RESUMO
BACKGROUND: Mitochondria play a critical role in the production of cell energy and the regulation of cell death. Therefore, mitochondria orchestrate numerous cell effector functions, including fine-tuning the immune system. While mitochondria are mainly found intracellularly, they can escape the confine of the cell during the process of extracellular vesicle release. Platelets patrol blood vessels to ensure vasculature integrity and to support the immune system. In blood, platelets are the primary source of circulating mitochondria. Activated platelets produce extracellular vesicles, including a subset of mitochondria-containing vesicles. STUDY DESIGN AND METHODS: We characterized mitochondrial functions in platelet-derived extracellular vesicles, and examined whether they could impact the bioenergetics of cellular immune recipients using an extracellular flux analyzer to measure real-time bioenergetics. RESULTS: We validated that extracellular vesicles derived from activated platelets contain the necessary mitochondrial machinery to respirate and generate energy. Moreover, neutrophils and monocytes efficiently captured platelet-derived extracellular vesicles, enhancing their mitochondrial fitness. This process required functional mitochondria from donor platelets, as it was abolished by the inactivation of extracellular mitochondria using mitochondrial poison. DISCUSSION: Together, the data suggest that extracellular mitochondria produced by platelets may support other metabolic functions through transcellular bioenergetics.
Assuntos
Plaquetas , Vesículas Extracelulares , Humanos , Plaquetas/metabolismo , Mitocôndrias/metabolismo , Metabolismo Energético/fisiologia , Vesículas Extracelulares/metabolismo , Exercício FísicoRESUMO
The mitochondrion is an intracellular organelle thought to originate from endosymbiosis between an ancestral eukaryotic cell and an α-proteobacterium. Mitochondria are the powerhouses of the cell, and can control several important processes within the cell, such as cell death. Conversely, dysregulation of mitochondria possibly contributes to the pathophysiology of several autoimmune diseases. Defects in mitochondria can be caused by mutations in the mitochondrial genome or by chronic exposure to pro-inflammatory cytokines, including type I interferons. Following the release of intact mitochondria or mitochondrial components into the cytosol or the extracellular space, the bacteria-like molecular motifs of mitochondria can elicit pro-inflammatory responses by the innate immune system. Moreover, antibodies can target mitochondria in autoimmune diseases, suggesting an interplay between the adaptive immune system and mitochondria. In this Review, we discuss the roles of mitochondria in rheumatic diseases such as systemic lupus erythematosus, antiphospholipid syndrome and rheumatoid arthritis. An understanding of the different contributions of mitochondria to distinct rheumatic diseases or manifestations could permit the development of novel therapeutic strategies and the use of mitochondria-derived biomarkers to inform pathogenesis.
Assuntos
Doenças Autoimunes , Interferon Tipo I , Lúpus Eritematoso Sistêmico , Doenças Reumáticas , Humanos , Mitocôndrias/metabolismo , Biomarcadores/metabolismo , Citocinas/metabolismo , Interferon Tipo I/metabolismoRESUMO
OBJECTIVE: Mitochondria are organelles that exhibit several bacterial features, such as a double-stranded genome with hypomethylated CpG islands, formylated proteins, and cardiolipin-containing membranes. In systemic lupus erythematosus (SLE), mitochondria and their inner components are released into the extracellular space, potentially eliciting a proinflammatory response from the immune system. While cardiolipin and mitochondrial DNA and RNA are confirmed targets of autoantibodies, other antigenic mitochondrial proteins in SLE remain to be identified. The present study was undertaken to characterize the protein repertoire recognized by antimitochondrial antibodies (AMAs) in patients with SLE. METHODS: Using shotgun proteomic profiling, we identified 1,345 proteins, 431 of which were associated with the mitochondrial proteome. Immunoreactivities to several of these candidate proteins were assessed in serum samples from a local cohort (n = 30 healthy donors and 87 patients with SLE) using enzyme-linked immunosorbent assay, and further analyzed for associations with demographic and disease characteristics. RESULTS: We determined that IgG antibodies to the complement component C1q binding protein were significantly elevated in the patients with SLE (P = 0.049) and were also associated with lupus anticoagulant positivity (P = 0.049). Elevated levels of IgG antibodies against mitochondrial protein mitofusin 1 (MFN-1) were promising predictors of SLE diagnosis in our cohort (adjusted odds ratio 2.99 [95% confidence interval 1.39-6.43], P = 0.0044). Moreover, increased levels of anti-MFN-1 were associated with the presence of antiphospholipids (P = 0.011) and anti-double-stranded DNA (P = 0.0005). CONCLUSION: In this study, we characterized the mitochondrial repertoire targeted by AMAs in the setting of SLE. Our results indicate that autoantibodies can recognize secreted and/or surface proteins of mitochondrial origin. Profiling of the AMA repertoire in large prospective cohorts may improve our knowledge of mitochondrial biomarkers and their usefulness for patient stratification.
