RESUMO
Transgenic mice expressing human major histocompatibility complex class II (MHCII) risk alleles are widely used in autoimmune disease research, but limitations arise due to non-physiologic expression. To address this, physiologically relevant mouse models are established via knock-in technology to explore the role of MHCII in diseases like rheumatoid arthritis. The gene sequences encoding the ectodomains are replaced with the human DRB1*04:01 and 04:02 alleles, DRA, and CD74 (invariant chain) in C57BL/6N mice. The collagen type II (Col2a1) gene is modified to mimic human COL2. Importantly, DRB1*04:01 knock-in mice display physiologic expression of human MHCII also on thymic epithelial cells, in contrast to DRB1*04:01 transgenic mice. Humanization of the invariant chain enhances MHCII expression on thymic epithelial cells, increases mature B cell numbers in spleen, and improves antigen presentation. To validate its functionality, the collagen-induced arthritis (CIA) model is used, where DRB1*04:01 expression led to a higher susceptibility to arthritis, as compared with mice expressing DRB1*04:02. In addition, the humanized T cell epitope on COL2 allows autoreactive T cell-mediated arthritis development. In conclusion, the humanized knock-in mouse faithfully expresses MHCII, confirming the DRB1*04:01 alleles role in rheumatoid arthritis and being also useful for studying MHCII-associated diseases.
Assuntos
Alelos , Antígenos de Diferenciação de Linfócitos B , Artrite Reumatoide , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Antígenos de Histocompatibilidade Classe II , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Animais , Camundongos , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Humanos , Técnicas de Introdução de Genes/métodos , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Artrite Experimental/genética , Artrite Experimental/imunologia , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Colágeno Tipo II/genética , Colágeno Tipo II/imunologiaRESUMO
Autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematous, are regulated by polymorphisms in genes contributing to the NOX2 complex. Mutations in both Ncf1 and Ncf4 affect development of arthritis in experimental models of RA, but the different regulatory pathways mediated by NOX2-derived reactive oxygen species (ROS) have not yet been clarified. Here we address the possibility that intracellular ROS, regulated by the NCF4 protein (earlier often denoted p40phox) which interacts with endosomal membranes, could play an important role in the oxidation of cysteine peptides in mononuclear phagocytic cells, thereby regulating antigen presentation and activation of arthritogenic T cells. To study the role of NCF4 we used mice with an amino acid replacing mutation (NCF4R58A), which is known to affect interaction with endosomal membranes, leading to decreased intracellular ROS production. To study the impact of NCF4 on T cell activation, we used the glucose phosphate isomerase peptide GPI325-339, which contains two cysteine residues (325-339c-c). Macrophages from mice with the NCF458A mutation efficiently presented the peptide when the two cysteines were intact and not crosslinked, leading to a strong arthritogenic T cell response. T cell priming occurred in the draining lymph nodes (LNs) within 8 days after immunization. Clodronate treatment, which depletes antigen-presenting mononuclear phagocytes, ameliorated arthritis severity, whereas treatment with FYT720, which traps activated T cells in LNs, prohibited arthritis. We conclude that NCF4-dependent intracellular ROS maintains cysteine peptides in an oxidized crosslinked state, which prevents presentation of peptides recognized by non-tolerized T cells and thereby protects against autoimmune arthritis.
Assuntos
Apresentação de Antígeno , Cisteína , Ativação Linfocitária , Oxirredução , Espécies Reativas de Oxigênio , Linfócitos T , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Cisteína/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Apresentação de Antígeno/imunologia , Ativação Linfocitária/imunologia , NADPH Oxidases/metabolismo , NADPH Oxidases/genética , Peptídeos/farmacologia , Peptídeos/imunologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Macrófagos/imunologia , Macrófagos/metabolismoRESUMO
A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.
