RESUMO
Cytokine release syndrome (CRS) is a phenomenon of immune hyperactivation described in the setting of immunotherapy. Unlike other immune-related adverse events, CRS triggered by immune checkpoint inhibitors (ICIs) is not well described. The clinical characteristics and course of 25 patients with ICI-induced CRS from 2 tertiary hospitals were abstracted retrospectively from the medical records and analyzed. CRS events were confirmed by 2 independent reviewers and graded using the Lee et al. scale. The median duration of CRS was 15.0 days (Q1; Q3 6.3; 29.8) and 10 (40.0%) had multiple episodes of CRS flares. Comparing the clinical factors and biomarkers in Grades 1-2 and 3-5 CRS, we found that patients with Grades 3-5 CRS had following: (i) had longer time to fever onset [25.0 days (Q1; Q3 13.0; 136.5) vs. 3.0 days (Q1; Q3 0.0; 18.0), p=0.027]; (ii) more cardiovascular (p=0.002), neurologic (p=0.001), pulmonary (p=0.044) and rheumatic (p=0.037) involvement; (iii) lower platelet count (p=0.041) and higher urea (p=0.041) at presentation compared to patients with Grades 1-2 CRS. 7 patients (28.0%) with Grades 1-2 CRS were rechallenged using ICIs without event. 9 patients (36.0%) were treated with pulse methylprednisolone and 6 patients (24.0%) were treated with tocilizumab. Despite this, 3 patients (50%) who received tocilizumab had fatal (Grade 5) outcomes from ICI-induced CRS. Longer time to fever onset, lower platelet count and higher urea at presentation were associated with Grade 3-5 CRS. These parameters may be used to predict which patients are likely to develop severe CRS.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Síndrome da Liberação de Citocina/induzido quimicamente , Síndrome da Liberação de Citocina/tratamento farmacológico , Inibidores de Checkpoint Imunológico/efeitos adversos , Imunoterapia/efeitos adversos , Metilprednisolona/administração & dosagem , Neoplasias/terapia , Índice de Gravidade de Doença , Idoso , Biomarcadores/sangue , Síndrome da Liberação de Citocina/sangue , Evolução Fatal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pulsoterapia/métodos , Estudos Retrospectivos , Centros de Atenção Terciária , Resultado do TratamentoRESUMO
Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.
Assuntos
Genes Dominantes , Transplante de Células-Tronco Hematopoéticas , Interleucina-1beta , Hepatopatias , Mutação , Inibidor de NF-kappaB alfa , Imunodeficiência Combinada Severa , Aloenxertos , Animais , Feminino , Células HEK293 , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Hepatopatias/genética , Hepatopatias/imunologia , Hepatopatias/terapia , Masculino , Camundongos , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/imunologia , Neutropenia/genética , Neutropenia/imunologia , Neutropenia/terapia , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/terapia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Within the last century, millions of lives have been lost to the four major Influenza pandemics. These influenza pandemics were all caused by Influenza Type A viruses (IAV) through their ability to undergo antigenic drifts and shifts. A greater understanding of IAV and host-pathogen interactions is required to develop effective therapeutics against future outbreaks. Annexin A1 (ANXA1) is a phospholipid binding, calcium-dependent protein known to play essential roles in multiple cellular functions including inflammation, proliferation, migration, and apoptosis. ANXA1 was previously shown to enhance apoptosis after IAV infection. The current study explores the role of ANXA1 in IAV infection of A549 lung epithelial cells further in the context of RIG-I-dependent signaling using A549 and Crispr/Cas9 ANXA1 deleted (A549∆ANXA1) cells. ANXA1 was found to enhance the expression of a cytoplasmic RNA sensor, RIG-I basally and post-infection. RIG-I activation by 5'ppp-RNA in A549 lung epithelial cell induces apoptotic cell death, which is inhibited when ANXA1 is deleted, and reversed when ANXA1 is re-expressed. RIG-I activation by 5'ppp-RNA stimulates the production of IFNß from lung epithelial cells to the same extent as monocytic cells, albeit very late after infection at 48-72 h, through IRF3 and STAT1 activation. ANXA1 deletion delays the phosphorylation of IRF3 and STAT1, leading to lower expression of interferon-stimulated genes, such as IFIT1, and silencing IFIT1 inhibited RIG-I-induced cell death. In all, these results suggest that ANXA1 plays a regulatory role in RIG-I signaling and cell death in A549 lung epithelial cells.
Assuntos
Anexina A1/metabolismo , Células Epiteliais/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Pulmão/metabolismo , Células A549 , Apoptose , Humanos , Transdução de Sinais , TransfecçãoRESUMO
Influenza viruses have been shown to use autophagy for their survival. However, the proteins and mechanisms involved in the autophagic process triggered by the influenza virus are unclear. Annexin-A1 (ANXA1) is an immunomodulatory protein involved in the regulation of the immune response and Influenza A virus (IAV) replication. In this study, using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated protein 9) deletion of ANXA1, combined with the next-generation sequencing, we systematically analyzed the critical role of ANXA1 in IAV infection as well as the detailed processes governing IAV infection, such as macroautophagy. A number of differentially expressed genes were uniquely expressed in influenza A virus-infected A549 parental cells and A549 ∆ANXA1 cells, which were enriched in the immune system and infection-related pathways. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed the role of ANXA1 in autophagy. To validate this, the effect of mechanistic target of rapamycin (mTOR) inhibitors, starvation and influenza infection on autophagy was determined, and our results demonstrate that ANXA1 enhances autophagy induced by conventional autophagy inducers and influenza virus. These results will help us to understand the underlying mechanisms of IAV infection and provide a potential therapeutic target for restricting influenza viral replication and infection.
Assuntos
Anexina A1/metabolismo , Autofagia/genética , Perfilação da Expressão Gênica , Vírus da Influenza A/fisiologia , Análise de Sequência de RNA , Células A549 , Animais , Anexina A1/genética , Autofagossomos/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Pulmão/patologia , Camundongos Endogâmicos BALB C , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The global increase in autoimmunity, together with the emerging autoimmune-related side effects of cancer immunotherapy, have furthered a need for understanding of immune tolerance and activation. Systemic lupus erythematosus (SLE) is the archetypical autoimmune disease, affecting multiple organs, and tissues. Studying SLE creates knowledge relevant not just for autoimmunity, but the immune system in general. Murine models and patient studies have provided increasing evidence for the innate immune toll like receptor-7 (TLR7) in disease initiation and progression. Here, we demonstrated that the kinase activity of the TLR7-downstream signaling molecule, interleukin-1 receptor associated kinase 4 (IRAK4), is essential for mild and severe autoimmune traits of the Sle1 and Sle1-TLR7 transgenic (Sle1Tg7) murine models, respectively. Elimination of IRAK4 signaling prevented all pathological traits associated with murine lupus, including splenomegaly with leukocyte expansion, detectable circulating antinuclear antibodies and glomerulonephritis, in both Sle1 and Sle1Tg7 mice. The expansion of germinal center B cells and increased effector memory T cell phenotypes that are typical of lupus-prone strains, were also prevented with IRAK4 kinase elimination. Analysis of renal leukocyte infiltrates confirmed our earlier findings of an expanded conventional dendritic cell (cDC) within the kidneys of nephritic mice, and this was prevented with IRAK4 kinase elimination. Analysis of TLR7 at the protein level revealed that the expression in immune cells is dependent on the TLR7-transgene itself and/or autoimmune disease factors in a cell-specific manner. Increased TLR7 protein expression in renal macrophages and cDCs correlated with disease parameters such as blood urea nitrogen (BUN) levels and the frequency of leukocytes infiltrating the kidney. These findings suggest that controlling the level of TLR7 or downstream signaling within myeloid populations may prevent chronic inflammation and severe nephritis.
Assuntos
Células Dendríticas/imunologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Rim/patologia , Leucócitos/fisiologia , Lúpus Eritematoso Sistêmico/metabolismo , Nefrite Lúpica/metabolismo , Macrófagos/imunologia , Receptor 7 Toll-Like/metabolismo , Animais , Anticorpos Antinucleares/sangue , Movimento Celular , Modelos Animais de Doenças , Glomerulonefrite , Humanos , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1/genética , Rim/metabolismo , Nefrite Lúpica/genética , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/genéticaRESUMO
OBJECTIVE: This study evaluates the utility of urinary pro-thrombotic molecules such as tissue factor (TF), anti-thrombotic molecules such as tissue factor pathway inhibitor (TFPI), and fibrinolytic molecules such as plasmin and d-dimer as biomarkers of lupus nephritis (LN). METHODS: Urine samples from 113 biopsy-proven LN patients (89 active LN and 24 inactive LN), 45 chronic kidney disease patients, and 41 healthy controls were examined for d-dimer, plasmin, TF, and TFPI levels by ELISA. The area under the receiver operating characteristic curve (AUC) analysis, multivariate regression analysis, and Bayesian network analysis were performed to assess the diagnostic value of the assayed molecules in LN. RESULTS: Although urinary d-dimer, plasmin, TF, and TFPI were all elevated in active LN compared to all control groups, and correlated with rSLEDAI and SLICC RAS disease activity indices, urine plasmin emerged as the strongest independent predictor of eGFR and renal disease status, by multivariate regression analysis and Bayesian network analysis. Whereas urine plasmin discriminated active LN from inactive disease with an AUC of 0.84, the combination of urine plasmin and TFPI discriminated ALN from ILN with an AUC of 0.86, with both surpassing the specificity and positive predictive value of traditional markers such as anti-dsDNA and complement C3. CONCLUSION: Both thrombogenic and thrombolytic cascades appear to be upregulated in lupus nephritis, with proteins from both cascades appearing in the urine. Of the coagulation cascade proteins surveyed, urine plasmin emerges as the strongest predictor of eGFR and clinical renal disease in patients with LN.
Assuntos
Biomarcadores/urina , Produtos de Degradação da Fibrina e do Fibrinogênio/urina , Fibrinolisina/urina , Lipoproteínas/urina , Nefrite Lúpica/urina , Tromboplastina/urina , Adulto , Teorema de Bayes , Feminino , Humanos , Nefrite Lúpica/diagnóstico , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Regressão , Sensibilidade e Especificidade , Adulto JovemRESUMO
Neutrophil extracellular traps (NETs) are web-like structures composed of decondensed chromatin and antimicrobial proteins that are released into the extracellular space during microbial infections. This active cell death program is known as NETosis. To date, florescence microscopy is the widely accepted method for visualization and quantification of NETs. However, this method is subjective, time consuming and yields low numbers of analyzed polymorphonuclear cells (PMNs) per sample. Increasing interest has emerged on the identification of NETs using flow cytometry techniques. However, flow cytometry analysis of NETs requires particular precautions for sample preparation to obtain reproducible data. Herein, we describe a flow cytometry-based assay for high-throughput detection and quantification of NETosis in mixed cell populations. We used fluorescent-labeled antibodies against cell markers on PMNs together with a combination of nucleic acid stains to measure NETosis in whole blood (WB) and purified PMNs. Using plasma membrane-impermeable DNA-binding dye, SYTOX Orange (SO), we found that cell-appendant DNA of NETting PMNs were positive for SO and DAPI. The combination of optimally diluted antibody and nucleic acid dyes required no washing and yielded low background fluorescence. Significant correlations were found for NETosis from WB and purified PMNs. We then validated the assay by comparing with time-lapse live cell fluorescence microscopy and determined very good intraassay and interassay variances. The assay was then applied to a disease associated with NETosis, systemic lupus erythematosus (SLE). We examined PMA-induced NETosis in peripheral PMNs from SLE patients and controls and in bone marrow PMNs from multiple murine models. In summary, this assay is observer-independent and allows for rapid assessment of a large number of PMNs per sample. Use of this assay does not require sophisticated microscopic equipment like imaging flow cytometers and may be a starting point to analyze extracellular trap formation from immune cells other than PMNs. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Assuntos
Armadilhas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Microscopia de Fluorescência/métodos , Neutrófilos/metabolismo , Animais , Células da Medula Óssea/metabolismo , DNA/análise , DNA/química , Modelos Animais de Doenças , Armadilhas Extracelulares/química , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Morte Celular Regulada/efeitos dos fármacos , Morte Celular Regulada/genéticaRESUMO
Macrophages are potent immune cells with well-established roles in the response to stress, injury, infection and inflammation. The classically activated macrophages (M1) are induced by lipopolysaccharide (LPS) and express a wide range of pro-inflammatory genes. M2 macrophages are induced by T helper type 2 cytokines such as interleukin-4 (IL4) and express high levels of anti-inflammatory and tissue repair genes. The strong association between macrophages and tumour cells as well as the high incidences of leukocyte infiltration in solid tumours have contributed to the discovery that tumour-associated macrophages (TAMs) are key to tumour progression. Here, we investigated the role of Annexin A1 (ANXA1), a well characterized immunomodulatory protein on macrophage polarization and the interaction between macrophages and breast cancer cells. Our results demonstrate that ANXA1 regulates macrophage polarization and activation. ANXA1 can act dually as an endogenous signalling molecule or as a secreted mediator which acts via its receptor, FPR2, to promote macrophage polarization. Furthermore, ANXA1 deficient mice exhibit reduced tumour growth and enhanced survival in vivo, possibly due to increased M1 macrophages within the tumor microenvironment. These results provide new insights into the molecular mechanisms of macrophage polarization with therapeutic potential to suppress breast cancer growth and metastasis.
Assuntos
Anexina A1/metabolismo , Movimento Celular , Proliferação de Células , Macrófagos/imunologia , Neoplasias Mamárias Animais/patologia , Microambiente Tumoral/imunologia , Animais , Anexina A1/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/metabolismo , Camundongos , NF-kappa B/metabolismo , Receptores de Formil Peptídeo/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.
Assuntos
Células Dendríticas , Antígenos , Humanos , Tolerância Imunológica , Linfócitos T ReguladoresRESUMO
The toll-like receptors (TLRs) are important innate receptors recognizing potentially pathogenic material. However, they also play a significant role in the development of Alzheimer's disease, cancer, autoimmunity and the susceptibility to viral infections. Macrophages are essential for an effective immune response to foreign material and the resolution of inflammation. In these studies, we examined the impact of different TLR ligands on macrophage cell function. We demonstrate that stimulation of all TLRs tested increases the phagocytosis of apoptotic cells by macrophages. TLR7 and TLR9 ligation decreased the levels of the surface co-expression molecules CD86 and MHCII, which was associated with a concomitant reduction in antigen presentation and proliferation of T cells. This down-regulation in macrophage function was not due to an increase in cell death. In fact, exposure to TLR7 or TLR9 ligands promoted cell viability for up to 9 days, in contrast to TLR3 or TLR4. Additionally, macrophages exposed to TLR7/TLR9 ligands had a significantly lower ratio of Il-12/Il-10 mRNA expression compared with those treated with the TLR4 ligand, LPS. Taken together, these data demonstrate that TLR7/TLR9 ligands push the macrophage into a phagocytic long-lived cell, with a decreased capacity of antigen presentation and reminiscent of the M2 polarized state.
Assuntos
Apresentação de Antígeno , Macrófagos/imunologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/imunologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Ligantes , Lipopolissacarídeos/toxicidade , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genéticaRESUMO
The three-prime repair exonuclease 1 (TREX1) is the most abundant exonuclease in mammalian cells. Mutations in Trex1 gene are being linked to the development of Aicardi-Goutières syndrome, an inflammatory disease of the brain, and systemic lupus erythematosus. In clinical cases and in a Trex1-deficient murine model, chronic production of type I IFN plays a pathogenic role. In this study, we demonstrate that Trex1(-/-) mice present inflammatory signatures in many different organs, including the brain. Trex1 is highly induced in macrophages in response to proinflammatory stimuli, including TLR7 and TLR9 ligands. Our findings show that, in the absence of Trex1, macrophages displayed an exacerbated proinflammatory response. More specifically, following proinflammatory stimulation, Trex1(-/-) macrophages exhibited an increased TNF-α and IFN-α production, higher levels of CD86, and increased Ag presentation to CD4(+) T cells, as well as an impaired apoptotic T cell clearance. These results evidence an unrevealed function of the Trex1 as a negative regulator of macrophage inflammatory activation and demonstrate that macrophages play a major role in diseases associated with Trex1 mutations, which contributes to the understanding of inflammatory signature in these diseases.
Assuntos
Exodesoxirribonucleases/fisiologia , Inflamação/imunologia , Ativação de Macrófagos/fisiologia , Fosfoproteínas/fisiologia , Animais , Apresentação de Antígeno , Apoptose , Antígeno B7-2/biossíntese , Antígeno B7-2/genética , Química Encefálica , Exodesoxirribonucleases/deficiência , Exodesoxirribonucleases/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/metabolismo , Interferon-alfa/biossíntese , Interferon-alfa/genética , Células Jurkat , Células L , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fagocitose , Fosfoproteínas/deficiência , Fosfoproteínas/imunologia , Proteínas Recombinantes/farmacologia , Linfócitos T/imunologia , Linfócitos T/patologia , Receptor Toll-Like 9/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-ß and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-ß and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-ß and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-ß and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.
Assuntos
Anexina A1/metabolismo , Interferon beta/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Anexina A1/biossíntese , Anexina A1/genética , Linhagem Celular , Quimiocina CXCL10/biossíntese , Células Dendríticas/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/metabolismo , Lipopolissacarídeos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosforilação , Poli I-C/farmacologia , Transdução de Sinais/imunologiaRESUMO
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by the production of antinuclear autoantibodies. Antinuclear autoantibody development is recognized as one of the initial stages of disease that often results in systemic inflammation, kidney disease, and death. The etiology is complex, but it is clear that innate pathways may play an important role in disease progression. Recent data have highlighted an important role for the TLR family, particularly TLR7, in both human disease and murine models. In this study, we have presented a low copy conditional TLR7 transgenic (Tg7) mouse strain that does not develop spontaneous autoimmunity. When we combine Tg7 with the Sle1 lupus susceptibility locus, the mice develop severe disease. Using the CD19(Cre) recombinase system, we normalized expression of TLR7 solely within the B cells. Using this method we demonstrated that overexpression of TLR7 within the B cell compartment reduces the marginal zone B cell compartment and increases B and T cell activation but not T follicular helper cell development. Moreover, this enhanced B cell TLR7 expression permits the specific development of Abs to RNA/protein complexes and exacerbates SLE disease.
Assuntos
Autoanticorpos/biossíntese , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/genética , Receptor 7 Toll-Like/genética , Animais , Autoanticorpos/efeitos adversos , Subpopulações de Linfócitos B/metabolismo , Progressão da Doença , Epistasia Genética/imunologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Receptor 7 Toll-Like/biossíntese , Receptor 7 Toll-Like/fisiologia , Transgenes/imunologiaRESUMO
OBJECTIVE: CXCR4 is a chemokine with multiple effects on the immune system. In murine lupus models, we demonstrated that monocytes, neutrophils, and B cells overexpressed CXCR4 and that its ligand, CXCL12, was up-regulated in diseased kidneys. We undertook this study to determine whether CXCR4 expression was increased in peripheral blood leukocytes from patients with systemic lupus erythematosus (SLE) and whether CXCL12 expression was increased in kidneys from patients with SLE. METHODS: Peripheral blood leukocytes from 31 SLE patients, 8 normal controls, and 9 patients with rheumatoid arthritis were prospectively analyzed by flow cytometry for CXCR4 expression. Biopsy samples (n = 14) from patients with lupus nephritis (LN) were immunostained with anti-CXCL12 antibody. RESULTS: CD19+ B cells and CD4+ T cells from SLE patients displayed a >2-fold increase (P = 0.0001) and >3-fold increase (P < 0.0001), respectively, in median CXCR4 expression compared with that in controls (n = 7-8). Moreover, CXCR4 expression on B cells was 1.61-fold higher in patients with SLE Disease Activity Index (SLEDAI) scores >10 (n = 8) than in patients with SLEDAI scores ≤10 (n = 16) (P = 0.0008), 1.71-fold higher in patients with class IV LN (n = 5) than in patients with other classes of LN (n = 7) (P = 0.02), and 1.40-fold higher in patients with active neuropsychiatric SLE (NPSLE) (n = 6) than in patients with inactive NPSLE (n = 18) (P = 0.01). CXCL12 was significantly up-regulated in the tubules and glomeruli of kidneys in patients with LN (n = 14), with the percentage of positive cells correlating positively with the severity of LN. CONCLUSION: CXCR4 appears to be up-regulated in multiple leukocyte subsets in SLE patients. The heightened expression of CXCR4 on B cells in active NPSLE and of CXCL12 in nephritic kidneys suggests that the CXCR4/CXCL12 axis might be a potential therapeutic target for SLE patients with kidney and/or central nervous system involvement.
Assuntos
Quimiocina CXCL12/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Receptores CXCR4/metabolismo , Adolescente , Adulto , Idoso , Quimiocina CXCL12/imunologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Leucócitos/imunologia , Leucócitos/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores CXCR4/imunologia , Regulação para Cima/imunologiaRESUMO
Among various surface molecules screened, CXCR4 was significantly up-regulated on monocytes, neutrophils, B cell subsets, and plasma cells in multiple murine models of lupus with active nephritis, including B6.Sle1Yaa, BXSB, and MRL.lpr. TLR-mediated signaling and inflammatory cytokines accounted in part for this increase. Increased CXCR4 expression was associated with functional consequences, including increased migration and enhanced B cell survival. Simultaneously, the ligand for CXCR4, CXCL12, was significantly up-regulated in the nephritic kidneys. Treatment with a peptide antagonist of CXCR4 prolonged survival and reduced serum autoantibodies, splenomegaly, intrarenal leukocyte trafficking, and end organ disease in a murine model of lupus. These findings underscore the pathogenic role of CXCR4/CXCL12 in lymphoproliferative lupus and lupus nephritis and highlight this axis as a promising therapeutic target in this disease.
Assuntos
Quimiocina CXCL12/biossíntese , Leucócitos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Receptores CXCR4/biossíntese , Animais , Quimiocina CXCL12/imunologia , Quimiotaxia de Leucócito/imunologia , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/imunologia , Inflamação/metabolismo , Leucócitos/metabolismo , Lúpus Eritematoso Sistêmico/complicações , Nefrite Lúpica/etiologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Masculino , Camundongos , Receptores CXCR4/imunologia , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Regulação para CimaRESUMO
The impact of IFN-alpha secretion on disease progression was assessed by comparing phenotypic changes in the lupus-prone B6.Sle1Sle2Sle3 (B6.Sle123) strain and the parental C57BL/6 (B6) congenic partner using an adenovirus (ADV) expression vector containing a recombinant IFN-alpha gene cassette (IFN-ADV). A comprehensive comparison of cell lineage composition and activation in young B6 and B6.Sle123 mice revealed a variety of cellular alterations in the presence and absence of systemic IFN-alpha. Most IFN-alpha-induced phenotypes were similar in B6 and B6.Sle123 mice; however, B6.Sle123 mice uniquely exhibited increased B1 and plasma cells after IFN-alpha exposure, although both strains had an overall loss of mature B cells in the bone marrow, spleen and periphery. Although most of the cellular effects of IFN-alpha were identical in both strains, severe glomerulonephritis occurred only in B6.Sle123 mice. Mice injected with IFN-ADV showed an increase in immune complex deposition in the kidney, together with an unexpected decrease in serum anti-nuclear antibody levels. In summary, the predominant impact of systemic IFN-alpha in this murine model is an exacerbation of mechanisms mediating end organ damage.
Assuntos
Linfócitos B/imunologia , Glomerulonefrite/imunologia , Interferon-alfa/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Animais , Complexo Antígeno-Anticorpo , Células Dendríticas/imunologia , Vetores Genéticos , Glomerulonefrite/metabolismo , Rim/patologia , Leucopenia/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Nefrite Lúpica/metabolismo , Ativação Linfocitária , Camundongos , Células Mieloides/citologia , Células Mieloides/imunologia , Esplenomegalia/imunologia , Linfócitos T/imunologiaRESUMO
DNA is a nuclear molecule that has both an intracellular and extracellular role. Inside the cell, it is the essential molecule of heredity while outside the cell it can have immunological activity, both alone and in the context of immune complexes. Furthermore, extracellular DNA has information content that can be mined by genomic techniques. Because of the association of extracellular DNA with clinical conditions marked by cell death, dead and dying cells have been considered the origin of this material. To investigate this process, in vitro and in vivo systems have been used to determine the release of DNA from cells, using Jurkat T cells as a model. Thus, in vitro, apoptotic Jurkat cells release DNA whereas necrotic cells do not. The presence of macrophages in these cultures, however, modifies the release process, causing release from necrotic cells as well. In in vivo experiments in which Jurkat cells are administered to normal mice, both apoptotic and necrotic cells give rise to DNA in the blood in a process that requires macrophages and can be modified by glucocorticoids. In this model, female and male mice differ in the extent of DNA release from the administered Jurkat cells. Together, these results indicate that, while apoptosis and necrosis can lead to a blood DNA response, this process requires macrophages and may be hormonally mediated.
Assuntos
Apoptose/imunologia , DNA/imunologia , Glucocorticoides/imunologia , Macrófagos/imunologia , Animais , Feminino , Genômica , Humanos , Células Jurkat , Masculino , Camundongos , Necrose/imunologiaRESUMO
IL-21 is a type I cytokine that influences the function of T cells, NK cells, and B cells. In this study, we report that IL-21 plays a major role in stimulating the differentiation of human B cells. When human B cells were stimulated through the BCR, IL-21 induced minimal proliferation, IgD down-modulation, and small numbers of plasma cells. In contrast, after CD40 engagement, IL-21 induced extensive proliferation, class switch recombination (CSR), and plasma cell differentiation. Upon cross-linking both BCR and CD40, IL-21 induced the largest numbers of plasma cells. IL-21 drove both postswitch memory cells as well as poorly responsive naive cord blood B cells to differentiate into plasma cells. The effect of IL-21 was more potent than the combination of IL-2 and IL-10, especially when responsiveness of cord blood B cells was examined. IL-21 costimulation potently induced the expression of both B lymphocyte-induced maturation protein-1 (BLIMP-1) and activation-induced cytidine deaminase as well as the production of large amounts of IgG from B cells. Despite the induction of activation-induced cytidine deaminase and CSR, IL-21 did not induce somatic hypermutation. Finally, IL-2 enhanced the effects of IL-21, whereas IL-4 inhibited IL-21-induced plasma cell differentiation. Taken together, our data show that IL-21 plays a central role in CSR and plasma cell differentiation during T cell-dependent B cell responses.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Interleucinas/farmacologia , Plasmócitos/metabolismo , Linfócitos B/imunologia , Antígenos CD40/metabolismo , Proliferação de Células , Humanos , Switching de Imunoglobulina , Memória Imunológica , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, expression of endogenous TTP is confined to the cytoplasm. Baseline expression of TTP was higher in monocytes than in lymphocytes or neutrophils. After in vitro incubation with lipopolysaccharide (LPS), leukocyte TTP levels increased rapidly, peaking after approximately 2 hours. Monocytes showed the greatest response to LPS stimulation and lymphocytes the least. TTP levels were also studied in leukocytes isolated from healthy volunteers infused with a bolus dose of LPS. TTP expression and initial upregulation in response to LPS infusion were consistent with the in vitro data. Neutrophil TTP levels responded first, reaching an initial peak within 1 hour, monocyte levels peaked next at 2 hours, followed by lymphocytes at 4 hours. This response paralleled plasma TNF levels, which peaked 2 hours after infusion and were no longer detectable after 12 hours. A second rise in intracellular TTP levels, which did not parallel plasma TNF levels, was observed in all leukocyte populations, starting 12 hours after infusion. These data establish the cytoplasmic location of TTP, supporting a major role for this protein in regulating TNF production, and suggest that TTP levels are not regulated solely by TNF.