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A balanced immune system is essential to maintain adequate host defense and effective self-tolerance. While an immune system that fails to generate appropriate response will permit infections to develop, uncontrolled activation may lead to autoinflammatory or autoimmune diseases. To identify drug candidates capable of modulating immune cell functions, we screened 1200 small molecules from the Prestwick Chemical Library for their property to inhibit innate or adaptive immune responses. Our studies focused specifically on drug interactions with T cells, B cells, and polymorphonuclear leukocytes (PMNs). Candidate drugs that were validated in vitro were examined in preclinical models to determine their immunomodulatory impact in chronic inflammatory diseases, here investigated in chronic inflammatory skin diseases. Using this approach, we identified several candidate drugs that were highly effective in preclinical models of chronic inflammatory disease. For example, we found that administration of pyrvinium pamoate, an FDA-approved over-the-counter anthelmintic drug, suppressed B cell activation in vitro and halted the progression of B cell-dependent experimental pemphigoid by reducing numbers of autoantigen-specific B cell responses. In addition, in studies performed in gene-deleted mouse strains provided additional insight into the mechanisms underlying these effects, for example, the receptor-dependent actions of tamoxifen that inhibit immune-complex-mediated activation of PMNs. Collectively, our methods and findings provide a vast resource that can be used to identify drugs that may be repurposed and used to promote or inhibit cellular immune responses.
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Immunological differences between hosts, such as diverse TCR repertoires, are widely credited for reducing the risk of pathogen spread and adaptation in a population. Within-host immunological diversity might likewise be important for robust pathogen control, but to what extent naive TCR repertoires differ across different locations in the same host is unclear. T cell zones (TCZs) in secondary lymphoid organs provide secluded microenvironmental niches. By harboring distinct TCRs, such niches could enhance within-host immunological diversity. In contrast, rapid T cell migration is expected to dilute such diversity. In this study, we combined tissue microdissection and deep sequencing of the TCR ß-chain to examine the extent to which TCR repertoires differ between TCZs in murine spleens. In the absence of Ag, we found little evidence for differences between TCZs of the same spleen. Yet, 3 d after immunization with sheep RBCs, we observed a >10-fold rise in the number of clones that appeared to localize to individual zones. Remarkably, these differences largely disappeared at 4 d after immunization, when hallmarks of an ongoing immune response were still observed. These data suggest that in the absence of Ag, any repertoire differences observed between TCZs of the same host can largely be attributed to random clone distribution. Upon Ag challenge, TCR repertoires in TCZs first segregate and then homogenize within days. Such "transient mosaic" dynamics could be an important barrier for pathogen adaptation and spread during an immune response.
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Linfocitos T/inmunología , Animales , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunización/métodos , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/inmunología , Ovinos , Bazo/inmunologíaRESUMEN
CD8αα TCRαß+ intestinal intraepithelial lymphocytes play a critical role in promoting intestinal homeostasis, although mechanisms controlling their development and peripheral homeostasis remain unclear. In this study, we examined the spatiotemporal role of Bim in the thymic selection of CD8αα precursors and the fate of these cells in the periphery. We found that T cell-specific expression of Bim during early/cortical, but not late/medullary, thymic development controls the agonist selection of CD8αα precursors and limits their private TCRß repertoire. During this process, agonist-selected double-positive cells lose CD4/8 coreceptor expression and masquerade as double-negative (DN) TCRαßhi thymocytes. Although these DN thymocytes fail to re-express coreceptors after OP9-DL1 culture, they eventually mature and accumulate in the spleen where TCR and IL-15/STAT5 signaling promotes their conversion to CD8αα cells and their expression of gut-homing receptors. Adoptive transfer of splenic DN cells gives rise to CD8αα cells in the gut, establishing their precursor relationship in vivo. Interestingly, Bim does not restrict the IL-15-driven maturation of CD8αα cells that is critical for intestinal homeostasis. Thus, we found a temporal and tissue-specific role for Bim in limiting thymic agonist selection of CD8αα precursors and their TCRß repertoire, but not in the maintenance of CD8αα intraepithelial lymphocytes in the intestine.