Assuntos
Artrite Reumatoide , Doenças Autoimunes , Animais , Camundongos , Vacinas de Subunidades Antigênicas , Linfócitos T Reguladores , AnticorposRESUMO
Although elevated levels of anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA), the in vivo functions of these antibodies remain unclear. Here, we have expressed monoclonal ACPAs derived from patients with RA, and analyzed their functions in mice, as well as their specificities. None of the ACPAs showed arthritogenicity nor induced pain-associated behavior in mice. However, one of the antibodies, clone E4, protected mice from antibody-induced arthritis. E4 showed a binding pattern restricted to skin, macrophages and dendritic cells in lymphoid tissue, and cartilage derived from mouse and human arthritic joints. Proteomic analysis confirmed that E4 strongly binds to macrophages and certain RA synovial fluid proteins such as α-enolase. The protective effect of E4 was epitope-specific and dependent on the interaction between E4-citrullinated α-enolase immune complexes with FCGR2B on macrophages, resulting in increased IL-10 secretion and reduced osteoclastogenesis. These findings suggest that a subset of ACPAs have therapeutic potential in RA.
Assuntos
Artrite Reumatoide , Autoanticorpos , Humanos , Animais , Camundongos , Proteômica , Fosfopiruvato HidrataseRESUMO
Low capacity to produce ROS because of mutations in neutrophil cytosolic factor 1 (NCF1/p47phox), a component of NADPH oxidase 2 (NOX2) complex, is strongly associated with systemic lupus erythematosus in both humans and mouse models. Here, we aimed to identify the key immune cell type(s) and cellular mechanisms driving lupus pathogenesis under the condition of NCF1-dependent ROS deficiency. Using cell-specific Cre-deleter, human NCF1-339 variant knockin, and transgenic mouse strains, we show that low ROS production in plasmacytoid dendritic cells (pDCs) exacerbated both pristane-induced lupus and a potentially new Y-linked autoimmune accelerating locus-related spontaneous model by promoting pDC accumulation in multiple organs during lupus development, accompanied by elevated IFN-α levels and expression of IFN-stimulated genes. Mechanistic studies revealed that ROS deficiency enhanced pDC generation through the AKT/mTOR pathway and CCR2-mediated migration to tissues, which together with hyperactivation of the redox-sensitive stimulator of interferon genes/IFN-α/JAK1/STAT1 cascade further augmented type I IFN responses. More importantly, by suppressing these pathways, restoration of NOX2-derived ROS specifically in pDCs protected against lupus. These discoveries explain the causative effect of dysfunctional NCF1 in lupus and demonstrate the protective role of pDC-derived ROS in disease development driven by NCF1-dependent ROS deficiency.
Assuntos
Interferon Tipo I , NADPH Oxidases , Camundongos , Animais , Humanos , Espécies Reativas de Oxigênio/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Interferon Tipo I/metabolismo , Interferon-alfa , Células DendríticasRESUMO
A breach of T cell tolerance is considered as a major step in the pathogenesis of rheumatoid arthritis. In collagen-induced arthritis (CIA) model, immunization with type II collagen (COL2) leads to arthritis in mice through T cells responding to the immunodominant COL2259-273 peptide. T cells could escape from thymus negative selection because endogenous COL2259-273 peptide only weakly binds to the major histocompatibility complex class II (MHCII) molecule Aq. To investigate the regulation of T cell tolerance, we used a new mouse strain BQ.Col2266E with homozygous D266E mutations in the Col2 gene leading to a replacement of the endogenous aspartic acid (D) to glutamic acid (E) at position 266 of the COL2259-273 peptide, resulting in stronger binding to Aq. We also established BQ.Col2264R mice carrying an additional K264R mutation changed the lysine (K) at position 264 to eliminate the major TCR recognition site. The BQ.Col2266E mice were fully resistant to CIA, while the BQ.Col2264R mice developed severe arthritis. Furthermore, we studied two of the most important non-MHCII genes associated with CIA, i.e., Ncf1 and Fcgr2b. Deficiency of either gene induced arthritis in BQ.Col2266E mice, and the downstream effects differ as Ncf1 deficiency reduced Tregs and was likely to decrease expression of autoimmune regulator (AIRE) while Fcgr2b did not. In conclusion, the new human-mimicking mouse model has strong T cell tolerance to COL2, which can be broken by deficiency of Fcgr2b or Ncf1, allowing activation of autoreactive T cells and development of arthritis.
Assuntos
Artrite Experimental , Doenças Autoimunes , NADPH Oxidases/metabolismo , Animais , Artrite Experimental/genética , Doenças Autoimunes/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Humanos , Tolerância Imunológica/genética , Camundongos , Receptores de IgG/genética , Linfócitos TRESUMO
Autoimmune murine disease models are vital tools for identifying novel targets and finding better treatments for human diseases. Complete Freund's adjuvant is commonly used to induce disease in autoimmune models, and the quality of the adjuvant/autoantigen emulsion is of critical importance in determining reproducibility. We have established an emulsification method using a standard homogenizer and specially designed receptacle. Emulsions are easy to prepare, form stable and uniform water-in-oil particles, are faster to make than the traditional syringe method, use less material and are designed to fill syringes with ease. In the present study, we have validated the emulsions for induction of experimental autoimmune encephalitis, collagen II induced arthritis, antigen induced arthritis, and delayed type hypersensitivity models. These models were induced consistently and reproducibly and, in some cases, the new method outperformed the traditional method. The method described herein is simple, cost-effective and will reduce variability, thereby requiring fewer animals for in vivo research involving animal models of autoimmune disease and in vaccine development.
Assuntos
Artrite Experimental , Doenças Autoimunes , Animais , Autoantígenos , Emulsões , Camundongos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
The most commonly used strains in experimental research, including genetically modified strains, are C57BL/6 mice. However, so far, no reliable model for rheumatoid arthritis is available, mainly due to the restriction by the MHC class II haplotype H-2b. Collagen-induced arthritis (CIA) is the most widely used animal model of rheumatoid arthritis, but C57BL/6 strain is resistant to CIA because there is no collagen II peptide associated with H-2b. To establish a rheumatoid arthritis model in C57BL/6 mice, we immunized C57BL/6NJ (B6N) mice with human cartilage oligomeric matrix protein (COMP), which induced severe arthritis with high incidence, accompanied by a strong auto-antibody response. Native COMP was required, as denatured COMP lost its ability to induce arthritis in B6N mice. An immunodominant COMP peptide was identified as the key T cell epitope, with a perfect fit into the Ab class II peptide binding pocket. A critical amino acid in this peptide was found to be phenylalanine at position 95. Recombinant COMP mutated at position 95 (COMP_F95S) lost its ability to induce arthritis or a strong immune response in the B6N mice. In conclusion, A new model for RA has been established using C57BL/6 mice through immunization with COMP, which is dependent on a COMP specific peptide binding Ab, thus in similarity with CIA in Aq expressing strains.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/fisiopatologia , Proteína de Matriz Oligomérica de Cartilagem/administração & dosagem , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Animais , Proteína de Matriz Oligomérica de Cartilagem/imunologia , Epitopos de Linfócito T/imunologia , Genes MHC da Classe II , Injeções Intradérmicas , Masculino , Camundongos , Peptídeos/administração & dosagem , Peptídeos/síntese químicaRESUMO
Systemic lupus erythematosus (SLE) is a common autoimmune disease. Recent findings have shown that a major single nucleotide variant predisposing to SLE is associated with low production of reactive oxygen species (ROS). A variant amino acid in a frequent NCF1 allele causing deficient ROS production leads to an exaggerated type I interferon (IFN) response, earlier disease onset, and higher susceptibility to SLE. It is the so far strongest identified single nucleotide variant, with an odds ratio (OR) of >3 and an allele frequency of >10%. Its functional role is in sharp contrast to the earlier belief that excessive ROS production is exclusively pathogenic rather than protective. It opens new possibilities to understand the pathogenesis of SLE and to develop novel diagnostics and treatment strategies.
Assuntos
Lúpus Eritematoso Sistêmico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Lúpus Eritematoso Sistêmico/genética , NADPH Oxidases/genética , NADPH Oxidases/metabolismoRESUMO
A single nucleotide polymorphism in Ncf1 has been found with a major effect on chronic inflammatory autoimmune diseases in the rat with the surprising observation that a lower reactive oxygen response led to more severe diseases. This finding was subsequently reproduced in the mouse and the effect operates in many different murine diseases through different pathogenic pathways; like models for rheumatoid arthritis, encephalomyelitis, lupus, gout, psoriasis and psoriatic arthritis. The human gene is located in an unstable region with many variable sequence repetitions, which means it has not been included in any genome wide associated screens so far. However, identification of copy number variations and single nucleotide polymorphisms has now clearly shown that major autoimmune diseases are strongly associated with the Ncf1 locus. In systemic lupus erythematosus the associated Ncf1 polymorphism (leading to an amino acid substitution at position 90) is the strongest locus and is associated with a lower reactive oxidative burst response. In addition, more precise mapping analysis of polymorphism of other NOX2 genes reveals that these are also associated with autoimmunity. The identified genetic association shows the importance of redox control and that ROS regulate chronic inflammation instead of promoting it. The genetic identification of Ncf1 polymorphisms now opens for relevant studies of the regulatory mechanisms involved, effects that will have severe consequences in many different pathogenic pathways and understanding of the origin of autoimmune diseases.
