In Vitro, Ex Vivo and In Vivo Models for the Study of Pemphigus

Pemphigus is a life-threatening autoimmune disease. Several phenotypic variants are part of this family of bullous disorders. The disease is mainly mediated by pathogenic autoantibodies, but is also directed against two desmosomal adhesion proteins, desmoglein 1 (DSG1) and 3 (DSG3), which are expressed in the skin and mucosae. By binding to their antigens, autoantibodies induce the separation of keratinocytes, in a process known as acantholysis. The two main Pemphigus variants are Pemphigus vulgaris and foliaceus. Several models of Pemphigus have been described: in vitro, ex vivo and in vivo, passive or active mouse models. Although no model is ideal, different models display specific characteristics that are useful for testing different hypotheses regarding the initiation of Pemphigus, or to evaluate the efficacy of experimental therapies. Different disease models also allow us to evaluate the pathogenicity of specific Pemphigus autoantibodies, or to investigate the role of previously not described autoantigens. The aim of this review is to provide an overview of Pemphigus disease models, with the main focus being on active models and their potential to reproduce different disease subgroups, based on the involvement of different autoantigens

A Novel In Vivo Active Pemphigus Model Targeting Desmoglein1 and Desmoglein3: A Tool Representing All Pemphigus Variants

: Background: Pemphigus is a life-threatening blistering autoimmune disease. Several forms, characterized by the presence of autoantibodies against different autoantigens, have been described. In Pemphigus Vulgaris (PV), autoantibodies target the cadherin Desmoglein 3 (DSG3), while in Pemphigus foliaceous (PF) autoantibodies target the cadherin Desmoglein 1 (DSG1). Another variant, mucocutaneous Pemphigus, is characterized by the presence of IgG against both DSG1 and DSG3. Moreover, other forms of Pemphigus characterized by the presence of autoantibodies against other autoantigens have been described. With regard to animal models, one can distinguish between passive models, where pathological IgG are transferred into neonatal mice, and active models, where B cells deriving from animals immunized against a specific autoantigen are transferred into immunodeficient mice that develop the disease. Active models recreate PV and a form of Pemphigus characterized by the presence of IgG against the cadherin Desmocollin 3 (DSC3). Further approaches allow to collect sera or B/T cells from mice immunized against a specific antigen to evaluate the mechanisms underlying the onset of the disease. Objective: To develop and characterize a new active model of Pemphigus where mice express auto antibodies against either DSG1 alone, or DSG1 and DSG3, thereby recapitulating PF and mucocutaneous Pemphigus, respectively. In addition to the existing models, with the active models reported in this work, it will be possible to recapitulate and mimic the main forms of pemphigus in adult mice, thus allowing a better understanding of the disease in the long term, including the benefit/risk ratio of new therapies. Results: The new DSG1 and the DSG1/DSG3 mixed models were developed as proposed. Immunized animals, and subsequently, animals that received splenocytes from the immunized donors produce a high concentration of circulating antibodies against the specific antigens. The severity of the disease was assessed by evaluating the PV score, evidencing that the DSG1/DSG3 mixed model exhibits the most severe symptoms among those analyzed. Alopecia, erosions, and blistering were observed in the skin of DSG1, DSG3 and DSG1/DSG3 models, while lesions in the mucosa were observed only in DSG3 and DSG1/DSG3 animals. The effectiveness of the corticosteroid Methyl-Prednisolone was evaluated in the DSG1 and DSG1/DSG3 models, that showed only partial responsiveness

Development of a Desmocollin-3 Active Mouse Model Recapitulating Human Atypical Pemphigus

Pemphigus vulgaris (PV) is a life-threatening mucocutaneous autoimmune blistering disease. It is often associated with autoantibodies to the desmosomal adhesion proteins Desmoglein 3 (DSG3) and Desmoglein 1 (DSG1). Recently, auto-antigens, such as desmocollins and others have been described in PV and in atypical pemphigus forms such as Pemphigus Herpetiformis (PH), Pemphigus Vegetans (PVeg), and Paraneoplastic Pemphigus (PP). Desmocollins belong to a cadherin subfamily that provides structure to the desmosomes and play an important role in cell-to-cell adhesion. In order to verify the pathogenic activity of anti-Desmocollin 3 (DSC3) antibodies, we developed an active disease model of pemphigus expressing anti-DSC3 autoantibodies or antiDSC3 and anti-DSG3 antibodies. This approach included the adoptive transfer of DSC3 and/or DSG3 lymphocytes to Rag2(-/-) immunodeficient mice that express DSC3 and DSG3. Our results show that the presence of anti-DSC3 auto-antibodies is sufficient to determine the appearance of a pathological phenotype relatable to pemphigus, but with features not completely super-imposable to those observed in the DSG3 active model, suggesting that the DSC3 active model might mimic the atypical pemphigus. Moreover, the presence of both anti-DSC3 and anti-DSG3 antibodies determines a more severe phenotype and a slower response to prednisolone. In conclusion, we have developed an adult DSC3 pemphigus mouse model that differs from the DSG3 model and supports the concept that antigens other than desmogleins may be responsible for different phenotypes in human pemphigus

KLF4 mediates the effect of 5-ASA on the b-catenin pathway in colon cancer cells

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing m-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores m-protocadherin expression and promotes the sequestration of b-catenin to the plasma membrane. Here, we show that 5-ASA–induced m-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA–mediated b-catenin inhibition is caused by m-protocadherin–dependent sequestration of b-catenin to the plasma membrane and by the direct binding of KLF4 to b-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA

In Vitro, Ex Vivo, and In Vivo Models for the Study of Pemphigus

Pemphigus is a life-threatening autoimmune disease. Several phenotypic variants are part of this family of bullous disorders. The disease is mainly mediated by pathogenic autoantibodies, but is also directed against two desmosomal adhesion proteins, desmoglein 1 (DSG1) and 3 (DSG3), which are expressed in the skin and mucosae. By binding to their antigens, autoantibodies induce the separation of keratinocytes, in a process known as acantholysis. The two main Pemphigus variants are Pemphigus vulgaris and foliaceus. Several models of Pemphigus have been described: in vitro, ex vivo and in vivo, passive or active mouse models. Although no model is ideal, different models display specific characteristics that are useful for testing different hypotheses regarding the initiation of Pemphigus, or to evaluate the efficacy of experimental therapies. Different disease models also allow us to evaluate the pathogenicity of specific Pemphigus autoantibodies, or to investigate the role of previously not described autoantigens. The aim of this review is to provide an overview of Pemphigus disease models, with the main focus being on active models and their potential to reproduce different disease subgroups, based on the involvement of different autoantigens

BTK Inhibitors Impair Platelet-Mediated Antifungal Activity

In recent years, the introduction of new drugs targeting Bruton’s tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients