Mareike Witte

Dimethylfumarate Impairs Neutrophil Functions

Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates-in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases.

T cells mediate autoantibody-induced cutaneous inflammation and blistering in epidermolysis bullosa acquisita

T cells are key players in autoimmune diseases by supporting the production of autoantibodies. However, their contribution to the effector phase of antibody-mediated autoimmune dermatoses, i.e., tissue injury and inflammation of the skin, has not been investigated. In this paper, we demonstrate that T cells amplify the development of autoantibody-induced tissue injury in a prototypical, organ-specific autoimmune disease, namely epidermolysis bullosa acquisita (EBA) – characterized and caused by autoantibodies targeting type VII collagen. Specifically, we show that immune complex (IC)-induced inflammation depends on the presence of T cells – a process facilitated by T cell receptor (TCR)γδ and NKT cells. Because tissue damage in IC-induced inflammation is neutrophil-dependent, we further analyze the interplay between T cells and neutrophils in an experimental model of EBA. We demonstrate that T cells not only enhance neutrophil recruitment into the site of inflammation but also interact with neutrophils in lymphatic organs. Collectively, this study shows that T cells amplify the effector phase of antibody-induced tissue inflammation.

Discovering potential drug-targets for personalized treatment of autoimmune disorders - what we learn from epidermolysis bullosa acquisita.

ABSTRACT Introduction: Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune bullous dermatosis (AIBD). Treatment of EBA is challenging and mostly relies on systemic immunosuppression. During the last decade, intensive research led to the identification of new potential therapeutic targets that interfere in different phases of disease progression. Therapeutic interventions acting upon these candidate drug targets in animal models of EBA, such as cytokine-modulating biologics and small molecules, have validated them as potential new therapeutic strategies for EBA patients. Areas covered: In this paper, we review the current treatments for EBA, describe the pathogenesis of the disease, and finally specify new drug candidates for the development of a more specific therapy with minimized side-effects for EBA and potentially other autoimmune diseases. Expert opinion: We currently understand EBA as a disease that evolves from the interplay of many different signaling pathways. These signaling pathways, which are described in this review, provide new targets for EBA treatment. The ultimate goal of this research field is the development of specific, pathogenesis-based therapeutic strategies. Through identification of up- or downregulated pathways that dominate disease progression in individual patients, we expect therapy in EBA to become more and more precise and move towards a patient-based therapy.

Radiosensitive Hematopoietic Cells Determine the Extent of Skin Inflammation in Experimental Epidermolysis Bullosa Acquisita

Animal models have enhanced our understanding of the pathogenesis of autoimmune diseases. For these models, genetically identical, inbred mice have commonly been used. Different inbred mouse strains, however, show a high variability in disease manifestation. Identifying the factors that influence this disease variability could provide unrecognized insights into pathogenesis. We established a novel Ab transfer-induced model of epidermolysis bullosa acquisita (EBA), an autoimmune disease characterized by (muco)-cutaneous blistering caused by anti-type VII collagen (COL7) autoantibodies. Blistering after anti-COL7 IgG (directed against the von Willebrand factor A–like domain 2) transfer showed clear variability among inbred mouse strains, that is, severe cutaneous blistering and inflammation in C57BL/6J and absence of skin lesions in MRL/MpJ mice. The transfer of anti-COL7 IgG into irradiated, EBA-resistant MRL/MpJ mice, rescued by transplantation with bone marrow from EBA-susceptible B6.AK-H2k mice, induced blistering. To the contrary, irradiated EBA-susceptible B6.AK-H2k mice that were rescued using MRL/MpJ bone marrow were devoid of blistering. In vitro, immune complex activation of neutrophils from C57BL/6J or MRL/MpJ mice showed an impaired reactive oxygen species release from the latter, whereas no differences were observed after PMA activation. This finding was paralleled by divergent expression profiles of immune complex–activated neutrophils from either C57BL/6J or MRL/MpJ mice. Collectively, we demonstrate that radiosensitive cells determine the varying extent of skin inflammation and blistering in the end-stage effector phase of EBA.

Dissecting genetics of cutaneous miRNA in a mouse model of an autoimmune blistering disease

BackgroundMicroRNAs (miRNAs) are small endogenous non-coding RNAs that control genes at post-transcriptional level. They are essential for development and tissue differentiation, and such altered miRNA expression patterns are linked to the pathogenesis of inflammation and cancer. There is evidence that miRNA expression is genetically controlled similar to the transcription of protein-coding genes and previous studies identified quantitative trait loci (QTL) for miRNA expression in the liver. So far, little attention has been paid to miRNA expression in the skin. Moreover, epistatic control of miRNA expression remains unknown. In this study, we characterize genetic regulation of cutaneous miRNA and their correlation with skin inflammation using a previously established murine autoimmune-prone advanced intercross line.ResultsWe identified in silico 42 eQTL controlling the expression of 38 cutaneous miRNAs and furthermore found two chromosomal hot-spots on chromosomes 2 and 8 that control the expression of multiple miRNAs. Moreover, for 8 miRNAs an interacting effect from pairs of SNPs was observed. Combining the constraints on genes from the statistical interaction of their loci and further using curated protein interaction networks, the number of candidate genes for association of miRNAs was reduced to a set of several genes. A cluster analysis identified miR-379 and miR-223 to be associated with EBA severity/onset, where miR-379 was observed to be associated to loci on chromosome 6.ConclusionThe murine advanced intercross line allowed us to identify the genetic loci regulating multiple miRNA in skin. The recurrence of trans-eQTL and epistasis suggest that cutaneous miRNAs are regulated by yet an unexplored complex gene networks. Further, using co-expression analysis of miRNA expression levels we showed that multiple miRNA contribute to multiple pathways that might be involved in pathogenesis of autoimmune skin blistering disease. Specifically, we provide evidence that miRNA such as miR-223 and miR-379 may play critical role in disease progression and severity.

Regulatory T Cells Suppress Inflammation and Blistering in Pemphigoid Diseases

Regulatory T cells (Tregs) are well known for their modulatory functions in adaptive immunity. Through regulation of T cell functions, Tregs have also been demonstrated to indirectly curb myeloid cell-driven inflammation. However, direct effects of Tregs on myeloid cell functions are insufficiently characterized, especially in the context of myeloid cell-mediated diseases, such as pemphigoid diseases (PDs). PDs are caused by autoantibodies targeting structural proteins of the skin. Autoantibody binding triggers myeloid cell activation through specific activation of Fc gamma receptors, leading to skin inflammation and subepidermal blistering. Here, we used mouse models to address the potential contribution of Tregs to PD pathogenesis in vivo. Depletion of Tregs induced excessive inflammation and blistering both clinically and histologically in two different PD mouse models. Of note, in the skin of Treg-depleted mice with PD, we detected increased expression of different cytokines, including Th2-specific IL-4, IL-10, and IL-13 as well as pro-inflammatory Th1 cytokine IFN-γ and the T cell chemoattractant CXCL-9. We next aimed to determine whether Tregs alter the migratory behavior of myeloid cells, dampen immune complex (IC)-induced myeloid cell activation, or both. In vitro experiments demonstrated that co-incubation of IC-activated myeloid cells with Tregs had no impact on the release of reactive oxygen species (ROS) but downregulated β2 integrin expression. Hence, Tregs mitigate PD by altering the migratory capabilities of myeloid cells rather than their release of ROS. Modulating cytokine expression by administering an excess of IL-10 or blocking IFN-γ may be used in clinical translation of these findings.

ptRNApred: computational identification and classification of post-transcriptional RNA

Non-coding RNAs (ncRNAs) are known to play important functional roles in the cell. However, their identification and recognition in genomic sequences remains challenging. In silico methods, such as classification tools, offer a fast and reliable way for such screening and multiple classifiers have already been developed to predict well-defined subfamilies of RNA. So far, however, out of all the ncRNAs, only tRNA, miRNA and snoRNA can be predicted with a satisfying sensitivity and specificity. We here present ptRNApred, a tool to detect and classify subclasses of non-coding RNA that are involved in the regulation of post-transcriptional modifications or DNA replication, which we here call post-transcriptional RNA (ptRNA). It (i) detects RNA sequences coding for post-transcriptional RNA from the genomic sequence with an overall sensitivity of 91% and a specificity of 94% and (ii) predicts ptRNA-subclasses that exist in eukaryotes: snRNA, snoRNA, RNase P, RNase MRP, Y RNA or telomerase RNA. AVAILABILITY: The ptRNApred software is open for public use on http://www.ptrnapred.org/.

Intravenous immunoglobulins for rituximab‐resistant mucous membrane pemphigoid

Mucous membrane pemphigoid (MMP) is an autoimmune blistering disease that is notoriously difficult to treat. Rituximab (anti-CD20) and high-dose intravenous immunoglobulins (IVIG) have both been reported to be effective in refractory MMP.1,2 Furthermore, treatment regimens based on the concomitant use of rituximab and IVIG in ocular MMP have been published.3,4 In the current report, we present 5 patients with treatment-resistant MMP who failed to respond to rituximab but rapidly achieved remission on IVIG. This article is protected by copyright. All rights reserved.

Immunohistochemical diagnosis of mucous membrane pemphigoid

BACKGROUND Mucous membrane pemphigoid (MMP) is an autoimmune subepithelial blistering disease with predominant involvement of mucosal surfaces. It is usually diagnosed by direct immunofluorescence microscopy of frozen biopsies, demonstrating linear deposits of complement, IgG or IgA along the basement membrane. The aim of this study was to investigate the utility of immunohistochemistry on formalin-fixed, paraffin-embedded tissue biopsies for the diagnosis of MMP and to compare its sensitivity to that of direct immunofluorescence microscopy. METHODS We examined 50 biopsies from 34 patients with immunologically confirmed MMP by immunohistochemistry for C3d, C4d, IgG and IgA. RESULTS Linear deposits of C3d were detected in 46% of biopsies, and 53% of patients had at least one biopsy positive for C3d. Linear deposits of C4d were detected in 52% of biopsies and 59% of patients had at least one biopsy positive for C4d. Overall, 56% of biopsies and 68% of patients were positive by either C3d or C4d or both stainings. The sensitivity of either staining in mucosal biopsies was lower than in skin samples. Basement membrane deposits of IgG or IgA could not be detected in any biopsy. CONCLUSIONS Our findings demonstrate that immunohistochemistry for C3d or C4d is a helpful screening procedure for cases of suspected MMP where frozen tissue is not readily available. Negative findings, however, do not exclude a possible diagnosis of MMP and should prompt an additional biopsy for direct immunofluorescence studies. Immunohistochemical detection of IgG or IgA cannot yet be used for the diagnosis of MMP.

Iatrogenic hypospadias classification: A new way to classify hypospadias caused by long-term catheterization

DOI: 10.1111/iju.13791 IH is an injury of the ventral male urethra caused by long-term catheterization. Pathomechanistically, a downward directed pressure of the transurethral catheter leads to compression of the ventral urethra and the development of pressure necrosis. Its incidence constantly increases due to the aging of the population and age-related indications, such as neurological and/or musculoskeletal disorders impairing movement, urinary retention, or the need to measure input and output of a patient. IH is virtually unknown to a high number of medical staff. Referral diagnosis is often one of the following: “cleft of the penis,” “penis injury,” “unknown penile condition,” “broken urethra” or “surgery required – penis looks weird.” This unawareness of the physiological process among nursing staff and medical doctors leads to tremendous costs of the healthcare system due to recurrent consultations of patients requesting therapy in urological departments. Therapeutically, a suprapubic catheter is mostly inserted at an early stage after development of IH. The hypothesis is that complications, especially higher rates of infection, can thereby be prevented. However, there is no standardized measure as to when to apply this treatment. Because of urinary leakage outside the catheter, a larger urethral catheter is often inserted. Conversely, an increase in the catheter size leads to a higher downward pressure, resulting in a vicious cycle of further necrosis of the urethral mucosa. The aim of the present study was to develop a classification system to enhance the awareness of IH, and set a standard for assessment of complications and treatment. The “IHC” was designed as follows (Fig. 1): IHC grade 1: Penile cleavage ranges from the meatus to the proximal part of the corona glans penis. IHC grade 2: Penile cleavage ranges from the meatus to the subcoronal part of the penis. IHC grade 3: Penile cleavage ranges from the meatus to the scrotum. IHC grade 4: The meatus is not affected, but pressure necrosis is seen along the penis shaft. The IHC was tested prospectively for accuracy using an online survey designed using SurveyMonkey (SurveyMonkey Inc., San Mateo, CA, USA). First, the participants were asked to memorize different drawings showing IH of different grades. Second, the drawings had to be assigned to the respective grade. It was only possible to assign exactly one picture to each grade. Each rater assessed four pictures. Furthermore, all participants had to assign one of the ranks “excellent,” “very good,” “good,” “fair” and “poor” to the properties “simplicity,” “clinical utility,” “applicability” and “comprehensibility” regarding the IHC. All answers were analyzed for mean values and standard deviations. The survey was sent to all active members of the national Society for Urology and to nonurological doctors from several hospitals in Germany. A total of 317 (256 urologists and 42 non-urologists) doctors participated in the survey. Out of these 317 medical doctors, 79.7% (n = 244), 82% (n = 251), 86.3% (n = 264) and 82% (n = 251) assigned the grades 1–4 according to the pictures correctly. The interrater reliability between urologists and non-urologists was high, with a j of 0.831, 1.0, 0.725 and 0.562 according to the grades 1–4 (P < 0.001), respectively. This shows an equivalent agreement rate among the participants. Regarding simplicity, the IHC was evaluated with “excellent” or “very good” in 71.8% (n = 214), with “good” in 25.2% (n = 75) and with “fair” or “poor” in 3.0% (n = 9; Fig. S1). Regarding clinical utility, the classification was evaluated with “excellent” or “very good” in 60.1% (n = 179), with “good” in 31.9% (n = 95) and with “fair” or “poor” in 8% (n = 24; Fig. S1). Regarding applicability, the classification was evaluated with “excellent” or “very good” in 63.1% (n = 188), with “good” in 30.5% (n = 91) and with “fair” or “poor” in 6.4% (n = 19; Fig. S1). Regarding comprehensibility, the classification was evaluated with “excellent” or “very good” in 62.7% (n = 187), with “good” in 30.5% (n = 91) and with “fair” or “poor” in 6.7% (n = 20; Fig. S1). Urological Notes