Assuntos
Doenças Autoimunes/genética , Doenças Autoimunes/patologia , NADPH Oxidase 2/genética , Polimorfismo de Nucleotídeo Único , Animais , Humanos , Transdução de SinaisRESUMO
Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism.
Assuntos
Artrite Experimental/imunologia , Tolerância Central/imunologia , Colágeno Tipo II/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Linfócitos T/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade/imunologia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Timócitos/imunologia , Timo/imunologiaRESUMO
OBJECTIVE: To assess serum levels of high mobility group box chromosomal protein 1 (HMGB-1) and the soluble receptor for advanced glycation end products (sRAGE) in patients with Sjögren's syndrome (SS) and explore correlations with disease activity. METHODS: Thirty-nine patients with SS and 21 healthy controls were included in this cross-sectional study. Clinical and laboratory values were obtained from all patients. Disease activity was assessed using the European League Against Rheumatism SS Disease Activity Index (ESSDAI). Serum samples were collected and HMGB-1 and sRAGE levels were measured using enzyme-linked immunosorbent assay (ELISA), and HMGB-1 concentrations were semiquantified by Western blotting. RESULTS: In ELISA, HMGB-1 serum levels did not differ between healthy controls and patients with SS (P = 0.783). When measured by semiquantitative Western blotting, HMGB-1 levels were increased in patients with SS compared to healthy controls (P = 0.012). HMGB-1 serum levels detected by Western blotting were higher in patients with extraglandular manifestations (P = 0.003) and were correlated with ESSDAI disease activity (r = 0.544, P < 0.0001). Furthermore, sRAGE was elevated in the sera of patients with SS (P = 0.003) compared to healthy controls and was also correlated with the ESSDAI (r = 0.545, P = 0.002). CONCLUSION: Serum levels of total HMGB-1 and sRAGE were elevated in patients with SS compared to healthy controls and correlated with disease activity as measured by the ESSDAI. Patients with extraglandular involvement had high serum levels of HMGB-1.
Assuntos
Produtos Finais de Glicação Avançada/sangue , Proteína HMGB1/sangue , Síndrome de Sjogren/sangue , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
Many effector mechanisms of neutrophils have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) have been assigned a particularly detrimental role. Here we investigated the functional impact of neutrophils and NETs on a mouse model of lupus triggered by intraperitoneal injection of the cell death-inducing alkane pristane. Pristane-induced lupus (PIL) was aggravated in 2 mouse strains with impaired induction of NET formation, i.e., NOX2-deficient (Ncf1-mutated) and peptidyl arginine deiminase 4-deficient (PAD4-deficient) mice, as seen from elevated levels of antinuclear autoantibodies (ANAs) and exacerbated glomerulonephritis. We observed a dramatically reduced ability to form pristane-induced NETs in vivo in both Ncf1-mutated and PAD4-deficient mice, accompanied by higher levels of inflammatory mediators in the peritoneum. Similarly, neutropenic Mcl-1ΔMyelo mice exhibited higher levels of ANAs, which indicates a regulatory function in lupus of NETs and neutrophils. Blood neutrophils from Ncf1-mutated and human individuals with SLE exhibited exuberant spontaneous NET formation. Treatment with specific chemical NOX2 activators induced NET formation and ameliorated PIL. Our findings suggest that aberrant NET is one of the factors promoting experimental lupus-like autoimmunity by uncontrolled release of inflammatory mediators.
RESUMO
OBJECTIVE: The mechanisms involved in breaking immunologic tolerance against nuclear autoantigens in systemic lupus erythematosus (SLE) are not fully understood. Our recent studies in nonautoimmune mice provided evidence of an important role of Toll-like receptor 2 (TLR-2) in antichromatin autoantibody induction by high mobility group box chromosomal protein 1-nucleosome complexes derived from apoptotic cells. The objective of this study was to investigate whether TLR-2 signaling is required for the induction of autoantibodies and the development of SLE-like disease in murine pristane-induced lupus. METHODS: Lupus-like disease in C57BL/6 and TLR-2(-/-) mice was induced by pristane injection. The numbers of immune cells and serum cytokine concentrations were determined by flow cytometry. Renal disease was assessed by quantification of proteinuria, histologic analyses, and enzyme-linked immunospot assay. RESULTS: Pristane-injected TLR-2(-/-) mice generated reduced numbers of splenic CD138+/cytoplasmic κL/λL chain-positive plasma cells and displayed diminished IgG responses against double-stranded DNA, histones, nucleosomes, some extractable nuclear autoantigens, and cardiolipin when compared with wild- type controls. TLR-2 deficiency prevented the pristane-induced systemic release of interleukin-6 (IL-6) and IL-10. The absence of TLR-2 attenuated peritoneal recruitment of CD11c+ cells and formation of lipogranulomas. Importantly, the renal disease that developed in pristane-treated TLR-2(-/-) mice was less severe than that in control mice, as reflected by milder proteinuria, reduced glomerular deposition of IgG and complement, and decreased renal infiltration of autoantibody-secreting cells. CONCLUSION: TLR-2 is required for the production of prototypical lupus autoantibodies and the development of renal disease in pristane-induced murine lupus. Interference with TLR-2 signaling may be a promising novel strategy for the treatment of SLE.
Assuntos
Autoanticorpos/imunologia , Citocinas/sangue , Proteína HMGB1/metabolismo , Nefropatias/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Terpenos/farmacologia , Receptor 2 Toll-Like/imunologia , Animais , Autoanticorpos/metabolismo , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/induzido quimicamente , Nefrite Lúpica/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteinúria , Receptor 2 Toll-Like/genéticaAssuntos
Proteínas de Fase Aguda/metabolismo , Artrite Reumatoide/sangue , Citocinas/sangue , Proteína HMGB1/sangue , Articulações/patologia , Pós-Menopausa/sangue , Corticosteroides/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrografia , Biomarcadores/sangue , Estudos Transversais , Feminino , Humanos , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Membrana Sinovial/diagnóstico por imagem , Membrana Sinovial/patologiaRESUMO
Autoantibodies to double-stranded (ds) DNA represent a serological hallmark of systemic lupus erythematosus (SLE) and may critically contribute to the pathogenesis of lupus nephritis. Self-reactive antibodies might be partially produced by long-lived plasma cells (PCs), which mainly reside within the bone marrow and spleen. In contrast to short-lived PCs, long-lived PCs are extremely resistant to therapy and may sustain refractory disease courses. Recently, antibody-secreting cells were found within the inflamed kidneys of New Zealand black/white (NZB/W) F1 lupus mice as well as of patients with SLE. To analyze the longevity of the IgG-producing cells present in nephritic kidneys of NZB/W F1 mice we performed in vivo BrdU-labeling. We identified a higher frequency of long-lived than short-lived renal PCs, indicating that survival niches for long-lived PCs also exist within inflamed kidneys. Using ELISPOT assays, we found that on average 31% of renal IgG-producing cells reacted with dsDNA and 24% with nucleolin. Moreover, the frequencies of IgG-secreting cells specific for the autoantigens dsDNA and nucleolin were higher in the kidneys compared with those in the spleen and bone marrow.
Assuntos
Anticorpos Antinucleares/biossíntese , Células Produtoras de Anticorpos/imunologia , Autoanticorpos/biossíntese , Rim/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Plasmócitos/imunologia , Animais , Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , Medula Óssea/imunologia , Bromodesoxiuridina/metabolismo , Sobrevivência Celular , ELISPOT , Feminino , Citometria de Fluxo , Imunoglobulina G/biossíntese , Rim/patologia , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos NZB , Fosfoproteínas/imunologia , Proteínas de Ligação a RNA/imunologia , Baço/imunologia , NucleolinaRESUMO
Systemic lupus erythematosus (SLE) is a complex disease resulting from inflammatory responses of the immune system against several autoantigens. Inflammation is conditioned by the continuous presence of autoantibodies and leaked autoantigens, e.g. from not properly cleared dying and dead cells. Various soluble molecules and biophysical properties of the surface of apoptotic cells play significant roles in the appropriate recognition and further processing of dying and dead cells. We exemplarily discuss how Milk fat globule epidermal growth factor 8 (MFG-E8), biophysical membrane alterations, High mobility group box 1 (HMGB1), C-reactive protein (CRP), and anti-nuclear autoantibodies may contribute to the etiopathogenesis of the disease. Up to date knowledge about these key elements may provide new insights that lead to the development of new treatment strategies of the disease.
