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T cell help is a crucial component of the normal humoral immune response, yet whether it promotes or restrains autoreactive B cell responses remains unclear. Here, we observe that autoreactive germinal centers require T cell help for their formation and persistence. Using retrogenic chimeras transduced with candidate TCRs, we demonstrate that a follicular T cell repertoire restricted to a single autoreactive TCR, but not a foreign antigen-specific TCR, is sufficient to initiate autoreactive germinal centers. Follicular T cell specificity influences the breadth of epitope spreading by regulating wild-type B cell entry into autoreactive germinal centers. These results demonstrate that TCR-dependent T cell help can promote loss of B cell tolerance and that epitope spreading is determined by TCR specificity.
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Linfócitos B , Linfócitos T , Receptores de Antígenos de Linfócitos T , Antígenos , EpitoposRESUMO
In systemic lupus erythematosus, recent findings highlight the extrafollicular (EF) pathway as prominent origin of autoantibody-secreting cells (ASCs). CD21loCD11c+ B cells, associated with aging, infection, and autoimmunity, are contributors to autoreactive EF ASCs but have an obscure developmental trajectory. To study EF kinetics of autoreactive B cell in tissue, we adoptively transferred WT and gene knockout B cell populations into the 564Igi mice - an autoreactive host enriched with autoantigens and T cell help. Time-stamped analyses revealed TLR7 dependence in early escape of peripheral B cell tolerance and establishment of a pre-ASC division program. We propose CD21lo cells as precursors to EF ASCs due to their elevated TLR7 sensitivity and proliferative nature. Blocking receptor function reversed CD21 loss and reduced effector cell generation, portraying CD21 as a differentiation initiator and a possible target for autoreactive B cell suppression. Repertoire analysis further delineated proto-autoreactive B cell selection and receptor evolution toward self-reactivity. This work elucidates receptor and clonal dynamics in EF development of autoreactive B cells, and establishes modular, native systems to probe mechanisms of autoreactivity.
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Among systemic lupus erythematosus (SLE) patients, neuropsychiatric symptoms are highly prevalent, being observed in up to 80% of adult and 95% of pediatric patients. Type 1 interferons, particularly interferon alpha (IFNα), have been implicated in the pathogenesis of SLE and its associated neuropsychiatric symptoms (NPSLE). However, it remains unclear how type 1 interferon signaling in the central nervous system (CNS) might result in neuropsychiatric sequelae. In this study, we validate an NPSLE mouse model and find an elevated peripheral type 1 interferon signature alongside clinically relevant NPSLE symptoms such as anxiety and fatigue. Unbiased single-nucleus sequencing of the hindbrain and hippocampus revealed that interferon-stimulated genes (ISGs) were among the most highly upregulated genes in both regions and that gene pathways involved in cellular interaction and neuronal development were generally repressed among astrocytes, oligodendrocytes, and neurons. Using image-based spatial transcriptomics, we found that the type 1 interferon signature is enriched as spatially distinct patches within the brain parenchyma of these mice. Our results suggest that type 1 interferon in the CNS may play an important mechanistic role in mediating NPSLE behavioral phenotypes by repressing general cellular communication pathways, and that type 1 interferon signaling modulators are a potential therapeutic option for NPSLE.
Assuntos
Lúpus Eritematoso Sistêmico , Vasculite Associada ao Lúpus do Sistema Nervoso Central , Adulto , Humanos , Criança , Animais , Camundongos , Vasculite Associada ao Lúpus do Sistema Nervoso Central/complicações , Vasculite Associada ao Lúpus do Sistema Nervoso Central/diagnóstico , Vasculite Associada ao Lúpus do Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Lúpus Eritematoso Sistêmico/complicações , Encéfalo/metabolismo , Interferon-alfa/metabolismoRESUMO
Affinity matured self-reactive antibodies are found in autoimmune diseases like systemic lupus erythematous. Here, we used fate-mapping reporter mice and single-cell transcriptomics coupled to antibody repertoire analysis to characterize the post-germinal center (GC) B cell compartment in a new mouse model of autoimmunity. Antibody-secreting cells (ASCs) and memory B cells (MemBs) from spontaneous GCs grouped into multiple subclusters. ASCs matured into two terminal clusters, with distinct secretion, antibody repertoire and metabolic profiles. MemBs contained FCRL5+ and CD23+ subsets, with different in vivo localization in the spleen. GC-derived FCRL5+ MemBs share transcriptomic and repertoire properties with atypical B cells found in aging and infection and localize to the marginal zone, suggesting a similar contribution to recall responses. While transcriptomically diverse, ASC and MemB subsets maintained an underlying clonal redundancy. Therefore, self-reactive clones could escape subset-targeting therapy by perpetuation of self-reactivity in distinct subsets.
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Doenças Autoimunes , Linfócitos B , Camundongos , Animais , Centro Germinativo , Autoimunidade , AutoanticorposRESUMO
bioRxiv has withdrawn this manuscript because it was posted without the consent of all authors. If you have any questions, please contact the corresponding author.
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The complement component 4 (C4) gene is linked to schizophrenia and synaptic refinement. In humans, greater expression of C4A in the brain is associated with an increased risk of schizophrenia. To investigate this genetic finding and address how C4A shapes brain circuits in vivo, here, we generated a mouse model with primate-lineage-specific isoforms of C4, human C4A and/or C4B. Human C4A bound synapses more efficiently than C4B. C4A (but not C4B) rescued the visual system synaptic refinement deficits of C4 knockout mice. Intriguingly, mice without C4 had normal numbers of cortical synapses, which suggests that complement is not required for normal developmental synaptic pruning. However, overexpressing C4A in mice reduced cortical synapse density, increased microglial engulfment of synapses and altered mouse behavior. These results suggest that increased C4A-mediated synaptic elimination results in abnormal brain circuits and behavior. Understanding pathological overpruning mechanisms has important therapeutic implications in disease conditions such as schizophrenia.
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Comportamento Animal , Complemento C4/genética , Esquizofrenia/genética , Psicologia do Esquizofrênico , Sinapses/patologia , Animais , Complemento C4/biossíntese , Espinhas Dendríticas/patologia , Depressão/psicologia , Feminino , Dosagem de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/patologia , Rede Nervosa/patologia , Desempenho Psicomotor , Esquizofrenia/patologia , Sinaptossomos/patologiaRESUMO
Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.
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Auxilinas/metabolismo , Vesículas Revestidas por Clatrina/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Auxilinas/genética , Células COS , Chlorocebus aethiops , Vesículas Revestidas por Clatrina/genética , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfatidilinositóis/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Transdução de Sinais , Fatores de TempoRESUMO
This article mainly reports the process of clinical diagnosis and treatment of a misdiagnosed Listeria monocytogenes meningoencephalitis. The patient's condition is aggravated because of the ineffective prophase therapy. In the later stage, we were mainly through combined antibiotics and given proper routes of administration, so that patient can recover quickly.
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Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.
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Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Auxilinas/metabolismo , Células COS , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Invaginações Revestidas da Membrana Celular/química , Endossomos/química , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/análise , Fosfatidilinositóis/química , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfotransferases/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Progressive multifocal leukoencephalopathy (PML) is a debilitating disease resulting from infection of oligodendrocytes by the JC polyomavirus (JCPyV). Currently, there is no anti-viral therapeutic available against JCPyV infection. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9) is a genome editing tool capable of introducing sequence specific breaks in double stranded DNA. Here we show that the CRISPR/Cas9 system can restrict the JCPyV life cycle in cultured cells. We utilized CRISPR/Cas9 to target the noncoding control region and the late gene open reading frame of the JCPyV genome. We found significant inhibition of virus replication and viral protein expression in cells recipient of Cas9 together with JCPyV-specific single-guide RNA delivered prior to or after JCPyV infection.
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Edição de Genes/métodos , Vírus JC/fisiologia , Leucoencefalopatia Multifocal Progressiva/virologia , Infecções por Polyomavirus/virologia , Sistemas CRISPR-Cas , Genoma Viral/efeitos dos fármacos , Células HEK293 , Humanos , Vírus JC/efeitos dos fármacos , Vírus JC/genética , Fases de Leitura Aberta/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/farmacologia , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
UNLABELLED: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE: The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.
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Antivirais/farmacologia , Benzamidas/farmacologia , Descoberta de Drogas , Tioureia/análogos & derivados , Internalização do Vírus/efeitos dos fármacos , Actinas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Clatrina/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/virologia , Técnicas de Inativação de Genes , Febre Hemorrágica Americana/genética , Febre Hemorrágica Americana/metabolismo , Febre Hemorrágica Americana/virologia , Humanos , Vírus Junin/efeitos dos fármacos , Vírus Junin/fisiologia , Camundongos , Ligação Proteica , Transporte Proteico , Proteólise , Ribonucleoproteínas/metabolismo , Tioureia/farmacologia , Carga Viral , Proteínas Virais/metabolismo , Ligação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. Using a BCR knock-in mouse strain, we identify a pathway by which B-cell selection to nucleolar self-antigens is complement dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B-cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles, and a greater propensity to form self-reactive GCs. Using mixed BM chimeras, we found that the myeloid compartment was sufficient to restore negative selection in the autoreactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self-reactive B-cell clones leading to increased spontaneous formation of GCs.
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Linfócitos B/imunologia , Complemento C4/imunologia , Tolerância Imunológica , Ribonucleoproteínas/imunologia , Animais , Apoptose , Autoantígenos/imunologia , Autoimunidade , Linfócitos B/metabolismo , Células da Medula Óssea/imunologia , Complemento C4/deficiência , Complemento C4/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Região Organizadora do Nucléolo/imunologia , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
Animals lacking complement factors C1q, C2, C3, or C4 have severely impaired Ab responses, suggesting a major role for the classic pathway. The classic pathway is primarily initiated by antigen-Ab complexes. Therefore, its role for primary Ab responses seems paradoxical because only low amounts of specific Abs are present in naive animals. A possible explanation could be that the classic pathway is initiated by IgM from naive mice, binding with sufficient avidity to the antigen. To test this hypothesis, a knock-in mouse strain, Cµ13, with a point mutation in the gene encoding the third constant domain of the µ-heavy chain was constructed. These mice produce IgM in which proline in position 436 is substituted with serine, a mutation previously shown to abrogate the ability of mouse IgM to activate complement. Unexpectedly, the Ab response to sheep erythrocytes and keyhole limpet hemocyanin in Cµ13 mice was similar to that in WT mice. Thus, although secreted IgM and the classic pathway activation are both required for the normal primary Ab response, this does not require that IgM activate C. This led us to test Ab responses in animals lacking one of three other endogenous activators of the classic pathway: specific intracellular adhesion molecule-grabbing nonintegrin R1, serum amyloid P component, and C-reactive protein. Ab responses were also normal in these animals.
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Formação de Anticorpos/imunologia , Via Clássica do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Cadeias mu de Imunoglobulina/imunologia , Animais , Anticorpos Monoclonais/imunologia , Proteína C-Reativa/imunologia , Cromatografia em Agarose , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Técnicas de Introdução de Genes , Cadeias mu de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Mutação de Sentido Incorreto/genética , Reação em Cadeia da Polimerase , Componente Amiloide P Sérico/imunologiaRESUMO
Complement receptors (CRs) CD21 and CD35 form a coreceptor with CD19 and CD81 on murine B cells that when coligated with the B-cell receptor lowers the threshold of activation by several orders of magnitude. This intrinsic signaling role is thought to explain the impaired humoral immunity of mice bearing deficiency in CRs. However, CRs have additional roles on B cells independent of CD19, such as transport of C3-coated immune complexes and regulation of C4 and C3 convertase. To test whether association of CR with CD19 is necessary for their intrinsic activation-enhancing role, knockin mice expressing mutant receptors, Cr2(Delta/Deltagfp), that bind C3 ligands but do not signal through CD19 were constructed. We found that uncoupling of CR and CD19 significantly diminishes survival of germinal center B cells and secondary antibody titers. However, B memory is less impaired relative to mice bearing a complete deficiency in CRs on B cells. These findings confirm the importance of interaction of CR and CD19 for coreceptor activity in humoral immunity but identify a role for CR in B-cell memory independent of CD19.
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Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Receptores de Complemento 3d/metabolismo , Animais , Formação de Anticorpos/imunologia , Antígenos CD19/genética , Linfócitos B/citologia , Linfócitos B/imunologia , Medula Óssea/imunologia , Medula Óssea/metabolismo , Transplante de Medula Óssea , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Haptenos , Hemocianinas/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imunização , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/metabolismo , Receptores de Complemento 3d/genética , Baço/imunologia , Baço/metabolismoRESUMO
Reperfusion injury of ischemic tissue represents an acute inflammatory response that can cause significant morbidity and mortality. The mechanism of injury is not fully elucidated, but recent studies indicate an important role for natural antibody and the classical pathway of complement. To test the hypothesis that injury is initiated by specific IgM, we have screened a panel of IgM-producing hybridomas prepared from peritoneal cells enriched in B-1 cells. One clone, CM22, was identified that could restore pathogenic injury in RAG-1(-/-) mice in an intestinal model of ischemia/reperfusion (I/R). In situ activation of the classical pathway of complement was evident by deposition of IgM, complement C4, and C3 in damaged tissue after passive transfer of CM22 IgM. Sequence analysis of CM22 Ig heavy and light chains showed germ-line configurations with high homology to a V(H) sequence from the B-1 repertoire and a V(K) of a known polyreactive natural IgM. These data provide definitive evidence that I/R injury can be initiated by clonally specific natural IgM that activates the classical pathway of complement. This finding opens an avenue for identification of I/R-specific self-antigen(s) and early prevention of injury.
Assuntos
Imunoglobulina M/imunologia , Intestinos/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Linfócitos B/imunologia , Proteínas de Homeodomínio/genética , Hibridomas/imunologia , Camundongos , Camundongos Knockout , Dados de Sequência MolecularRESUMO
Complement and Fc receptor (FcR)-positive cells mediate effector functions of antibodies. Antibody-dependent immunity against the melanosome membrane glycoprotein gp75/tyrosinase-related protein-1 (TYRP-1) of melanocytes leads to autoimmune hypopigmentation (vitiligo) in mice. Hypopigmentation occurred in mice deficient in activating FcR containing the common gamma subunit (Fc gamma R gamma(-/-)) and in mice deficient in the C3 complement component. Mice doubly deficient in both Fc gamma R gamma and C3 did not develop hypopigmentation, suggesting that complement and Fc gamma R formed redundant mechanisms. Following passive immunization with antibody, no further adaptive immune responses were required. Chimeric Fc gamma R gamma(-/-),C3(-/-) mice reconstituted with bone marrow from either Fc gamma R gamma(-/-) or C3(-/-) mice or adoptively transferred with Fc gamma R gamma(+/-) macrophages did develop antibody-mediated hypopigmentation. Thus, either complement or macrophages expressing activating Fc gamma R can independently and alternatively mediate disease in a model of autoimmune vitiligo.
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Ativação do Complemento/fisiologia , Glicoproteínas de Membrana/imunologia , Oxirredutases , Receptores Fc/fisiologia , Vitiligo/imunologia , Transferência Adotiva , Animais , Complemento C3/imunologia , Genótipo , Imunização Passiva , Macrófagos/imunologia , Melanócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes de Fusão/imunologiaRESUMO
The role of complement C3 in mediating systemic lupus erythematosus (SLE) was examined using a double-knockout C3(null)C4(null) Fas (CD95)-deficient mouse model. Results from this study reveal significant lymphadenopathy, splenomegaly, elevated titers of anti-nuclear Abs and anti-dsDNA Abs, an increased number of anti-dsDNA-producing cells in ELISPOT assay, as well as severe glomerulonephritis in the double-deficient mice. Based on these clinical, serological, and histological parameters, we find that autoimmune disease in the double-knockout group is similar in severity to that in C4(null) lpr mice, but not to that in C3(null) lpr mice. The development of severe SLE in the absence of both classical and alternative complement pathways suggests that it is the absence of C4, and not the presence of C3, that is critical in SLE pathogenesis. Thus, complement C4 provides an important protective role against the development of SLE.