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Proteína 11 Similar a Bcl2/biosíntesis , Diferenciación Celular/inmunología , Subgrupos de Linfocitos T/citología , Timocitos/citología , Traslado Adoptivo , Animales , Proteína 11 Similar a Bcl2/inmunología , Citometría de Flujo , Intestinos/citología , Intestinos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Subgrupos de Linfocitos T/inmunología , Timocitos/inmunologíaRESUMEN
Endogenous circadian clocks regulate 24-h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse-chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro, caspase activity was assayed in Per1/2-specific small interfering RNA-transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.-Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.
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Ritmo Circadiano , Homeostasis , Enfermedades Inflamatorias del Intestino/metabolismo , Animales , Caspasas/genética , Caspasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Imidazoles/farmacología , Indoles/farmacología , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Ratones , Ratones Mutantes , Mutación , Necrosis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/efectos adversos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Next generation sequencing (NGS) technologies enable studies and analyses of the diversity of both T and B cell receptors (TCR and BCR) in human and animal systems to elucidate immune functions in health and disease. Over the last few years, several algorithms and tools have been developed to support respective analyses of raw sequencing data of the immune repertoire. These tools focus on distinct aspects of the data processing and require a strong bioinformatics background. To facilitate the analysis of T and B cell repertoires by less experienced users, software is needed that combines the most common tools for repertoire analysis. RESULTS: We introduce a graphical user interface (GUI) providing a complete analysis pipeline for processing raw NGS data for human and animal TCR and BCR clonotype determination and advanced differential repertoire studies. It provides two applications. ClonoCalc prepares the raw data for downstream analyses. It combines a demultiplexer for barcode splitting and employs MiXCR for paired-end read merging and the extraction of human and animal TCR/BCR sequences. ClonoPlot wraps the R package tcR and further contributes self-developed plots for the descriptive comparative investigation of immune repertoires. CONCLUSION: This workflow reduces the amount of programming required to perform the respective analyses and supports both communication and training between scientists and technicians, and across scientific disciplines. The Open Source development in Java and R is modular and invites advanced users to extend its functionality. Software and documentation are freely available at https://bitbucket.org/ClonoSuite/clonocalc-plot .
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Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Interfaz Usuario-Computador , Algoritmos , Animales , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Internet , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Secuencia de ADNRESUMEN
Epidermolysis bullosa acquisita (EBA) is a difficult-to-treat subepidermal autoimmune blistering skin disease (AIBD) with circulating and tissue-bound anti-type VII collagen antibodies. Different reports have indicated an increased concentration of tumor necrosis factor alpha (TNF) in the serum and blister fluid of patients with subepidermal AIBDs. Furthermore, successful anti-TNF treatment has been reported for individual patients with AIBDs. Here, we show that in mice, induction of experimental EBA by repeated injections of rabbit-anti mouse type VII collagen antibodies led to increased expression of TNF in skin, as determined by real-time PCR and immunohistochemistry. To investigate if the increased TNF expression is of functional relevance in experimental EBA, we inhibited TNF function using the soluble TNF receptor fusion protein etanercept (Enbrel®) or a monoclonal antibody to murine TNF. Interestingly, mice receiving either of these two treatments showed significantly milder disease progression than controls. In addition, immunohistochemical staining demonstrated reduced numbers of macrophages in lesional skin in mice treated with TNF inhibitors compared to controls. Furthermore, etanercept treatment significantly reduced the disease progression in immunization-induced EBA. In conclusion, the increased expression of TNF in experimental EBA is of functional relevance, as both the prophylactic blockade of TNF and the therapeutic use of etanercept impaired the induction and progression of experimental EBA. Thus, TNF is likely to serve as a new therapeutic target for EBA and AIBDs with a similar pathogenesis.
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Although advances in medical care have significantly improved sepsis survival, sepsis remains the leading cause of death in the ICU. This is likely due to a lack of complete understanding of the pathophysiologic mechanisms that lead to dysfunctional immunity. Neutrophil derived microparticles (NDMPs) have been shown to be the predominant microparticle present at infectious and inflamed foci in human models, however their effect on the immune response to inflammation and infection is sepsis has not been fully elucidated. As NDMPs may be a potential diagnostic and therapeutic target, we sought to determine the impact NDMPs on the immune response to a murine polymicrobial sepsis. We found that peritoneal neutrophil numbers, bacterial loads, and NDMPs were increased in our abdominal sepsis model. When NDMPs were injected into septic mice, we observed increased bacterial load, decreased neutrophil recruitment, increased expression of IL-10 and worsened mortality. Furthermore, the NDMPs express phosphatidylserine and are ingested by F4/80 macrophages via a Tim-4 and MFG-E8 dependent mechanism. Finally, upon treatment, NDMPs decrease macrophage activation, increase IL-10 release and decrease macrophage numbers. Altogether, these data suggest that NDMPs enhance immune dysfunction in sepsis by blunting the function of neutrophils and macrophages, two key cell populations involved in the early immune response to infection. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.
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Micropartículas Derivadas de Células/inmunología , Neutrófilos/inmunología , Sepsis/inmunología , Animales , Carga Bacteriana , Micropartículas Derivadas de Células/patología , Micropartículas Derivadas de Células/trasplante , Modelos Animales de Enfermedad , Humanos , Interleucina-10/inmunología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Proteínas de la Membrana/inmunología , Ratones , Neutrófilos/patología , Fosfatidilserinas/inmunología , Sepsis/microbiología , Sepsis/patologíaRESUMEN
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.
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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
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.
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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
Although reports documented aberrant cytokine expression in autoimmune bullous dermatoses (AIBDs), cytokine-targeting therapies have not been established in these disorders. We showed previously that IL-6 treatment protected against tissue destruction in experimental epidermolysis bullosa acquisita (EBA), an AIBD caused by autoantibodies to type VII collagen (COL7). The anti-inflammatory effects of IL-6 were mediated by induction of IL-1ra, and prophylactic IL-1ra administration prevented blistering. In this article, we demonstrate elevated serum concentrations of IL-1ß in both mice with experimental EBA induced by injection of anti-COL7 IgG and in EBA patients. Increased IL-1α and IL-1ß expression also was observed in the skin of anti-COL7 IgG-injected wild-type mice compared with the significantly less diseased IL-1R-deficient or wild-type mice treated with the IL-1R antagonist anakinra or anti-IL-1ß. These findings suggested that IL-1 contributed to recruitment of inflammatory cells into the skin. Accordingly, the expression of ICAM-1 was decreased in IL-1R-deficient and anakinra-treated mice injected with anti-COL7. This effect appeared to be specifically attributable to IL-1 because anakinra blocked the upregulation of different endothelial adhesion molecules on IL-1-stimulated, but not on TNF-α-stimulated, cultured endothelial cells. Interestingly, injection of caspase-1/11-deficient mice with anti-COL7 IgG led to the same extent of skin lesions as in wild-type mice. Collectively, our data suggest that IL-1, independently of caspase-1, contributes to the pathogenesis of EBA. Because anti-IL-1ß in a prophylactic setting and anakinra in a quasi-therapeutic setting (i.e., when skin lesions had already developed) improved experimental EBA, IL-1 appears to be a potential therapeutic target for EBA and related AIBDs.
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Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Vesícula/inmunología , Caspasa 1/inmunología , Epidermólisis Ampollosa Adquirida/inmunología , Inmunoglobulina G/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Interleucina-1beta/inmunología , Animales , Autoanticuerpos/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Vesícula/genética , Caspasa 1/genética , Caspasas/genética , Caspasas/inmunología , Caspasas Iniciadoras , Colágeno Tipo VII/genética , Colágeno Tipo VII/inmunología , Epidermólisis Ampollosa Adquirida/genética , Inmunoglobulina G/genética , Molécula 1 de Adhesión Intercelular/genética , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/genética , Ratones , Ratones Noqueados , Piel/inmunología , Piel/patología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
BACKGROUND: Plasmacytosis (ie, an expansion of plasma cell populations to much greater than the homeostatic level) occurs in the context of various immune disorders and plasma cell neoplasia. This condition is often associated with immunodeficiency that causes increased susceptibility to severe infections. Yet a causative link between plasmacytosis and immunodeficiency has not been established. OBJECTIVE: Because recent studies have identified plasma cells as a relevant source of the immunosuppressive cytokine IL-10, we sought to investigate the role of IL-10 during conditions of polyclonal and neoplastic plasmacytosis for the regulation of immunity and its effect on inflammation and immunodeficiency. METHODS: We used flow cytometry, IL-10 reporter (Vert-X) and B cell-specific IL-10 knockout mice, migration assays, and antibody-mediated IL-10 receptor blockade to study plasmacytosis-associated IL-10 expression and its effect on inflammation and Streptococcus pneumoniae infection in mice. ELISA was used to quantify IL-10 levels in patients with myeloma. RESULTS: IL-10 production was a common feature of normal and neoplastic plasma cells in mice, and IL-10 levels increased with myeloma progression in patients. IL-10 directly inhibited neutrophil migration toward the anaphylatoxin C5a and suppressed neutrophil-dependent inflammation in a murine model of autoimmune disease. MOPC.315.BM murine myeloma leads to an increased incidence of bacterial infection in the airways, which was reversed after IL-10 receptor blockade. CONCLUSION: We provide evidence that plasmacytosis-associated overexpression of IL-10 inhibits neutrophil migration and neutrophil-mediated inflammation but also promotes immunodeficiency.
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Interleucina-10/inmunología , Células Plasmáticas/inmunología , Animales , Línea Celular Tumoral , Complemento C5a/inmunología , Humanos , Enfermedades del Sistema Inmune/inmunología , Inflamación/inmunología , Interleucina-10/genética , Trastornos Leucocíticos/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Mieloma Múltiple/inmunología , Neutrófilos/inmunología , Infecciones Neumocócicas/inmunologíaRESUMEN
Microfibrillar-associated protein 4 (MFAP4) is an extracellular protein belonging to the fibrinogen-related protein superfamily and is recognized as an integrin ligand with suggested functions in pulmonary and vascular tissue homeostasis. MFAP4 expression in the spleen is increased during infections; however, the significance of MFAP4 for the function of the spleen is unknown. Immunohistochemistry, morphometry and real-time RT-PCR were used to analyze wild-type and MFAP4-deficient spleens. In addition, they were compared with splenic tissue, which was newly formed 8 weeks after avascular implantation into adult mice in order to obtain information about the role of MFAP4 in the formation of splenic tissue during ontogeny and adult life. The present study shows that MFAP4 is co-localized with laminin in the B- and T-cell zones of the spleen, in addition to capsular and trabecular expression. MFAP4 is most likely produced by fibroblastic reticulum cells and follicular dendritic cells of the spleen but can also be imported via the blood from other tissues. The development of splenic tissue is not disturbed in MFAP4-deficient mice. However, in splenic tissue regenerating under MFAP4-deficient conditions, the number of FDCs is significantly decreased but is corrected by MFAP4 imported from other tissues. No differences were observed for lymphocyte numbers or splenic structure. The data indicate that MFAP4 promotes FDC development in regenerating splenic tissue and warrant further investigations regarding the MFAP4 dependency of splenic B-cell maturation.
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Envejecimiento/metabolismo , Proteínas Portadoras/metabolismo , Embrión de Mamíferos/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Bazo/embriología , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Proliferación Celular , Femenino , Centro Germinal/citología , Laminina/metabolismo , Ratones Endogámicos C57BL , Regeneración , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune blistering skin disease characterized by autoantibodies against type VII collagen (COL7). Immunization of SJL/J mice with recombinant murine COL7 results in break of tolerance and skin blisters. Strikingly, despite circulating autoantibodies, the same genetic background and identical environmental conditions, 20% of mice remain healthy. To elucidate the regulation of the transition from the presence of autoantibodies to overt autoimmune disease, we characterized the innate and adaptive immune response of mice that remain healthy after immunization and compared it to mice that developed skin disease. Both clinically healthy and diseased SJL/J mice showed circulating autoantibodies and deposition of complement-ï¬xing IgG2c autoantibodies and C3 at the dermal-epidermal junction. However, only in diseased animals significant neutrophil infiltration and increase in FcgRIV expression were observed in the skin. In contrast, the expression of T cell signature cytokines in the T cell zone of the draining lymph node was comparable between clinically healthy and diseased animals after immunization. Surprisingly, health was associated with a decreased expression of CD11c, TNFA and KC (CXCL1) in the skin prior to immunization and could be predicted with a negative predictive value of >80%. Furthermore, mice that did not develop clinical disease showed a significantly higher richness and distinctly clustered diversity of their skin microbiota before immunization. Our data indicate that the decision whether blisters develop in the presence of autoantibodies is governed in the skin rather than in the lymph node, and that a greater richness of cutaneous bacterial species appears to be protective.
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Autoanticuerpos/inmunología , Epidermólisis Ampollosa Adquirida/etiología , Epidermólisis Ampollosa Adquirida/patología , Microbiota , Animales , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Interacciones Huésped-Patógeno , Inmunidad Innata , Inmunización , Metagenómica , Ratones , Curva ROC , Piel/inmunología , Piel/microbiología , Piel/patologíaRESUMEN
Genetic studies have added to the understanding of complex diseases. Here, we used a combined genetic approach for risk-loci identification in a prototypic, organ-specific, autoimmune disease, namely experimental epidermolysis bullosa acquisita (EBA), in which autoantibodies to type VII collagen (COL7) and neutrophil activation cause mucocutaneous blisters. Anti-COL7 IgG induced moderate blistering in most inbred mouse strains, while some showed severe disease or were completely protected. Using publicly available genotyping data, we identified haplotype blocks that control blistering and confirmed two haplotype blocks in outbred mice. To identify the blistering-associated genes, haplotype blocks encoding genes that are differentially expressed in EBA-affected skin were considered. This procedure identified nine genes, including retinoid-related orphan receptor alpha (RORα), known to be involved in neurological development and function. After anti-COL7 IgG injection, RORα+/- mice showed reduced blistering and homozygous mice were completely resistant to EBA induction. Furthermore, pharmacological RORα inhibition dose-dependently blocked reactive oxygen species (ROS) release from activated neutrophils but did not affect migration or phagocytosis. Thus, forward genomics combined with multiple validation steps identifies RORα to be essential to drive inflammation in experimental EBA.
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Epidermólisis Ampollosa Adquirida/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Piel/metabolismo , Animales , Autoanticuerpos/inmunología , Colágeno Tipo VII/inmunología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Agonismo Parcial de Drogas , Epidermólisis Ampollosa Adquirida/genética , Epidermólisis Ampollosa Adquirida/inmunología , Epidermólisis Ampollosa Adquirida/patología , Predisposición Genética a la Enfermedad , Genómica/métodos , Haplotipos , Heterocigoto , Homocigoto , Inmunoglobulina G/inmunología , Ratones Noqueados , Activación Neutrófila , Neutrófilos/inmunología , Neutrófilos/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/deficiencia , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Especificidad de la Especie , Sulfonamidas/farmacología , Tiofenos/farmacología , Factores de TiempoRESUMEN
CD4(+) T (helper) cells migrate in huge numbers through lymphoid organs. However, little is known about traffic routes and kinetics of CD4(+) T-cell subsets within different organ compartments. Such information is important because there are indications that CD4(+) T cells may influence the function of microenvironments depending on their developmental stage. Therefore, we investigated the migration of resting (naïve), activated, and recently activated (memory) CD4(+) T cells through the different compartments of the spleen. Resting and recently activated CD4(+) T cells were separated from thoracic duct lymph and activated CD4(+) T cells were generated in vitro by cross-linking the T-cell receptor and CD28. The present study shows that all three CD4(+) T-cell subsets selectively accumulate in the T-cell zone of the spleen. However, only activated T cells induce the formation of germinal centers (GCs) and autoantibodies in rats and mice. Our results suggest that in a two-step process they first activate B cells independent of the T-cell receptor repertoire and CD40 ligand (CD154) expression. The activated B cells then form GCs whereby CD154-dependent T-cell help is needed. Thus, activated T cells may contribute to the development of autoimmune diseases by activating autoreactive B cells in an Ag-independent manner.
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Autoanticuerpos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Centro Germinal/inmunología , Bazo/inmunología , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Efecto Espectador , Ligando de CD40/genética , Células Cultivadas , Memoria Inmunológica , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Endogámicas LewRESUMEN
In autoimmune bullous dermatoses (AIBD), autoantibodies induce blisters on skin or mucous membranes, or both. Mechanisms of continued autoantibody production and blistering have been well characterized using AIBD animal models. Mechanisms leading to the initial autoantibody production, however, have not been investigated in detail. Epidermolysis bullosa acquisita (EBA) is an AIBD associated with autoantibodies to type VII collagen (COL7). The majority of EBA patients' sera recognize the noncollagenous domain 1, including the von Willebrand factor A-like domain 2 (vWFA2). In experimental EBA induced by immunization with GST-COL7, disease manifestation depended on the genetic background, a Th1 polarization, and the GST-tag. In this model, nude mice neither produced autoantibodies nor blisters. It has remained uncertain which APC and T cell subsets are required for EBA induction. We established a novel EBA model by immunization with vWFA2 fused to intein (lacking the GST-tag). All tested mouse strains developed autoantibodies, but blisters were exclusively observed in mice carrying H2s. In immunized mice, CD4 T cells specific for vWFA2 were detected, and their induction required presence of B cells, dendritic cells, and macrophages. Anti-vWFA2 autoantibodies located at the lamina densa bound to the dermal side of salt-split skin and induced blisters when transferred into healthy mice. Absence of CD8 T cells at time of immunization had no effect, whereas depletion of CD4 T cells during the same time period delayed autoantibody production and blisters. Collectively, we demonstrate the pathogenic relevance of Abs targeting the vWFA2 domain of COL7 and show the requirement of APC-induced CD4 T cells to induce experimental EBA.
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Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Epidermólisis Ampollosa Adquirida/inmunología , Macrófagos/inmunología , Animales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoinmunidad/inmunología , Biomarcadores de Tumor/inmunología , Proteínas de Unión al Calcio , Colágeno Tipo VII/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Matriz Extracelular/inmunología , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Estructura Terciaria de Proteína , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND & AIMS: Dysregulated energy homeostasis in the intestinal mucosa frequently is observed in patients with ulcerative colitis (UC). Intestinal tissues from these patients have reduced activity of the mitochondrial oxidative phosphorylation (OXPHOS) complex, so mitochondrial dysfunction could contribute to the pathogenesis of UC. However, little is known about the mechanisms by which OXPHOS activity could be altered. We used conplastic mice, which have identical nuclear but different mitochondrial genomes, to investigate activities of the OXPHOS complex. METHODS: Colitis was induced in C57BL/6J wild-type (B6.B6) and 3 strains of conplastic mice (B6.NZB, B6.NOD, and B6.AKR) by administration of dextran sodium sulfate or rectal application of trinitrobenzene sulfonate. Colon tissues were collected and analyzed by histopathology, immunohistochemical analysis, and immunoblot analysis; we also measured mucosal levels of adenosine triphosphate (ATP) and reactive oxygen species, OXPHOS complex activity, and epithelial cell proliferation and apoptosis. RESULTS: We identified mice with increased mucosal OXPHOS complex activities and levels of ATP. These mice developed less-severe colitis after administration of dextran sodium sulfate or trinitrobenzene sulfonate than mice with lower mucosal levels of ATP. Colon tissues from these mice also had increased enterocyte proliferation and transcription factor nuclear factor-κB activity, which have been shown to protect the mucosal barrier-defects in these processes have been associated with inflammatory bowel disease. CONCLUSIONS: Variants in mitochondrial DNA that increase mucosal levels of ATP protect mice from colitis. Increasing mitochondrial ATP synthesis in intestinal epithelial cells could be a therapeutic approach for UC.
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Colitis/genética , ADN Mitocondrial/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo Genético/genética , Adenosina Trifosfato/metabolismo , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos NZB , Especies Reactivas de Oxígeno/metabolismo , Ácido Trinitrobencenosulfónico/efectos adversosRESUMEN
Epidermolysis bullosa acquisita (EBA) is a muco-cutaneous autoimmune disease characterized and caused by autoantibodies targeting type VII collagen (COL7). The treatment of EBA is notoriously difficult, with a median time to remission of 9 months. In preclinical EBA models, we previously discovered that depletion of regulatory T cells (Treg) enhances autoantibody-induced, neutrophil-mediated inflammation and blistering. Increased EBA severity in Treg-depleted mice was accompanied by an increased cutaneous expression of interferon gamma (IFN-γ). The functional relevance of IFN-γ in EBA pathogenesis had been unknown. Given that emapalumab, an anti-IFN-γ antibody, is approved for primary hemophagocytic lymphohistiocytosis patients, we sought to assess the therapeutic potential of IFN-γ inhibition in EBA. Specifically, we evaluated if IFN-γ inhibition has modulatory effects on skin inflammation in a pre-clinical EBA model, based on the transfer of COL7 antibodies into mice. Compared to isotype control antibody, anti-IFN-γ treatment significantly reduced clinical disease manifestation in experimental EBA. Clinical improvement was associated with a reduced dermal infiltrate, especially Ly6G+ neutrophils. On the molecular level, we noted few changes. Apart from reduced CXCL1 serum concentrations, which has been demonstrated to promote skin inflammation in EBA, the expression of cytokines was unaltered in the serum and skin following IFN-γ blockade. This validates IFN-γ as a potential therapeutic target in EBA, and possibly other diseases with a similar pathogenesis, such as bullous pemphigoid and mucous membrane pemphigoid.