Assuntos
Antígenos de Superfície/imunologia , Apoptose , Autoanticorpos/imunologia , Proteínas HMGB/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas do Leite/imunologia , RNA de Cadeia Dupla/imunologia , Animais , Antígenos de Superfície/química , Proteínas HMGB/química , Humanos , Proteínas do Leite/químicaRESUMO
The architectural chromosomal protein high-mobility group box 1 protein (HMGB1) acts as an alarmin when released from cells. It is involved in the pathogenesis of inflammatory and autoimmune diseases. HMGB1 can undergo post-translational modifications including oxidation. However, the mechanisms and functional relevance of HMGB1 oxidation are not yet understood. Increased concentrations of reactive oxygen species (ROS) have been reported during apoptosis and necrosis. Hence, we investigated the oxidative status of HMGB1 in dead cells. Immunoblot analyses under reducing and non-reducing conditions revealed that HMGB1 is oxidized in dead cells. Moreover, tagging of oxidized cysteine residues by a maleimide moiety linked to polyethylene glycol showed that HMGB1 passively released from primary and secondary necrotic cells was predominantly oxidized. Also HMGB1 in plasma of patients with systemic lupus was reversibly oxidized. In conclusion, HMGB1 undergoes reversible oxidative modifications at cysteine residues during cell death, which may modulate its biological properties.
Assuntos
Apoptose/imunologia , Proteína HMGB1/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Cisteína/metabolismo , Proteína HMGB1/imunologia , Humanos , Células Jurkat , Lúpus Eritematoso Sistêmico/imunologia , Necrose , Oxirredução , Processamento de Proteína Pós-TraducionalRESUMO
The functional spectrum of human galectins is currently explored, with a wide range of activities being described. The role of galectin-3 as adhesin for bacteria is based on its strong binding to lipopolysaccharides (LPSs), which brings the possibility of such a contamination in galectin preparations to awareness. This assumption was verified in three independent functional assay systems using polymyxin B as inhibitor of LPS-dependent effects. Moreover, a commercial LPS quantification kit also revealed LPS in galectin preparations. Chromatography was effective in removing LPS, suggesting that such a technique needs to be applied to prevent assigning cellular responses to galectins rather than LPS.
Assuntos
Galectinas/química , Lipopolissacarídeos/análise , Proteínas Recombinantes/química , Animais , Linhagem Celular , Cromatografia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Galectina 3/biossíntese , Galectina 3/farmacologia , Galectinas/biossíntese , Galectinas/farmacologia , Humanos , Lipopolissacarídeos/isolamento & purificação , Camundongos , Polimixina B/farmacologia , Ligação Proteica , Kit de Reagentes para Diagnóstico , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the mechanisms involved in breaking the immunological tolerance against these poorly immunogenic nuclear components are not fully understood. Impaired phagocytosis of apoptotic cells with consecutive release of nuclear antigens may contribute to the immune pathogenesis. The architectural chromosomal protein and proinflammatory mediator high mobility group box protein 1 (HMGB1) is tightly attached to the chromatin of apoptotic cells. We demonstrate that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. HMGB1-nucleosome complexes were also detected in plasma from SLE patients. HMGB1-containing nucleosomes from apoptotic cells induced secretion of interleukin (IL) 1beta, IL-6, IL-10, and tumor necrosis factor (TNF) alpha and expression of costimulatory molecules in macrophages and dendritic cells (DC), respectively. Neither HMGB1-free nucleosomes from viable cells nor nucleosomes from apoptotic cells lacking HMGB1 induced cytokine production or DC activation. HMGB1-containing nucleosomes from apoptotic cells induced anti-dsDNA and antihistone IgG responses in a Toll-like receptor (TLR) 2-dependent manner, whereas nucleosomes from living cells did not. In conclusion, HMGB1-nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the pathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA.