cohort of pemphigus patients

Lichen planus – a clinical guide

Lichen planus (LP) is a chronic lichenoid inflammatory disorder of the skin, mucosa and of the appendages. LP is classically characterized by the presence of a rich infiltration of inflammatory T cells, which migrate in the upper part of the dermis, arranged in a band-like pattern. Different sub types of the disease have been so far described. Albeit LP is clinically well defined, the disease still represents a therapeutic enigma. Especially with regard to mucosal or scalp affecting LP types, which often present a recalcitrant and treatment unresponsive course, efficacious therapeutic options are still lacking. Thus, LP represents a disease with a high psychosocial burden. Yet, development in the deciphering of LP pathogenesis reveals possible new druggable targets, thus paving the way for future therapeutic options. In this clinical guide, we summarize the current clinical knowledge and therapeutic standards and discuss the future perspective for the management of LP

Pemphigus Foliaceus Autoantibodies Induce Redistribution Primarily of Extradesmosomal Desmoglein 1 in the Cell Membrane

The autoimmune dermatosis pemphigus foliaceus (PF) is predominantly caused by IgG autoantibodies against the desmosomal cadherin desmoglein (Dsg) 1. The exact mechanisms that lead to the characteristic epidermal blistering are not yet fully understood. In the present study, we used a variety of biophysical methods to examine the fate of membrane-bound Dsg1 after incubation with PF patients' IgG. Dispase-based dissociation assays confirmed that PF-IgG used for this study reduced intercellular adhesion in a manner dependent on phospholipase C (PLC)/Ca2+ and extracellular signal-regulated kinase (ERK) 1/2 signaling. Atomic force microscopy (AFM) revealed that Dsg1 binding on single molecule level paralleled effects on keratinocyte adhesion under the different conditions. Stimulated emission depletion (STED) super-resolution microscopy was used to investigate the localization of Dsg1 after PF-IgG incubation for 24 h. Under control conditions, Dsg1 was found to be in part co-localized with desmoplakin and thus inside of desmosomes as well as extra-desmosomal along the cell border. Incubation with PF-IgG reduced the extra-desmosomal Dsg1 fraction. In line with this, fluorescence recovery after photobleaching (FRAP) experiments demonstrated a strongly reduced mobility of Dsg1 in the cell membrane after PF-IgG treatment indicating remaining Dsg1 molecules were primarily located inside desmosomes. Mechanistically, experiments confirmed the involvement of PLC/Ca2+ since inhibition of PLC or 1,4,5-trisphosphate (IP3) receptor to reduce cytosolic Ca2+ reverted the effects of PF-IgG on Dsg1 intra-membrane mobility and localization. Taken together, our findings suggest that during the first 24 h PF-IgG induce redistribution predominantly of membrane-bound extradesmosomal Dsg1 in a PLC/Ca2+ dependent manner whereas Dsg1-containing desmosomes remain

Arginase 1+ IL-10+ polymorphonuclear myeloid-derived suppressor cells are elevated in patients with active pemphigus and correlate with an increased Th2/Th1 response

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which are characterized by their capability to suppress T-cell responses. While MDSCs have been traditionally associated with cancer diseases, their role as regulators of autoimmune diseases is emerging. Pemphigus is a chronic autoimmune blistering skin disease characterized by dysregulated T-cell responses and autoantibody production. The role of MDSCs in pemphigus disease has not been defined yet. The aim of this study was to characterize MDSCs in pemphigus patients and to dissect their relationship with CD4(+) T-cell subsets and clinical disease assessments. For this purpose, we performed a cross-sectional analysis of 20 patients with pemphigus. Our results indicate that a population of CD66b(+)CD11b(+) polymorphonuclear-like MDSCs (PMN-MDSCs) is expanded in the peripheral blood mononuclear cell fraction of pemphigus patients compared to age-matched healthy donors. These PMN-MDSCs have the capability of suppressing allogeneic T-cell proliferation in vitro and show increased expression of characteristic effector molecules such as arginase I and interleukin-10. We further demonstrate that PMN-MDSCs are especially expanded in patients with active pemphigus, but not in patients in remission. Moreover, MDSC frequencies correlate with an increased Th2/Th1 cell ratio. In conclusion, the identification of a functional PMN-MDSC population suggests a possible role of these cells as regulators of Th cell responses in pemphigus

The inflammation in cutaneous lichen planus is dominated by IFN‐ϒ and IL‐21—A basis for therapeutic JAK1 inhibition

Cutaneous lichen planus (CLP) and psoriasis (PSO) are both common chronic inflammatory skin diseases for which development of new treatments requires the identification of key targets. While PSO is a typical Th17/IL-17-disorder, there is some evidence that Th1/IFN-ɣ dominate the inflammatory process in CLP. Nonetheless, the immunopathogenesis of CLP is not fully explained and key immunological factors still have to be recognized. In this study, we compared the immune signature of CLP lesions with the well-characterized inflammation present in PSO skin. First, we analysed the histological and immunohistological characteristics of CLP and PSO. Second, we assessed the cytokine expression (IL1A, IL1B, IL4, IL6, IL8, IL10, IL17A, IL19, IL21, IL22, IL23A, IL13, IFNG, TNF, IL12A, IL12B and IL36G) of lesional skin of CLP with PSO by qPCR. Histology revealed a similar epidermal thickness in CLP and PSO. Immunohistochemically, both diseases presented with an inflammatory infiltrate mainly composed by CD3+CD4+ T cells rather than CD3+CD8+. Importantly, mRNA analysis showed a distinct cytokine signature: while levels of IL12B, IL1A, IL6 and IL23 were similar between the two groups, the characteristic PSO-associated cytokines IL8, IL17A, IL22, IL19 and IL36G were expressed at very low levels in CLP. In contrast, CLP lesional skin was dominated by the expression of IFNG, IL21, IL4, IL12A and TNF. Immunohistochemistry confirmed the dominance of IL-21, IFN-ɣ and also pSTAT1 in the dermal infiltrate of CLP, while IL-17A was more present in PSO. Collectively, this study improves our understanding of the immunological factors dominating CLP. The dominating cytokines and signalling proteins identified suggest that anti-cytokine therapeutics like JAK inhibitors may be beneficial in CLP

Development and validation of serological immunoassays in laminin γ-1 pemphigoid and epidermolysis bullosa acquisita

Pemphigoid diseases encompass a heterogeneous group of subepidermal autoimmune bullous diseases of the skin and mucosa. These disorders are characterized by autoantibodies that target distinct structural proteins of the basement membrane zone which are crucial for the integrity of the skin. Targeting of these proteins leads to skin fragility, detachment of the epidermis and eventually formation of blisters and skin inflammation. The clinical presentation of the pemphigoids is heterogeneous, the subtypes are divided by clinical symptoms, target antigens and response to treatment. A crucial step for the proper classification is the serological detection of the autoantigens directed against distinct components of the dermal epidermal junction. Thus, it is crucial to develop reliable and reproducible diagnostic methods which help clinicians to differentiate between different entities and therefore to select the best treatment. This thesis is focused on Laminin γ-1 pemphigoid and epidermolysis bullosa acquisita, the study of their clinical appearance and the development and validation of serological assays to correctly diagnose these two entities. Both autoimmune blistering diseases are rare and often hard to diagnose with standard serological assays. In the first study presented in this thesis, I discuss the development of an immunoblot assay for the detection of autoantibodies against Laminin γ-1. Laminin γ-1 pemphigoid is a novel subepidermal blistering disease which has been first described in 1996. Since then, different immune serological methods for the detection of circulating autoantibodies have been described, however these are often not reproducible for most of the centers worldwide, since often advanced lab skills are required. In our study, we present a novel immunoblot assay based on three commercially available Laminin γ-1 recombinants, which can be reproduced in almost every laboratory with basic skills. In the second study, we thought to serologically identify for the best immunoassay for the detection of serum IgG autoantibodies against anti-human collagen VII in patients with epidermolysis bullosa acquisita. Epidermolysis bullosa acquisita is a severe blistering diseases characterized by circulating IgG autoantibodies targeting human collagen VII. In this study, four different diagnostic assays (immunoblot, indirect immunofluorescence with saline split human skin and 2 enzyme linked immunosorbent assays) have been tested and compared with regard to their sensitivity and specificity in a large cohort of epidermolysis bullosa acquisita sera (n=95). In summary, our study showed that enzyme linked immunosorbent assays based on recombinant human collagen VII NC1-NC2 or collagen VII-NC1 proteins provide the highest sensitivity and specificity for the detection of anticollagen VII IgG and should be preferred to immunoblot analysis or indirect immunofluorescence on saline split human skin in making the diagnosis of epidermolysis bullosa acquisita