RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Systemic lupus erythematosus (SLE) is an incurable autoimmune disease characterized by autoantibody deposition in tissues such as kidney, skin and lungs. Notably, up to 75% of patients with SLE experience neuropsychiatric symptoms that range from anxiety, depression and cognitive impairment to seizures and, in rare cases, psychosis-collectively this is referred to as central nervous system (CNS) lupus. In some cases, certain autoantibodies, such as anti-NMDAR or anti-phospholipid antibodies, promote CNS lupus. However, in most patients, the mechanisms that underlie these symptoms are unknown. CNS lupus typically presents at lupus diagnosis or within the first year, suggesting that early factors contributing to peripheral autoimmunity may promote CNS lupus symptoms. Here we report behavioural phenotypes and synapse loss in lupus-prone mice that are prevented by blocking type I interferon (IFN) signalling. Furthermore, we show that type I IFN stimulates microglia to become reactive and engulf neuronal and synaptic material in lupus-prone mice. These findings and our observation of increased type I IFN signalling in post-mortem hippocampal brain sections from patients with SLE may instruct the evaluation of ongoing clinical trials of anifrolumab, a type I IFN-receptor antagonist. Moreover, identification of IFN-driven microglia-dependent synapse loss, along with microglia transcriptome data, connects CNS lupus with other CNS diseases and provides an explanation for the neurological symptoms observed in some patients with SLE.
Assuntos
Interferon Tipo I/imunologia , Vasculite Associada ao Lúpus do Sistema Nervoso Central/imunologia , Vasculite Associada ao Lúpus do Sistema Nervoso Central/patologia , Microglia/imunologia , Microglia/patologia , Sinapses/patologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Comportamento Animal , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Interferon Tipo I/antagonistas & inibidores , Vasculite Associada ao Lúpus do Sistema Nervoso Central/psicologia , Masculino , Camundongos , Microglia/metabolismo , Fenótipo , Transdução de Sinais , Sinapses/imunologia , TranscriptomaRESUMO
The classical and lectin pathways of the complement system are important for the elimination of pathogens and apoptotic cells and stimulation of the adaptive immune system. Upon activation of these pathways, complement component C4 is proteolytically cleaved, and the major product C4b is deposited on the activator, enabling assembly of a C3 convertase and downstream alternative pathway amplification. Although excessive activation of the lectin and classical pathways contributes to multiple autoimmune and inflammatory diseases and overexpression of a C4 isoform has recently been linked to schizophrenia, a C4 inhibitor and structural characterization of the convertase formed by C4b is lacking. In this study, we present the nanobody hC4Nb8 that binds with picomolar affinity to human C4b and potently inhibits in vitro complement C3 deposition through the classical and lectin pathways in human serum and in mouse serum. The crystal structure of the C4b:hC4Nb8 complex and a three-dimensional reconstruction of the C4bC2 proconvertase obtained by electron microscopy together rationalize how hC4Nb8 prevents proconvertase assembly through recognition of a neoepitope exposed in C4b and reveals a unique C2 conformation compared with the alternative pathway proconvertase. On human induced pluripotent stem cell-derived neurons, the nanobody prevents C3 deposition through the classical pathway. Furthermore, hC4Nb8 inhibits the classical pathway-mediated immune complex delivery to follicular dendritic cells in vivo. The hC4Nb8 represents a novel ultrahigh-affinity inhibitor of the classical and lectin pathways of the complement cascade under both in vitro and in vivo conditions.
Assuntos
Convertases de Complemento C3-C5 da Via Clássica/metabolismo , Complemento C3/metabolismo , Complemento C4b/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurônios/fisiologia , Esquizofrenia/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/metabolismo , Diferenciação Celular , Células Cultivadas , Ativação do Complemento , Complemento C4b/genética , Complemento C4b/imunologia , Humanos , Camundongos , Camundongos Knockout , Multimerização Proteica , Regulação para CimaRESUMO
Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia's strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.
