Type 2 T-Cell Responses against Distinct Epitopes of the Desmoglein 3 Ectodomain in Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is an autoimmune blistering disorder of the skin and/or mucous membranes caused by IgG autoantibodies that predominantly target two transmembrane desmosomal cadherins: desmoglein (DSG)1 and DSG3. DSG-specific T cells play a central role in PV pathogenesis because they provide help to autoreactive B cells for autoantibody production. In this study, we characterized DSG3-specific peripheral T cells in a cohort of 52 patients with PV and 41 healthy controls with regard to cytokine profile and epitope specificity. By ELISpot analysis, type 2 T cells reactive with the DSG3 ectodomain were significantly increased in patients with PV compared with those in healthy controls. By dextramer analysis, CD4+ T cells specific for an epitope within the extracellular domain of DSG3, DSG3(206-220), were found at significantly higher frequencies in patients with PV than in HLA-matched healthy controls. T-cell recognition of two distinct DSG3 epitopes, that is, DSG3(206-220) and DSG3(378-392), correlated significantly, suggesting a synergistic effect in B-cell help. Immunization of HLA-DRB1∗04:02-transgenic mice with PV with the same set of DSG3 peptides induced pathogenic DSG3-specific IgG antibodies, which induced loss of keratinocyte adhesion in vitro. Thus, DSG3 peptide-specific T cells are of particular interest as surrogate markers of disease activity and potential therapeutic targets in PV.

Mast cell deficiency prevents BCR::ABL1 induced splenomegaly and cytokine elevation in a CML mouse model.

The persistence of leukemic stem cells (LSCs) represents a problem in the therapy of chronic myeloid leukemia (CML). Hence, it is of utmost importance to explore the underlying mechanisms to develop new therapeutic approaches to cure CML. Using the genetically engineered ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML, we previously demonstrated that the loss of the docking protein GAB2 counteracts the infiltration of mast cells (MCs) in the bone marrow (BM) of BCR::ABL1 positive mice. Here, we show for the first time that BCR::ABL1 drives the cytokine independent expansion of BM derived MCs and sensitizes them for FcεRI triggered degranulation. Importantly, we demonstrate that genetic mast cell deficiency conferred by the Cpa3Cre allele prevents BCR::ABL1 induced splenomegaly and impairs the production of pro-inflammatory cytokines. Furthermore, we show in CML patients that splenomegaly is associated with high BM MC counts and that upregulation of pro-inflammatory cytokines in patient serum samples correlates with tryptase levels. Finally, MC-associated transcripts were elevated in human CML BM samples. Thus, our study identifies MCs as essential contributors to disease progression and suggests considering them as an additional target in CML therapy. Mast cells play a key role in the pro-inflammatory tumor microenvironment of the bone marrow. Shown is a cartoon summarizing our results from the mouse model. BCR::ABL1 transformed MCs, as part of the malignant clone, are essential for the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression.

Survival and prognostic factors in bullous pemphigoid: A retrospective cohort study.

Background Bullous pemphigoid is the most common subepidermal autoimmune blistering disease. Till now, the reported prognostic factors in bullous pemphigoid vary considerably. Aims The purpose of this study was to determine the overall survival rate and prognostic factors in bullous pemphigoid. Methods We conducted a retrospective cohort study on newly diagnosed bullous pemphigoid patients between July 2001 and November 2019 in a referral unit for autoimmune blistering skin diseases in Romania. Results One hundred forty-eight patients were included in the study. The Kaplan-Meier overall survival rates at 1, 3, 5 and 10 years were respectively 74.2% (95% confidence interval, 67.5-81.6%), 53.4% (45.7-62.2%), 43.6% (35.9-53%) and 31.3% (23.5-41.7%). The median follow-up among survivors was 48 months (interquartile range: 11-150). Ninety (60.8%) patients died during the follow-up period; of them, 38 (42.2%) had active disease at the time of death. Advanced age, neurological diseases, valvular heart disease, malignancies, use of statins, skin infections and extensive cutaneous involvement were linked to poorer outcomes, while the use of topical corticosteroids was associated with increased overall survival. Limitations This study lacks a control cohort to validate the obtained results. It was conducted in a retrospective manner in a single centre. In addition, indirect immunofluorescence microscopy was not performed in all patients. Conclusion Beyond ageing and neurological comorbidities, the prognosis of bullous pemphigoid patients was significantly influenced by the presence of skin infections, valvular heart disease, use of statins and extensive cutaneous involvement. Topical corticosteroid treatment was associated with increased survival in these patients.

