Bullous pemphigoid induced by IgG targeting type XVII collagen non-NC16A/NC15A extracellular domains is driven by Fc gamma receptor- and complement-mediated effector mechanisms and is ameliorated by neonatal Fc receptor blockade.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies targeting type XVII collagen (Col17) with the noncollagenous 16A (NC16A) ectodomain representing the immunodominant site. The role of additional extracellular targets of Col17 outside NC16A has not been unequivocally demonstrated. In this study, we showed that Col17 ectodomain-reactive patient sera depleted in NC16A IgG induced dermal-epidermal separation in a cryosection model indicating the pathogenic potential of anti-Col17 non-NC16A extracellular IgG. Moreover, injection of IgG targeting the murine Col17 NC14-1 domains (downstream of NC15A, the murine homologue of human NC16A) into C57BL/6J mice resulted in erythematous skin lesions and erosions. Clinical findings were accompanied by IgG/C3 deposits along the basement membrane and subepidermal blistering with inflammatory infiltrates. Disease development was significantly reduced in either Fc-gamma receptor (FcγR)- or complement-5a receptor-1 (C5aR1)-deficient mice. Inhibition of the neonatal FcR (FcRn), an atypical FcγR regulating IgG homeostasis, with the murine Fc fragment IgG2c-ABDEG, a derivative of efgartigimod, reduced anti-NC14-1 IgG levels, resulting in ameliorated skin inflammation compared with isotype-treated controls. These data demonstrate that the pathogenic effects of IgG targeting the Col17 domain outside human NC16A/murine NC15A are partly attributable to antibody-mediated FcγR- and C5aR1 effector mechanisms while pharmacological inhibition of the FcRn represents a promising treatment for BP. The mouse model of BP will be instrumental in further investigating the role of Col17 non-NC16A/NC15A extracellular epitopes and validating new therapies for this disease. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Rabbit neurospheres as a novel in vitro tool for studying neurodevelopmental effects induced by intrauterine growth restriction.

The aim of this study was to develop a rabbit neurosphere culture to characterize differences in basic processes of neurogenesis induced by intrauterine growth restriction (IUGR). A novel in vitro neurosphere culture has been established using fresh or frozen neural progenitor cells from newborn (PND0) rabbit brains. After surgical IUGR induction in pregnant rabbits and cesarean section 5 days later, neural progenitor cells from both control and IUGR groups were isolated and directly cultured or frozen at -80°C. These neural progenitor cells spontaneously formed neurospheres after 7 days in culture. The ability of control and IUGR neurospheres to migrate, proliferate, differentiate to neurons, astrocytes, or oligodendrocytes was compared and the possibility to modulate their responses was tested by exposure to several positive and negative controls. Neurospheres obtained from IUGR brains have a significant impairment in oligodendrocyte differentiation, whereas no significant differences are observed in other basic processes of neurogenesis. This impairment can be reverted by in vitro exposure of IUGR neurospheres to thyroid hormone, which is known to play an essential role in white matter maturation in vivo. Our new rabbit neurosphere model and the results of this study open the possibility to test several substances in vitro as neuroprotective candidates against IUGR induced neurodevelopmental damage while decreasing the number of animals and resources and allowing a more mechanistic approach at a cellular functional level.

Treatment with anti-neonatal Fc receptor (FcRn) antibody ameliorates experimental epidermolysis bullosa acquisita in mice.

BACKGROUND AND PURPOSE: Pemphigus and pemphigoid diseases are characterized and caused predominantly by IgG autoantibodies targeting structural proteins of the skin. Their current treatment relies on general and prolonged immunosuppression that causes severe adverse events, including death. Hence, novel safe and more effective treatments are urgently needed. Due to its' physiological functions, the neonatal Fc receptor (FcRn) has emerged as a potential therapeutic target for pemphigus and pemphigoid, primarily because IgG is protected from proteolysis after uptake into endothelial cells. Thus, blockade of FcRn would reduce circulating autoantibody concentrations. However, long-term effects of pharmacological FcRn inhibition in therapeutic settings of autoimmune diseases are unknown. EXPERIMENTAL APPROACH: Therapeutic effects of FcRn blockade were investigated in a murine model of the prototypical autoantibody-mediated pemphigoid disease, epidermolysis bullosa acquisita (EBA). B6.SJL-H2s C3c/1CyJ mice with clinically active disease were randomized to receive either an anti-FcRn monoclonal antibody (4470) or an isotype control over 4 weeks. KEY RESULTS: While clinical disease continued to worsen in isotype control-treated mice, overall disease severity continuously decreased in mice injected with 4470, leading to almost complete remission in over 25% of treated mice. These clinical findings were paralleled by a reduction of autoantibody concentrations. Reduction of autoantibody concentrations, rather than modulating neutrophil activation, was responsible for the observed therapeutic effects. CONCLUSION AND IMPLICATIONS: The clinical efficacy of anti-FcRn treatment in this prototypical autoantibody-mediated disease encourages further development of anti-FcRn antibodies for clinical use in pemphigoid diseases and potentially in other autoantibody mediated diseases.