Assuntos
Complemento C4/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Esquizofrenia/genética , Alelos , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Complemento C4/química , Via Clássica do Complemento , Dendritos/metabolismo , Dosagem de Genes/genética , Regulação da Expressão Gênica/genética , Haplótipos/genética , Humanos , Complexo Principal de Histocompatibilidade/genética , Camundongos , Modelos Animais , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Risco , Esquizofrenia/patologia , Sinapses/metabolismoRESUMO
RATIONALE: After a stroke, patients frequently experience altered systemic immunity resulting in peripheral immunosuppression and higher susceptibility to infections, which is at least partly attributed to lymphopenia. The mechanisms that profoundly change the systemic leukocyte repertoire after stroke are incompletely understood. Emerging evidence indicates that stroke alters hematopoietic output of the bone marrow. OBJECTIVE: To explore the mechanisms that lead to defects of B lymphopoiesis after ischemic stroke. METHODS AND RESULTS: We here report that ischemic stroke triggers brain-bone marrow communication via hormonal long-range signals that regulate hematopoietic B lineage decisions. Bone marrow fluorescence-activated cell sorter analyses and serial intravital microscopy indicate that transient middle cerebral artery occlusion in mice arrests B-cell development beginning at the pro-B-cell stage. This phenotype was not rescued in Myd88-/- and TLR4-/- mice with disrupted TLR (Toll-like receptor) signaling or after blockage of peripheral sympathetic nerves. Mechanistically, we identified stroke-induced glucocorticoid release as the main instigator of B lymphopoiesis defects. B-cell lineage-specific deletion of the GR (glucocorticoid receptor) in CD19-Cre loxP Nr3c1 mice attenuated lymphocytopenia after transient middle cerebral artery. In 20 patients with acute stroke, increased cortisol levels inversely correlated with blood lymphocyte numbers. CONCLUSIONS: Our data demonstrate that the hypothalamic-pituitary-adrenal axis mediates B lymphopoiesis defects after ischemic stroke.
Assuntos
Corticosteroides/sangue , Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Linfopoese , Receptores de Glucocorticoides/sangue , Acidente Vascular Cerebral/sangue , Idoso , Animais , Linfócitos B/citologia , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Feminino , Humanos , Sistema Hipotálamo-Hipofisário/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Sistema Hipófise-Suprarrenal/fisiopatologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/fisiopatologiaRESUMO
According to the current view, each microRNA regulates hundreds of genes. Computational tools aim at identifying microRNA targets, usually selecting evolutionarily conserved microRNA binding sites. While the false positive rates have been evaluated for some prediction programs, that information is rarely put forward in studies making use of their predictions. Here, we provide evidence that such predictions are often biologically irrelevant. Focusing on miR-223-guided repression, we observed that it is often smaller than inter-individual variability in gene expression among wild-type mice, suggesting that most predicted targets are functionally insensitive to that microRNA. Furthermore, we found that human haplo-insufficient genes tend to bear the most highly conserved microRNA binding sites. It thus appears that biological functionality of microRNA binding sites depends on the dose-sensitivity of their host gene and that, conversely, it is unlikely that every predicted microRNA target is dose-sensitive enough to be functionally regulated by microRNAs. We also observed that some mRNAs can efficiently titrate microRNAs, providing a reason for microRNA binding site conservation for inefficiently repressed targets. Finally, many conserved microRNA binding sites are conserved in a microRNA-independent fashion: Sequence elements may be conserved for other reasons, while being fortuitously complementary to microRNAs. Collectively, our data suggest that the role of microRNAs in normal and pathological conditions has been overestimated due to the frequent overlooking of false positive rates.
Assuntos
Regulação da Expressão Gênica/genética , MicroRNAs/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Algoritmos , Animais , Sítios de Ligação , Biologia Computacional , Humanos , Camundongos , MicroRNAs/metabolismoRESUMO
Metabolic changes drive monocyte differentiation and fate. Although abnormal mitochondria metabolism and innate immune responses participate in the pathogenesis of many inflammatory disorders, molecular events regulating mitochondrial activity to control life and death in monocytes remain poorly understood. We show here that, in human monocytes, microRNA-125b (miR-125b) attenuates the mitochondrial respiration through the silencing of the BH3-only proapoptotic protein BIK and promotes the elongation of the mitochondrial network through the targeting of the mitochondrial fission process 1 protein MTP18, leading to apoptosis. Proinflammatory activation of monocyte-derived macrophages is associated with a concomitant increase in miR-125b expression and decrease in BIK and MTP18 expression, which lead to reduced oxidative phosphorylation and enhanced mitochondrial fusion. In a chronic inflammatory systemic disorder, CD14+ blood monocytes display reduced miR-125b expression as compared with healthy controls, inversely correlated with BIK and MTP18 messenger RNA expression. Our findings not only identify BIK and MTP18 as novel targets for miR-125b that control mitochondrial metabolism and dynamics, respectively, but also reveal a novel function for miR-125b in regulating metabolic adaptation of monocytes to inflammation. Together, these data unravel new molecular mechanisms for a proapoptotic role of miR-125b in monocytes and identify potential targets for interfering with excessive inflammatory activation of monocytes in inflammatory disorders.
Assuntos
Inflamação/genética , Inflamação/patologia , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Monócitos/metabolismo , Monócitos/patologia , Idoso , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Polaridade Celular/genética , Respiração Celular/genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Receptor 4 Toll-Like/metabolismoRESUMO
FCGR3A presents a single nucleotide polymorphism at location 158 (V/F), which affects its binding to the fragment crystallizable (Fc) of antibodies (Abs). FcγRIIIa-158 V allotype has the highest affinity and is associated with a better clinical response to IgG1 monoclonal Abs (mAb) treatment. We compared the allele frequency of FCGR3A-F158V polymorphism in cohorts of patients with B-cell lymphoproliferative disorders, including multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), non-Hodgkin lymphoma (NHL), and B-cell chronic leukemia (B-CLL). FCGR3A-158F homozygous were enriched and tended to be in MM and MGUS patients, respectively; but neither in B-CLL nor in NHL patients. We identified a significantly lower concentration of CD8 T-cells and resting memory CD4 T-cells in MM patients bone marrow with the F/F genotype, associated with an increase in the macrophage percentage. In contrast, natural killer cells increased in V/V homozygous patients. This suggests a deregulation of the immune microenvironment in FCGR3A-F/F homozygous patients. However, we did not observe difference in response following treatment combining chemotherapy associated or not with daratumumab, an IgG1 mAb direct against CD38. Our findings suggest that FCGR3A F158V polymorphism can regulate the immune environment and affect the development of tumor plasma cells.
Assuntos
Frequência do Gene , Mieloma Múltiplo , Polimorfismo de Nucleotídeo Único , Receptores de IgG , Humanos , Receptores de IgG/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/imunologia , GenótipoRESUMO
BACKGROUND: Natural killer (NK) cell therapy is considered an attractive and safe strategy for anticancer therapy. Nevertheless, when autologous or allogenic NK cells are used alone, the clinical benefit has been disappointing. This is partially due to the lack of target specificity. Recently, CD19-specific chimeric antigen receptor (CAR)-NK cells have proven to be safe and potent in patients with B-cell tumors. However, the generation of CAR-NK cells is a complicated manufacturing process. We aim at developing a targeted NK cell therapy without the need for cellular genetic modifications. We took advantage of the natural expression of the IgG Fc receptor CD16a (FcγRIIIa) to induce strong antigen-specific effector functions through antibody-dependent cell-mediated cytotoxicity (ADCC). We have generated the new technology "Pin", which enables the arming of modified monoclonal antibodies (mAbs) onto the CD16a of ex vivo expanded NK (eNK) cells. Methods Ex vivo eNK were prepared from umbilical cord blood cells and expanded using interleukin (IL)-2/IL-15 and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid feeder cells. mAbs were engineered with four substitutions called Pin mutations to increase their affinity to CD16a. eNK were incubated with anti-CD20 or anti-CD19 Pin-mAbs to generate "armed" eNK and were used to assess effector functions in vitro on cancer cell lines, lymphoma patient cells and in vivo. RESULTS: CD16a/Pin-mAb interaction is stable for several days and Pin-mAb eNK inherit the mAb specificity and exclusively induce ADCC against targets expressing the cognate antigen. Hence, Pin-mAbs confer long-term selectivity to eNK, which allows specific elimination of the target cells in several in vivo mouse models. Finally, we showed that it is possible to arm eNK with at least two Pin-mAbs simultaneously, to increase efficacy against heterogenous cancer cell populations. CONCLUSIONS: The Pin technology provides an off-the-shelf NK cell therapy platform to generate CAR-like NK cells, without genetic modifications, that easily target multiple tumor antigens.
Assuntos
Células Matadoras Naturais , Receptores de IgG , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Humanos , Animais , Camundongos , Receptores de IgG/metabolismo , Receptores de IgG/imunologia , Imunoterapia Adotiva/métodos , Linhagem Celular Tumoral , Antígenos CD19/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/farmacologiaRESUMO
OBJECTIVES: Nicotinamide phosphoribosyltransferase (NAMPT)/pre-B-cell colony-enhancing factor/visfatin exerts multiple functions and has been implicated in the pathogenesis of rheumatoid arthritis. To gain insight into its role in arthritis and given that NAMPT is identified as a novel mediator of innate immunity, we addressed the function of monocyte-derived NAMPT in experimental arthritis by selective gene knockdown in inflammatory monocytes. METHODS: siRNA uptake and NAMPT expression were determined in Ly6Chigh and Ly6Clow monocyte subsets following intravenous injection of siRNA against NAMPT (siNAMPT) or non-targeting siRNA (siCT) formulated with the DMAPAP cationic liposome into mice. Mice with established collagen-induced arthritis (CIA) were treated weekly after disease onset with siNAMPT or siCT and clinical features were assessed. T-helper cell frequencies, cytokine production and percentage of IL-6-producing Ly6Chigh monocytes were analysed. Using a co-culture system consisting of purified CD14 monocytes and autologous CD4 T cells, NAMPT and cytokine production, and the percentage of IL-17-producing CD4 T cells, were determined following transfection of CD14 monocytes with siCT or siNAMPT. RESULTS: On intravenous injection, siRNA was preferentially engulfed by Ly6Chigh monocytes, and siRNA-mediated silencing of NAMPT expression in Ly6Chigh monocytes inhibited CIA progression. This effect was associated with reduced IL-6 production by Ly6Chigh monocytes, reduced proportion of Th17 cells and autoantibody titers, and decreased activation and infiltration of monocytes/macrophages and neutrophils in arthritic joints. Moreover, NAMPT-RNAi-silenced CD14 monocytes were found to reduce the percentage of IL-17-producing CD4 T cells in vitro. CONCLUSIONS: Our results show that the expression of NAMPT in Ly6Chigh monocytes promotes many downstream effects involved in inflammatory arthritis and demonstrate the utility of targeting disease-causing genes, such as NAMPT, in Ly6Chigh monocytes for therapeutic intervention in arthritis.
Assuntos
Artrite Experimental/imunologia , Citocinas/imunologia , Monócitos/imunologia , Nicotinamida Fosforribosiltransferase/imunologia , Animais , Artrite Experimental/prevenção & controle , Linfócitos T CD4-Positivos/imunologia , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/genética , Inativação Gênica , Humanos , Imunomodulação/imunologia , Interleucina-6/biossíntese , Receptores de Lipopolissacarídeos/análise , Camundongos , Camundongos Endogâmicos DBA , Nicotinamida Fosforribosiltransferase/genética , RNA Interferente Pequeno/genética , Células Th17/imunologiaRESUMO
COVID-19 is caused by the infection of the lungs by SARS-CoV-2. Monoclonal antibodies, such as sotrovimab, showed great efficiency in neutralizing the virus before its internalization by lung epithelial cells. However, parenteral routes are still the preferred route of administration, even for local infections, which requires injection of high doses of antibody to reach efficacious concentrations in the lungs. Lung administration of antibodies would be more relevant requiring lower doses, thus reducing the costs and the side effects. But aerosolization of therapeutic proteins is very challenging, as the different processes available are harsh and trigger protein aggregation and conformational changes. This decreases the efficiency of the treatment, and can increase its immunogenicity. To address those issues, we developed a series of new excipients composed of a trehalose core, a succinyl side chain and a hydrophobic carbon chain (from 8 to 16 carbons). Succinylation increased the solubility of the excipients, allowing their use at relevant concentrations for protein stabilization. In particular, the excipient with 16 carbons (C16TreSuc) used at 5.6 mM was able to preserve colloidal stability and antigen-binding ability of sotrovimab during the nebulization process. It could also be used as a cryoprotectant, allowing storage of sotrovimab in a lyophilized form during weeks. Finally, we demonstrated that C16TreSuc could be used as an excipient to stabilize antibodies for the treatment against COVID-19, by in vitro and in vivo assays. The presence of C16TreSuc during nebulization preserved the neutralization capacity of sotrovimab against SARS-CoV-2 in vitro; an increase of its efficacy was even observed, compared to the non-nebulized control. The in vivo study also showed the wide distribution of sotrovimab in mice lungs, after nebulization with 5.6 mM of excipient. This work brings a solution to stabilize therapeutic proteins during storage and nebulization, making pulmonary immunotherapy possible in the treatment of COVID-19 and other lung diseases.
Assuntos
COVID-19 , Excipientes , Camundongos , Animais , Excipientes/química , Trealose/química , SARS-CoV-2 , Anticorpos AntiviraisRESUMO
Cells from the mononuclear phagocyte system (MPS) act as systemic and local amplifiers that contribute to the progression of chronic inflammatory disorders. Transforming growth factor-ß-activated kinase 1 (TAK1) is a pivotal upstream mitogen-activated protein kinase-kinase-kinase acting as a mediator of cytokine expression. It remains critical to determine in vivo the implication of TAK1 in controlling the innate immune system. Here, we describe a vehicle tailored to selectively deliver siRNAs into MPS cells after intravenous administration, and validate in vivo the potential of the RNAi-mediated TAK1 knock down for immunomodulation. In a mouse model of immune-mediated inflammatory disorder, we show that anti-TAK1 siRNA lipoplexes efficiently alleviate inflammation, severely impair the downstream c-Jun N-terminal kinase and nuclear factor-κB signaling pathways, and decrease the expression of proinflammatory mediators. Importantly, the systemic TAK1 gene silencing decreases the frequency of Th1 and Th17 cells, both mediating autoimmunity in experimental arthritis, demonstrating the immunomodulatory potential of TAK1. Finally, in vitro inhibition of TAK1 in myeloid cells decreases interferon-γ-producing T cells, suggesting that a delivery system able to target MPS cells and to silence TAK1 impacts on pathogenic T effector cells in autoimmunity.
Assuntos
MAP Quinase Quinase Quinases/genética , Células Mieloides/imunologia , Interferência de RNA , Células Th1/imunologia , Células Th17/imunologia , Animais , Artrite/terapia , Linhagem Celular , Inflamação/terapia , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Lipopolissacarídeos/imunologia , MAP Quinase Quinase Quinases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/imunologia , Monócitos/imunologia , NF-kappa B/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
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.
Assuntos
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
Systemic lupus erythematosus (SLE) is a severe autoimmune disease mediated by pathogenic autoantibodies. While complement protein C4 is associated with SLE, its isoforms (C4A and C4B) are not equal in their impact. Despite being 99% homologous, genetic studies identified C4A as more protective than C4B. By generating gene-edited mouse strains expressing either human C4A or C4B and crossing these with the 564lgi lupus strain, we show that, overall, C4A-like 564Igi mice develop less humoral autoimmunity than C4B-like 564Igi mice. This includes a decrease in the number of GCs, autoreactive B cells, autoantibodies, and memory B cells. The higher efficiency of C4A in inducing self-antigen clearance is associated with the follicular exclusion of autoreactive B cells. These results explain how the C4A isoform is protective in lupus and suggest C4A as a possible replacement therapy in lupus.
Assuntos
Linfócitos B/imunologia , Complemento C4a/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Sequência de Aminoácidos , Animais , Apoptose , Autoanticorpos/metabolismo , Autoantígenos/metabolismo , Sequência de Bases , Complemento C4a/química , Complemento C4b/química , Complemento C4b/metabolismo , Modelos Animais de Doenças , Edição de Genes , Humanos , Tolerância Imunológica , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
In this study we evaluated the potential of expanded NK cells (eNKs) from two sources combined with the mAbs daratumumab and pembrolizumab to target primary multiple myeloma (MM) cells ex vivo. In order to ascertain the best source of NK cells, we expanded and activated NK cells from peripheral blood (PB) of healthy adult donors and from umbilical cord blood (UCB). The resulting expanded NK (eNK) cells express CD16, necessary for carrying out antibody-dependent cellular cytotoxicity (ADCC). Cytotoxicity assays were performed on bone marrow aspirates of 18 MM patients and 4 patients with monoclonal gammopathy of undetermined significance (MGUS). Expression levels of PD-1 on eNKs and PD-L1 on MM and MGUS cells were also quantified. Results indicate that most eNKs obtained using our expansion protocol express a low percentage of PD-1+ cells. UCB eNKs were highly cytotoxic against MM cells and addition of daratumumab or pembrolizumab did not further increase their cytotoxicity. PB eNKs, while effective against MM cells, were significantly more cytotoxic when combined with daratumumab. In a minority of cases, eNK cells showed a detectable population of PD1+ cells. This correlated with low cytotoxic activity, particularly in UCB eNKs. Addition of pembrolizumab did not restore their activity. Results indicate that UCB eNKs are to be preferentially used against MM in the absence of daratumumab while PB eNKs have significant cytotoxic advantage when combined with this mAb.
Assuntos
Mieloma Múltiplo , Adulto , Anticorpos Monoclonais/farmacologia , Sangue Fetal , Humanos , Células Matadoras Naturais , Mieloma Múltiplo/tratamento farmacológicoRESUMO
Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6Chigh and non-classical Ly6Clow monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored. Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6Chigh monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a-/- mice. Results: We showed that the expression of miR-146a is reduced in the Ly6Chigh subset of CIA mice and in the analogous monocyte subset (CD14+CD16-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6Chigh monocytes, and not to Ly6Clow monocytes, rescues bone erosion in miR-146a-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a-/- Ly6Chigh monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6Chigh, and not Ly6Clow, monocytes into osteoclasts under arthritic conditions. Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6Chigh monocytes to prevent joint destruction while sparing inflammation in arthritis.
Assuntos
Antígenos Ly/análise , Artrite/patologia , Osso e Ossos/patologia , Diferenciação Celular , MicroRNAs/análise , Monócitos/fisiologia , Osteoclastos/fisiologia , Animais , Artrite/induzido quimicamente , Artrite/terapia , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , MicroRNAs/administração & dosagem , Monócitos/químicaRESUMO
Recent discoveries implicate the classical complement cascade in normal brain development and in disease. Complement proteins C1q, C3, and C4 participate in synapse elimination, tagging inappropriate synaptic connections between neurons for removal by phagocytic microglia that exist in a special, highly phagocytic state during the synaptic pruning period. Several neurodevelopmental disorders, such as schizophrenia and autism, are thought to be caused by an imbalance in synaptic pruning, and recent studies suggest that dysregulation of complement could promote this synaptic pruning imbalance. Moreover, in the mature brain, complement can be aberrantly activated in early stages of neurodegenerative diseases to stimulate synapse loss. Similar pathways can also be activated in response to inflammation, as in West Nile Virus infection or in lupus, where peripheral inflammation can promote microglia-mediated synapse loss. Whether synapse loss in disease is a true reactivation of developmental synaptic pruning programs remains unclear; nonetheless, complement proteins represent potential therapeutic targets for both neurodevelopmental and neurodegenerative diseases.
Assuntos
Proteínas do Sistema Complemento/imunologia , Rede Nervosa/imunologia , Neurogênese/imunologia , Plasticidade Neuronal/imunologia , Sinapses/imunologia , Animais , Transtorno Autístico/genética , Transtorno Autístico/imunologia , Transtorno Autístico/patologia , Proteínas do Sistema Complemento/genética , Epilepsia/genética , Epilepsia/imunologia , Epilepsia/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Microglia/imunologia , Microglia/patologia , Rede Nervosa/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Neurogênese/genética , Plasticidade Neuronal/genética , Neurônios/imunologia , Neurônios/patologia , Esquizofrenia/genética , Esquizofrenia/imunologia , Esquizofrenia/patologia , Sinapses/genética , Sinapses/patologia , Transmissão SinápticaRESUMO
OBJECTIVE: The nuclear protein heterogeneous nuclear RNP A2/B1 (hnRNP A2/B1) is involved in posttranscriptional regulation of gene expression. It is constitutively expressed in lymphoid organs and highly up-regulated in the synovial tissue of patients with rheumatoid arthritis (RA), who may also generate autoantibodies to this protein. This study was undertaken to investigate the potential involvement of hnRNP A2/B1 in the pathogenesis of autoimmune arthritis, by silencing hnRNP A2/B1 expression in 2 animal models of RA. METHODS: Collagen-induced arthritis (CIA) and the K/BxN serum-transfer model were used as animal models of RA. Efficient silencing of hnRNP A2/B1 was achieved using a liposome-based carrier system for delivery of small interfering RNAs. Expression of hnRNP A2/B1 was analyzed by flow cytometry, reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The number of osteoclasts was determined by tartrate-resistant acid phosphatase staining. Cytokine levels and anticollagen antibody levels were measured by enzyme-linked immunosorbent assay. RESULTS: Efficient silencing of hnRNP A2/B1 was achieved in all lymphoid organs. In both experimental models, the incidence and severity of arthritis were largely reduced and bone erosion was not detectable as compared to the control groups. Down-modulation of hnRNP A2/B1 significantly interfered with the production of proinflammatory cytokines from monocyte/macrophages, but not from T cells. Consistent with these findings, production of T cell cytokines was not impaired when cells were restimulated in vitro with type II collagen. Furthermore, levels of anticollagen antibodies were not affected by hnRNP A2/B1 silencing. CONCLUSION: Our findings suggest that hnRNP A2/B1 has an important role in regulation of the innate immune system, especially at the level of monocyte/macrophage activation. Therefore, down-modulation of hnRNP A2/B1 seems to affect primarily the effector phase of autoimmune arthritis.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Citocinas/imunologia , Inativação Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Macrófagos/imunologia , Interferência de RNA , Animais , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Modelos Animais de Doenças , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/imunologia , Inflamação/genética , Inflamação/imunologia , Ativação de Macrófagos/imunologia , Camundongos , Monócitos/imunologia , Linfócitos T/imunologiaRESUMO
MicroRNAs (miRNAs) are small noncoding RNA molecules that negatively regulate gene expression at the post-transcriptional level. Currently, there are 939 mature human miRNA sequences listed in the Sanger updated miRNA registry. There are approximately 1500 predicted miRNAs in the human genome that may regulate the expression of one third of our genes. By controlling the accumulation of the target protein(s) in cells, these regulatory RNA molecules participate in key functions in many physiological networks and their deregulation has been implicated in the pathogenesis of serious human disorders, such as cancer and infection. The implication of miRNAs in immune-mediated disorders such as rheumatoid arthritis (RA) has recently emerged suggesting that miRNA-based therapeutic approaches may have a promising potential in these diseases. Here, we provide an overview of the state-of-the-art on miRNAs in RA, focusing on both systemic and local features of the pathology.