Anti-FcαRI Monoclonal Antibodies Resolve IgA Autoantibody-Mediated Disease.

Immunoglobulin A (IgA) is generally considered as a non-inflammatory regulator of mucosal immunity, and its importance in diversifying the gut microbiota is increasingly appreciated. IgA autoantibodies have been found in several autoimmune or chronic inflammatory diseases, but their role in pathophysiology is ill-understood. IgA can interact with the Fc receptor FcαRI on immune cells. We now established a novel IgA autoimmune blistering model, which closely resembles the human disease linear IgA bullous disease (LABD) by using genetically modified mice that produce human IgA and express human FcαRI. Intravital microscopy demonstrated that presence of IgA anti-collagen XVII, - the auto-antigen in LABD-, resulted in neutrophil activation and extravasation from blood vessels into skin tissue. Continued exposure to anti-collagen XVII IgA led to massive neutrophil accumulation, severe tissue damage and blister formation. Importantly, treatment with anti-FcαRI monoclonal antibodies not only prevented disease, but was also able to resolve existing inflammation and tissue damage. Collectively, our data reveal a novel role of neutrophil FcαRI in IgA autoantibody-mediated disease and identify FcαRI as promising new therapeutic target to resolve chronic inflammation and tissue damage.

Challenges and pitfalls between lichen planus pemphigoides and bullous lichen planus.

Lichen planus pemphigoides (LPP) and bullous lichen planus (BLP) are rare dermatoses, which are characterised by blisters and lichenoid lesions. Their clinical presentation is heterogenous, displaying overlapping features or mimicking other dermatological diseases. Therefore, diagnosis can often be challenging, requiring a thorough dermatological examination along with distinctive histological and immunopathological characteristics. Lichenoid degeneration of the basal epidermis exposes various antigens of the dermal-epidermal junction in LPP, resulting in the breakdown of immune tolerance, hence, the production of autoantibodies against type XVII collagen. Conversely, no pathogenic autoantibodies are detected in BLP. However, some cases of mucosal lichen planus might display immunopathological features suggestive of autoimmune blistering diseases. Therefore, a better understanding of the pathophysiology of these two distinct dermatoses is imperative. The aim of this review was to provide a summary of the current knowledge on the clinical hallmarks, diagnosis and available therapeutic options in LPP and BLP.

Autoreactive Peripheral Blood T Helper Cell Responses in Bullous Pemphigoid and Elderly Patients With Pruritic Disorders.

Bullous pemphigoid (BP) is a prototypic autoimmune disorder of the elderly, characterized by serum IgG autoantibodies, namely anti-BP180 and anti-BP230, directed against components of the basal membrane zone that lead to sub-epidermal loss of adhesion. Pruritus may be indicative of a pre-clinical stage of BP, since a subset of these patients shows serum IgG autoantibodies against BP230 and/or BP180 while chronic pruritus is increasingly common in the elderly population and is associated with a variety of dermatoses. Clinical and experimental evidence further suggests that pruritus of the elderly may be linked to autoimmunity with loss of self-tolerance against cutaneous autoantigens. Thus, the objective of this study was to determine autoreactive T cell responses against BP180 in elderly patients in comparison to patients with BP. A total of 22 elderly patients with pruritic disorders, 34 patients with bullous or non-bullous BP and 34 age-matched healthy controls were included in this study. The level of anti-BP180 and anti-BP230 IgG serum autoantibodies, Bullous Pemphigoid Disease Area Index (BPDAI), and pruritus severity were assessed for all patients and controls. For characterization of the autoreactive T cell response, peripheral blood mononuclear cells were stimulated ex vivo with recombinant BP180 proteins (NH2- and COOH-terminal domains) and the frequencies of BP180-specific T cells producing interferon-γ, interleukin (IL)-5 or IL-17 were subsequently determined by ELISpot assay. Patients with BP showed a mixed Th1/Th2 response against BP180 while autoreactive Th1 cells were identified in a minor subset of elderly patients with pruritic disorders. Furthermore, our T cell characterization revealed that therapeutic application of topical clobetasol propionate ointment in BP patients significantly reduced peripheral blood BP180-specific T cells, along with clinically improved symptoms, strongly suggesting a systemic immunosuppressive effect of this treatment.

Spread of Terbinafine-Resistant Trichophyton mentagrophytes Type VIII (India) in Germany-"The Tip of the Iceberg?"

Chronic recalcitrant dermatophytoses, due to Trichophyton (T.) mentagrophytes Type VIII are on the rise in India and are noteworthy for their predominance. It would not be wrong to assume that travel and migration would be responsible for the spread of T. mentagrophytes Type VIII from India, with many strains resistant to terbinafine, to other parts of the world. From September 2016 until March 2020, a total of 29 strains of T. mentagrophytes Type VIII (India) were isolated. All patients were residents of Germany: 12 females, 15 males and the gender of the remaining two was not assignable. Patients originated from India (11), Pakistan (two), Bangladesh (one), Iraq (two), Bahrain (one), Libya (one) and other unspecified countries (10). At least two patients were German-born residents. Most samples (21) were collected in 2019 and 2020. All 29 T. mentagrophytes isolates were sequenced (internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1-α)). All were identified as genotype VIII (India) of T. mentagrophytes. In vitro resistance testing revealed 13/29 strains (45%) to be terbinafine-resistant with minimum inhibitory concentration (MIC) breakpoints ≥0.2 µg/mL. The remaining 16 strains (55%) were terbinafine-sensitive. Point mutation analysis revealed that 10/13 resistant strains exhibited Phe397Leu amino acid substitution of squalene epoxidase (SQLE), indicative for in vitro resistance to terbinafine. Two resistant strains showed combined Phe397Leu and Ala448Thr amino acid substitutions, and one strain a single Leu393Phe amino acid substitution. Out of 16 terbinafine-sensitive strains, in eight Ala448Thr, and in one Ala448Thr +, new Val444 Ile amino acid substitutions were detected. Resistance to both itraconazole and voriconazole was observed in three out of 13 analyzed strains. Treatment included topical ciclopirox olamine plus topical miconazole or sertaconazole. Oral itraconazole 200 mg twice daily for four to eight weeks was found to be adequate. Terbinafine-resistant T. mentagrophytes Type VIII are being increasingly isolated. In Germany, transmission of T. mentagrophytes Type VIII from the Indian subcontinent to Europe should be viewed as a significant public health issue.

Complement-Activating Capacity of Autoantibodies Correlates With Disease Activity in Bullous Pemphigoid Patients.

Background: Bullous pemphigoid is a subepidermal blistering skin disease, associated with autoantibodies to hemidesmosomal proteins, complement activation at the dermal-epidermal junction, and dermal granulocyte infiltration. Clinical and experimental laboratory findings support conflicting hypotheses regarding the role of complement activation for the skin blistering induced by pemphigoid autoantibodies. In-depth studies on the pathogenic relevance of autoimmune complement activation in patients are largely lacking. Therefore, the aim of this study was to investigate the pathogenic relevance of complement activation in patients with bullous pemphigoid. Complement activation by autoantibodies in vivo as measured by the intensity of complement C3 deposits in the patients' skin and ex vivo by the complement-fixation assay in serum was correlated with the clinical disease activity, evaluated by Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) and Bullous Pemphigoid Disease Area Index (BPDAI), as well as, with further immunopathological findings in patients with bullous pemphigoid. Results: Complement-activation capacity of autoantibodies ex vivo, but not deposition of complement in the perilesional skin of patients, correlates with the extent of skin disease (measured by ABSIS and BPDAI) and with levels of autoantibodies. Conclusions: Our study provides for the first time evidence in patients for a pathogenic role of complement activation in bullous pemphigoid and should greatly facilitate the development of novel diagnostic tools and of more specific therapies for complement-dependent autoimmune injury.

Molecular diagnosis of anti-laminin 332 (epiligrin) mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid is a group of chronic subepithelial autoimmune blistering diseases that mainly affect mucous membranes. Laminin 332-specific autoantibodies are present in approximately 1/3 of the patients, being associated with an increased risk of malignancy. Because of the severe complications, an early recognition of the disease allowing a timely therapy is essential. The gold standard methods for detection of laminin 332-specific autoantibodies, including the immunoprecipitation and immunoblotting are non-quantitative, laborious and restricted to a few specialized laboratories worldwide. In addition, the use of radioimmunoassays, although highly sensitive and specific, are laborious, expensive and tightly regulated. Therefore, there is a stringent need for a quantitative immunoassay for the routine detection of laminin 332-specific autoantibodies more broadly available to diagnostic laboratories. The aim of this study was to compare different antigenic substrates, including native, recombinant laminin 332 and laminin 332-rich keratinocyte extracellular matrix, for development of an ELISA to detect autoantibodies in mucous membrane pemphigoid. RESULTS: Using a relatively large number of sera from MMP patients with well-characterized autoantibody reactivity we show the suitability of ELISA systems using laminin 332 preparations as adjunct diagnostic tools in MMP. While glycosylation of laminin 332 does not appear to influence its recognition by MMP autoantibodies, ELISA systems using both purified, native and recombinant laminin 332 demonstrated a high sensitivity and good correlation with the detection of autoantibodies by immunoblotting. ELISA systems using different laminin 332 preparations represent a feasible and more accessible alternative for a broad range of laboratories. CONCLUSIONS: Our findings qualify the use of immunoassays with the laminin 332-rich preparations as an ancillary diagnostic tool in mucous membrane pemphigoid.

Immunological markers as predictors of developing steroid-induced diabetes mellitus in pemphigus vulgaris patients: An observational study.

The aim of this study was to evaluate the clinical importance of autoantibodies in pemphigus vulgaris patients who developed steroid-induced diabetes mellitus (SID) because of the glucocorticoid therapy of pemphigus.A total of 137 patients with pemphigus vulgaris were studied. Patients with SID and pemphigus were compared with those that had only pemphigus. The variables recorded were: age at diagnosis, sex, body mass index, presence of diabetes mellitus (DM), cumulative cortisone dose, treatment duration, value of anti-desmoglein 1 and 3, and anti-glutamic acid decarboxylase autoantibodies.A total of 31 patients (22.62%) that developed steroid-induced DM were identified. Anti-glutamic acid decarboxylase autoantibodies were positive in 20.75% of patients with pemphigus vulgaris and in 25.75% of patients with pemphigus vulgaris and SID.The overall anti-glutamic acid decarboxylase autoantibodies prevalence in pemphigus patients was high, and the risk of developing DM in patients with pemphigus remains a serious problem, being associated with increased risk of mortality.

The Syk Tyrosine Kinase Is Required for Skin Inflammation in an In Vivo Mouse Model of Epidermolysis Bullosa Acquisita.

The inflammatory form of epidermolysis bullosa acquisita is caused by autoantibodies against type VII collagen (C7), a component of the dermal-epidermal junction. We have previously shown that myeloid Src family kinases mediate skin inflammation triggered by anti-C7 antibodies. Here we identify the Syk tyrosine kinase as a critical component of autoantibody-induced skin inflammation downstream of Src family kinases. Immobilized C7-anti-C7 immune complexes triggered neutrophil activation and Syk phosphorylation in a Src family kinase-dependent manner. Bone marrow chimeric mice lacking Syk in their hematopoietic compartment were completely protected from skin inflammation triggered by anti-C7 antibodies despite normal circulating anti-C7 levels. Syk deficiency abrogated the accumulation of CXCL2, IL-1ß, and leukotriene B4 at the site of inflammation and resulted in defective in vivo neutrophil recruitment. Syk-/- neutrophils had a normal intrinsic migratory capacity but failed to release CXCL2 or leukotriene B4 upon activation by immobilized C7-anti-C7 immune complexes, indicating a role for Syk in the amplification of the inflammation process. These results identify Syk as a critical component of skin inflammation in a mouse model of epidermolysis bullosa acquisita and as a potential therapeutic target in epidermolysis bullosa acquisita and other mechanistically related inflammatory skin diseases such as bullous pemphigoid.

Nanocarriers as Tools in Delivering Active Compounds for Immune System Related Pathologies.

BACKGROUND: Immune system related pathologies affect an increasing number of patients every year, having potentially lethal consequences and requiring expensive and difficult tolerated treatments. This area of pathologies offers an excellent potential for the development of innovative solutions such as nanoformulations; this review aims to describe the main categories of nanostructured drug delivery platforms that have been developed in order to achieve a more effective, low toxic treatment in autoimmune pathologies. METHODS: We conducted a systematic search of peer-reviewed original studies focusing on nanoformulations suitable for use against autoimmune disorders. Each study was evaluated by at least two reviewers who applied inclusion and exclusion criteria in order to establish the quality of the research reported in the selected papers. The systematic screening of the literature also identified the most recent patents issued in the field of inflammatory/autoimmune diseases which were included in the current paper. RESULTS: One hundred ninety-seven bibliographic titles were included in the review of which fifty-three recently published patents. Twenty-one papers were selected to define the interaction between nanoformulations and the immune system triggering either immunosuppression or immunostimulation. Thirty-seven papers outlined the characteristics of liposomal formulations that have been reported as treatment for several autoimmune diseases including multiple sclerosis. Twentythree articles provided data on the main lipid nanoparticles of the first and second generation which may include physiological lipids with low cytotoxicity. Gold nanoparticles were described by thirteen papers which highlighted the advantages of the new hybrid organic-inorganic nanoparticles vs. the classical ionic gold compounds. Polymeric nanoformulations were the subject of fifty-nine papers that described their preparation as well as various advantages in the treatment of inflammatory disorders; dendrimers and polymeric micelles were included in the study. Thirty-three studies were selected to outline the benefits provided by the use of cyclodextrins as carriers for active drugs. The review also identified the possibility of producing combined nanoformulations (e.g. polymer/cyclodextrin, liposome/polymer) thus achieving superior therapeutic results. CONCLUSION: Nanoformulations offer multiple advantages over the classical pharmaceutical formulations, the main ones being the precise targeted therapy with small doses of active drugs, longer circulating time in the bloodstream, and high loading capacity for diagnostic and therapeutic molecules; moreover, the nanoparticles may be functionalized in order to act as nanotheranostics within the larger frame of personalized medicine.

Autoimmunity against laminins.

Laminins are ubiquitous constituents of the basement membranes with major architectural and functional role as supported by the fact that absence or mutations of laminins lead to either lethal or severely impairing phenotypes. Besides genetic defects, laminins are involved in a wide range of human diseases including cancer, infections, and inflammatory diseases, as well as autoimmune disorders. A growing body of evidence implicates several laminin chains as autoantigens in blistering skin diseases, collagenoses, vasculitis, or post-infectious autoimmunity. The current paper reviews the existing knowledge on autoimmunity against laminins referring to both experimental and clinical data, and on therapeutic implications of anti-laminin antibodies. Further investigation of relevant laminin epitopes in pathogenic autoimmunity would facilitate the development of appropriate diagnostic tools for thorough characterization of patients' antibody specificities and should decisively contribute to designing more specific therapeutic interventions.

Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo.

Neutrophils are terminally differentiated cells with limited transcriptional activity. The biological function of their gene expression changes is poorly understood. CARD9 regulates transcription during antifungal immunity but its role in sterile inflammation is unclear. Here we show that neutrophil CARD9 mediates pro-inflammatory chemokine/cytokine but not lipid mediator release during non-infectious inflammation. Genetic deficiency of CARD9 suppresses autoantibody-induced arthritis and dermatitis in mice. Neutrophil-specific deletion of CARD9 is sufficient to induce that phenotype. Card9(-/-) neutrophils show defective immune complex-induced gene expression changes and pro-inflammatory chemokine/cytokine release but normal LTB4 production and other short-term responses. In vivo deletion of CARD9 reduces tissue levels of pro-inflammatory chemokines and cytokines but not LTB4. The CARD9-mediated signalling pathway involves Src-family kinases, Syk, PLCγ2, Bcl10/Malt1 and NFκB. Collectively, CARD9-mediated gene expression changes within neutrophils play important roles during non-infectious inflammation in vivo and CARD9 acts as a divergence point between chemokine/cytokine and lipid mediator release.

Generation of a Functional Non-Shedding Collagen XVII Mouse Model: Relevance of Collagen XVII Shedding in Wound Healing.

Collagen XVII is a hemidesmosomal anchorage molecule of basal keratinocytes that promotes stable epidermal-dermal adhesion. One unique feature of collagen XVII is that its collagenous ectodomain is constitutively released from the cell surface by a disintegrin and metalloproteinases (ADAMs) through cleavage within its juxtamembranous linker domain. The responsivity of shedding to environmental stimuli and the high stability of the released ectodomain in several tissues suggests functions in cell detachment during epidermal morphogenesis, differentiation, and regeneration, but its physiologic relevance remained elusive. To investigate this, we generated knock-in mice, which express a functional non-sheddable collagen XVII mutant, with a 41 amino acid deletion in the linker domain spanning all ADAM cleavage sites. These mice showed no macroscopic phenotypic changes, were fertile, and had a normal lifespan. Prevention of collagen XVII shedding interfered neither with skin development nor with epidermal adhesion and differentiation. However, it led to faster wound closure due to accelerated re-epithelialization at the wound edges where shedding of wild-type collagen XVII was strongly induced. Taken together, we have successfully generated a functional non-shedding collagen XVII mouse model, which represents a powerful tool to investigate the pathophysiologic relevance of ectodomain shedding during wound regeneration and cancer invasion.

Granular C3 Dermatosis.

There has been no previous systematic study of bullous skin diseases with granular basement membrane zone deposition exclusively of C3. In this study we collected 20 such patients, none of whom showed cutaneous vasculitis histopathologically. Oral dapsone and topical steroids were effective. Various serological tests detected no autoantibodies or autoantigens. Direct immunofluorescence for various complement components revealed deposition only of C3 and C5-C9, indicating that no known complement pathways were involved. Studies of in situ hybridization and micro-dissection with quantitative RT-PCR revealed a slight reduction in expression of C3 in patient epidermis. These patients may represent a new disease entity, for which we propose the term "granular C3 dermatosis". The mechanism for granular C3 deposition in these patients is unknown, but it is possible that the condition is caused by autoantibodies to skin or aberrant C3 expression in epidermal keratinocytes.

Role of physical factors in the pathogenesis of bullous pemphigoid: Case report series and a comprehensive review of the published work.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease affecting mainly the elderly. The subtype of the disease induced by physical agents represents a rare and, therefore, insufficiently characterized form. In the present study, we aimed to contribute to a better understanding of the pathogenetic mechanisms involved in the onset of BP induced by different trigger factors. We have retrospectively analyzed nine cases of BP. All patients were characterized based on clinical, epidemiological and immunological parameters. For each case, the trigger factor involved was specified. In addition to our retrospective analysis, a comprehensive review of the 59 published cases was conducted, regarding the involvement of trigger factor in BP, and clinical, epidemiological and immunological data were collected. In the local study, conducted on nine patients diagnosed with BP, various trigger factors were identified: contrast substance injection, surgical procedure, mechanical trauma, insect bite, thermal burn, radiotherapy and ultraviolet exposure associated with pre-existing psoriasis. The autoantibodies from all patients were shown to activate granulocytes and induce dermal-epidermal split. Different hypotheses regarding the pathogenetic mechanism involving the trigger factors have been discussed. In regard of the pathogenetic mechanism, we believe that the most reliable hypothesis is that BP patients already have low titers of anti-basement membrane autoantibodies which activate the granulocytes. However, more studies are needed for a better understanding of the pathogenetic mechanism of the intervention of trigger factors.