Immunoadsorption of Desmoglein-3-Specific IgG Abolishes the Blister-Inducing Capacity of Pemphigus Vulgaris IgG in Neonatal Mice.

Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease which is associated with autoantibodies directed against two desmosomal proteins, desmoglein (Dsg) 3 and 1. Treatment of PV is rather challenging and relies on the long-term use of systemic corticosteroids and additional immunosuppressants. More recently, autoantibody-depleting therapies such as rituximab, high-dose intravenous immunoglobulins, and immunoadsorption were shown to be valuable treatment options in PV. Specific removal of pathogenic autoantibodies would further increase efficacy and usability of immunoadsorption. Here, we tested the capacity of our recently developed prototypic Dsg1- and Dsg3-specific adsorbers to remove circulating pathogenic autoantibodies from three different PV patients. The pathogenic potential of the Dsg3/1-depleted IgG fractions and the anti-Dsg3-specific IgG was explored in two different in vitro assays based on cultured human keratinocytes, the desmosome degradation assay and the dispase-based dissociation assay. In addition, the neonatal mouse model of PV was used. In both in vitro assays, no difference between the pathogenic effect of total PV IgG and anti-Dsg3-specific IgG was seen, while Dsg3/1-depleted and control IgG were not pathogenic. For the samples of all 3 PV patients, depletion of anti-Dsg3/1 IgG resulted in a complete loss of pathogenicity when injected into neonatal mice. In contrast, injection of anti-Dsg3-specific IgG, eluted from the column, induced gross blistering in the mice. Our data clearly show that anti-Dsg3-specific IgG alone is pathogenic in vitro and in vivo, whereas Dsg3/1-depletion results in a complete loss of pathogenicity. Furthermore, our data suggest that Dsg-specific adsorption may be a suitable therapeutic modality to efficiently reduce pathogenic autoantibodies in patients with severe PV.

Comparative performance analysis of human iPSC-derived and primary neural progenitor cells (NPC) grown as neurospheres in vitro.

Developmental neurotoxicity (DNT) testing performed in rats is resource-intensive (costs, time, animals) and bears the issue of species extrapolation. Thus, reliable alternative human-based approaches are needed for predicting neurodevelopmental toxicity. Human induced pluripotent stem cells (hiPSCs) represent a basis for an alternative method possibly being part of an alternative DNT testing strategy. Here, we compared two hiPSC neural induction protocols resulting in 3D neurospheres: one using noggin and one cultivating cells in neural induction medium (NIM protocol). Performance of Nestin+/SOX2+ hiPSC-derived neural progenitor cells (NPCs) was compared to primary human NPCs. Generally, primary hNPCs first differentiate into Nestin+ and/or GFAP+ radial glia-like cells, while the hiPSC-derived NPCs (hiPSC-NPC) first differentiate into ßIII-Tubulin+ neurons suggesting an earlier developmental stage of hiPSC-NPC. In the 'Neurosphere Assay', NIM generated hiPSC-NPC produced neurons with higher performance than with the noggin protocol. After long-term differentiation, hiPSC-NPC form neuronal networks, which become electrically active on microelectrode arrays after 85days. Finally, methylmercury chloride inhibits hiPSC-NPC and hNPC migration with similar potencies. hiPSC-NPCs-derived neurospheres seem to be useful for DNT evaluation representing early neural development in vitro. More system characterization by compound testing is needed to gain higher confidence in this method.