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Colágeno Tipo VII , Modelos Animales de Enfermedad , Epidermólisis Ampollosa Adquirida , Interferón gamma , Animales , Epidermólisis Ampollosa Adquirida/inmunología , Epidermólisis Ampollosa Adquirida/tratamiento farmacológico , Interferón gamma/metabolismo , Ratones , Colágeno Tipo VII/inmunología , Piel/inmunología , Piel/patología , Piel/metabolismo , Autoanticuerpos/inmunología , Femenino , Linfocitos T Reguladores/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismoRESUMEN
Cytokines are key regulators of physiological inflammatory responses, while aberrant cytokine expression contributes to pathogenesis of autoimmune diseases. We noted increased IL-6 levels in human and murine epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune bullous dermatoses (AIBD) induced by autoantibodies to type VII collagen (COL7). In contrast to rheumatoid arthritis, blockade of IL-6 led to strikingly enhanced experimental EBA, while treatment with recombinant IL-6 was protective. This was due to classical IL-6 signalling and independent of IL-6 trans-signalling, as treatment of mice with sgp130Fc had no impact on EBA manifestation. Induction of EBA in mice led to increased IL-1ra levels in skin and serum, while blockade of IL-6 completely inhibited IL-1ra expression induced by autoantibodies to COL7. In line, treatment of mice with EBA with recombinant IL-6 induced IL-1ra concentrations exceeding those of untreated animals with EBA, and IL-1ra (anakinra) administration significantly impaired experimental EBA induction. We here identified a novel anti-inflammatory pathway in an organ-specific autoimmune disease. Modulation of this IL-1ra pathway by classical IL-6 signalling demonstrates anti-inflammatory and protective activities of IL-6 in vivo.
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Enfermedades Autoinmunes/prevención & control , Epidermólisis Ampollosa Adquirida/prevención & control , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-6/farmacología , Animales , Apoptosis , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/sangre , Colágeno Tipo VII/inmunología , Citocinas/sangre , Modelos Animales de Enfermedad , Epidermólisis Ampollosa Adquirida/sangre , Epidermólisis Ampollosa Adquirida/inmunología , Humanos , Inflamación , Proteína Antagonista del Receptor de Interleucina 1/biosíntesis , Proteína Antagonista del Receptor de Interleucina 1/sangre , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/farmacología , Transducción de Señal/inmunología , Piel/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/inmunologíaRESUMEN
Autoantibody-mediated diseases are clinically heterogeneous and often fail conventional therapeutic strategies. Gene expression profiling has helped to identify new molecular pathways in these diseases, although their potential as treatment targets largely remains to be functionally validated. Based on weighted gene co-expression network analysis, we determined the transcriptional network in experimental epidermolysis bullosa acquisita (EBA), a paradigm of an antibody-mediated organ-specific autoimmune disease characterized by autoantibodies directed against type VII collagen. We identified 33 distinct and differentially expressed modules, including Fcγ receptor (FcγR) IV and components of the neutrophil-associated enzyme system in autoantibody transfer-induced EBA. Validation experiments, including functional analysis, demonstrated that FcγRIV expression on neutrophils crucially contributes to autoantibody-induced tissue injury in the transfer model of EBA. Mice lacking the common γ-chain of activating FcγRs, deficient in FcγRIV or treated with FcγRIV function blocking antibody, but not mice deficient in FcγRI, FcγRIIB, FcγRIII or both FcγRI and FcγRIII, were effectively protected from EBA. Skin disease was restored in γ-chain-deficient mice locally reconstituted with neutrophils from wild-type, but not from γ-chain-deficient, mice. Our findings both genetically and functionally identify a novel disease-related molecule, FcγRIV, in an autoantibody-mediated disorder, which may be of importance for the development of novel targeted therapies.