RESUMEN
BACKGROUND AND OBJECTIVES: Autoantibodies against the protein leucine-rich glioma inactivated 1 (LGI1) cause the most common subtype of autoimmune encephalitis with predominant involvement of the limbic system, associated with seizures and memory deficits. LGI1 and its receptor ADAM22 are part of a transsynaptic protein complex that includes several proteins involved in presynaptic neurotransmitter release and postsynaptic glutamate sensing. Autoantibodies against LGI1 increase excitatory synaptic strength, but studies that genetically disrupt the LGI1-ADAM22 complex report a reduction in postsynaptic glutamate receptor-mediated responses. Thus, the mechanisms underlying the increased synaptic strength induced by LGI1 autoantibodies remain elusive, and the contributions of presynaptic molecules to the LGI1-transsynaptic complex remain unclear. We therefore investigated the presynaptic mechanisms that mediate autoantibody-induced synaptic strengthening. METHODS: We studied the effects of patient-derived purified polyclonal LGI1 autoantibodies on synaptic structure and function by combining direct patch-clamp recordings from presynaptic boutons and somata of hippocampal neurons with super-resolution light and electron microscopy of hippocampal cultures and brain slices. We also identified the protein domain mediating the presynaptic effect using domain-specific patient-derived monoclonal antibodies. RESULTS: LGI1 autoantibodies dose-dependently increased short-term depression during high-frequency transmission, consistent with increased release probability. The increased neurotransmission was not related to presynaptic calcium channels because presynaptic Cav2.1 channel density, calcium current amplitude, and calcium channel gating were unaffected by LGI1 autoantibodies. By contrast, application of LGI1 autoantibodies homogeneously reduced Kv1.1 and Kv1.2 channel density on the surface of presynaptic boutons. Direct presynaptic patch-clamp recordings revealed that LGI1 autoantibodies cause a pronounced broadening of the presynaptic action potential. Domain-specific effects of LGI1 autoantibodies were analyzed at the neuronal soma. Somatic action potential broadening was induced by polyclonal LGI1 autoantibodies and patient-derived monoclonal autoantibodies targeting the epitempin domain, but not the leucin-rich repeat domain. DISCUSSION: Our results indicate that LGI1 autoantibodies reduce the density of both Kv1.1 and Kv1.2 on presynaptic boutons, without actions on calcium channel density or function, thereby broadening the presynaptic action potential and increasing neurotransmitter release. This study provides a molecular explanation for the neuronal hyperactivity observed in patients with LGI1 autoantibodies.
Asunto(s)
Potenciales de Acción , Autoanticuerpos , Péptidos y Proteínas de Señalización Intracelular , Terminales Presinápticos , Transmisión Sináptica , Autoanticuerpos/inmunología , Autoanticuerpos/farmacología , Humanos , Animales , Transmisión Sináptica/fisiología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Terminales Presinápticos/metabolismo , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de los fármacos , Hipocampo/metabolismo , Ratas , Canal de Potasio Kv.1.1/inmunología , Proteínas/inmunología , Proteínas/metabolismo , Masculino , Células CultivadasRESUMEN
Epidermolysis bullosa acquisita is an autoimmune skin disease characterized by subepidermal blisters. The pathogenesis is mediated by deposits of autoantibodies directed against type VII collagen in the skin, but the sequence of events regulating the localization of skin blisters is not fully understood. In this study, using the immunization-induced mouse model of epidermolysis bullosa acquisita, we demonstrate that epidermal disruption induces not only an infiltration of CD4+ T cells but also a T helper type 1 phenotype as it has been described for delayed-type hypersensitivity reactions. This T helper type 1 reaction was not found when different antigens were applied. Deep T-cell receptor ß profiling revealed shifts in the V/J gene usage only in epidermolysis bullosa acquisita, suggesting an infiltration of autoantigen-specific T cells. To target these autoantigen-specific T cells, we established an approach with which skin inflammation could be prevented without impairing the functionality of autoantibodies. We conclude that T-cell involvement in skin blistering diseases such as epidermolysis bullosa acquisita relates not only to T-cell help for B cells that produce pathogenic autoantibodies but also to autoreactive T helper type 1 effector cells that migrate into injured skin sites, exacerbate inflammation through production of inflammatory cytokines such as IFNγ, and prevent wound healing.
Asunto(s)
Autoanticuerpos/inmunología , Epidermis/patología , Epidermólisis Ampollosa Adquirida/inmunología , Células TH1/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/metabolismo , Movimiento Celular/inmunología , Colágeno Tipo VII/administración & dosificación , Colágeno Tipo VII/inmunología , Modelos Animales de Enfermedad , Epidermis/inmunología , Epidermólisis Ampollosa Adquirida/sangre , Epidermólisis Ampollosa Adquirida/patología , Femenino , Humanos , Interferón gamma/metabolismo , Ratones , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Células TH1/metabolismo , Cicatrización de Heridas/inmunologíaRESUMEN
Previous reports have demonstrated that cell-derived nanoparticles (CDNPs) composed of bovine or porcine protein complexes exerted therapeutic effects against viral infections and cancer in mice and humans. Based on these observations, we asked whether CDNPs would improve inflammatory skin disorders. To address this, we utilized two distinct mouse models of cutaneous inflammation: the autoimmune skin-blistering disease epidermolysis bullosa acquisita (EBA) as an example of an autoantibody-induced cutaneous inflammation, and Leishmania major (L. major) infection as an example of a pathogen-induced cutaneous inflammation. In both models, we observed that CDNPs increased mRNA expression of the Th2 cytokine IL-4. Clinically, CDNPs decreased inflammation due to EBA and increased L. major-specific IgG1 levels without major effects on infected skin lesions. In addition, CDNPs supported the growth of keratinocytes in human skin cultures. In vitro studies revealed that CDNPs were taken up predominantly by macrophages, leading to a shift towards the expression of anti-inflammatory cytokine genes. Altogether, our data demonstrate that treatment with porcine CDNPs may be a new therapeutic option for the control of autoimmune-mediated inflammatory skin disorders.
Asunto(s)
Micropartículas Derivadas de Células/trasplante , Epidermólisis Ampollosa Adquirida/terapia , Leishmaniasis Cutánea/terapia , Repitelización , Células Th2/fisiología , Adulto , Animales , Diferenciación Celular , Epidermólisis Ampollosa Adquirida/inmunología , Femenino , Humanos , Interleucina-4/metabolismo , Leishmania major , Leishmaniasis Cutánea/inmunología , Ganglios Linfáticos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Porcinos , Adulto JovenRESUMEN
Autoimmune diseases affect a large fraction of the population in Western countries. To elucidate the underlying causes, autoantibody transfer-induced mouse models have been established that greatly contributed to the understanding of the pathophysiology of these diseases. However, the role of a potentially co-occurring murine xenogeneic immune response to commonly utilized rabbit anti-mouse IgG remains poorly understood. Using the established rabbit anti-mouse type VII collagen (COL7) IgG-induced mouse model of the mucocutaneous blistering disorder epidermolysis bullosa acquisita (EBA), we found in this study a profound T- and B-cell response along with an altered cytokine expression profile in draining lymph nodes of mice injected with the xenogeneic IgG. This was associated with the formation of circulating and skin-bound mouse anti-rabbit IgG in wild-type but not CD154-deficient or B-cell-deficient JHT mice challenged with pathogenic rabbit IgG. Development of EBA skin lesions was attenuated in the two mouse strains lacking a B-cell response at later observation time points, but was not affected in mice treated with the T-cell trafficking blocker FTY720. Collectively, our results implicate a host's xenoreactive immune response to rabbit anti-mouse COL7 IgG, a confounding effect that may contribute to immune complex-driven inflammation and tissue damage in this antibody transfer-induced EBA mouse model, especially at later time points. In this regard, it may be recommended to finish the evaluation of results obtained by experiments employing antibody-transferred mouse models within the first 2 weeks after the pathogenic antibody injection.
Asunto(s)
Anticuerpos Heterófilos/metabolismo , Colágeno Tipo VII/inmunología , Epidermólisis Ampollosa Adquirida/inmunología , Animales , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Ganglios Linfáticos/inmunología , Ratones Endogámicos C57BL , Conejos , Linfocitos T/fisiologíaRESUMEN
Understanding of particle strain and drop breakage is relevant for various technical applications. To analyze it, single drop experiments in a breakage cell and evolving drop size distributions in an agitated system are studied. The mechanisms for particle strain and drop breakage are assumed to be comparable for the investigated turbulent flow regime. The agitation process is simulated using a population balance model. This model provides transient prediction capacities at different scales and can be used for scale-up/down projects. The number and the size distributions of daughter fragments for single drops have been studied. The results clearly support the assumption of binary breakage. The most common assumption of a Gaussian distribution for the daughter drop size distribution could not be supported. The evolution of a breakage-dominated toluene/water system was then simulated using different daughter drop size distributions from literature. The computational results were compared with experimental values. All simulations were able to predict the transient Sauter mean diameter excellently but varied strongly in the results on the shape of the distribution. In agreement with the experimental single drop results, the use of a bimodal or a very broad bell-shaped distribution of the daughter drops is proposed for the simulations. Although these results were obtained in a particular vessel for a specific phase system, it can be applied to simulate transient multiphase systems at different scales. We would expect that the general trends observed in this study are comparable to various applications in multiphase bioreactors.