Assuntos
Proteínas de Transporte , GTP Fosfo-Hidrolases , Lúpus Eritematoso Sistêmico , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais , Autoanticorpos , Cardiolipinas , Proteínas de Transporte/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Imunoglobulina G , Lúpus Eritematoso Sistêmico/metabolismo , Mitocôndrias , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Estudos Prospectivos , ProteômicaRESUMO
The accumulation of DNA and nuclear components in blood and their recognition by autoantibodies play a central role in the pathophysiology of systemic lupus erythematosus (SLE). Despite the efforts, the sources of circulating autoantigens in SLE are still unclear. Here, we show that in SLE, platelets release mitochondrial DNA, the majority of which is associated with the extracellular mitochondrial organelle. Mitochondrial release in patients with SLE correlates with platelet degranulation. This process requires the stimulation of platelet FcγRIIA, a receptor for immune complexes. Because mice lack FcγRIIA and murine platelets are completely devoid of receptor capable of binding IgG-containing immune complexes, we used transgenic mice expressing FcγRIIA for our in vivo investigations. FcγRIIA expression in lupus-prone mice led to the recruitment of platelets in kidneys and to the release of mitochondria in vivo. Using a reporter mouse with red fluorescent protein targeted to the mitochondrion, we confirmed platelets as a source of extracellular mitochondria driven by FcγRIIA and its cosignaling by the fibrinogen receptor α2bß3 in vivo. These findings suggest that platelets might be a key source of mitochondrial antigens in SLE and might be a therapeutic target for treating SLE.
Assuntos
Plaquetas , Lúpus Eritematoso Sistêmico , Animais , Complexo Antígeno-Anticorpo , Autoanticorpos/metabolismo , Plaquetas/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Mitocôndrias , Receptores de IgG/metabolismoRESUMO
BACKGROUND: Mitochondria are intracellular organelles of bacterial origin capable of stimulating the immune system when released into the extracellular milieu. We previously reported the expression of anti-mitochondrial antibodies (AMA) targeting whole organelles (AwMA), mitochondrial DNA (AmtDNA) or mitochondrial RNA (AmtRNA) in patients with systemic lupus erythematosus (SLE). Antiphospholipid syndrome (APS) is an autoimmune condition that may be independent of, or associated with, other diseases, usually SLE. This study aimed to detect AMA in patients with APS and to explore the association with clinical features of APS. METHODS: AwMA-, AmtDNA- and AmtRNA-IgG and -IgM were detected in a pilot study (healthy controls n = 30 and APS patients n = 24) by direct ELISA, and their levels were associated with demographic and disease characteristics. RESULTS: AmtDNA-IgM and AmtRNA-IgG and IgM were elevated in APS compared to healthy controls (p = 0.009, p = 0.0005 and p = 0.01, respectively). AwMA-IgG were increased in patients positive for lupus anticoagulant (median ± interquartile range = 0.36 ± 0.31 vs. 0.14 ± 0.08, p = 0.008), and optical density values for AwMA-IgM were correlated with titres of IgM against cardiolipin (rs = 0.51, p = 0.01). An increment of 0.1 unit of AmtDNA-IgM levels was associated with reduced prior reporting of arterial events (odds ratio = 0.86; 95% confidence interval 0.74-1.00; p = 0.047). CONCLUSION: Our pilot study suggests that AMA are represented within the autoantibody repertoire in APS and may display different associations with the clinical manifestations of the disease. Further studies should focus on reproducing these preliminary results by following AMA levels through time in larger prospective cohorts.
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Síndrome Antifosfolipídica/imunologia , Autoanticorpos/imunologia , DNA Mitocondrial/imunologia , RNA Mitocondrial/imunologia , Adulto , Idoso , Autoanticorpos/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/imunologia , Projetos PilotoRESUMO
The mitochondrion supplies energy to the cell and regulates apoptosis. Unlike other mammalian organelles, mitochondria are formed by binary fission and cannot be directly produced by the cell. They contain numerous copies of a compact circular genome that encodes RNA molecules and proteins involved in mitochondrial oxidative phosphorylation. Whereas, mitochondrial DNA (mtDNA) activates the innate immune system if present in the cytosol or the extracellular milieu, it is also the target of circulating autoantibodies in systemic lupus erythematosus (SLE). However, it is not known whether mitochondrial RNA is also recognized by autoantibodies in SLE. In the present study, we evaluated the presence of autoantibodies targeting mitochondrial RNA (AmtRNA) in SLE. We quantified AmtRNA in an inducible model of murine SLE. The AmtRNA were also determined in SLE patients and healthy volunteers. AmtRNA titers were measured in both our induced model of murine SLE and in human SLE, and biostatistical analyses were performed to determine whether the presence and/or levels of AmtRNA were associated with clinical features expressed by SLE patients. Both IgG and IgM classes of AmtRNA were increased in SLE patients (n = 86) compared to healthy controls (n = 30) (p < 0.0001 and p = 0.0493, respectively). AmtRNA IgG levels correlated with anti-mtDNA-IgG titers (rs = 0.54, p < 0.0001) as well as with both IgG and IgM against ß-2-glycoprotein I (anti-ß2GPI; rs = 0.22, p = 0.05), and AmtRNA-IgG antibodies were present at higher levels when patients were positive for autoantibodies to double-stranded-genomic DNA (p < 0.0001). AmtRNA-IgG were able to specifically discriminate SLE patients from healthy controls, and were negatively associated with plaque formation (p = 0.04) and lupus nephritis (p = 0.03). Conversely, AmtRNA-IgM titers correlated with those of anti-ß2GPI-IgM (rs = 0.48, p < 0.0001). AmtRNA-IgM were higher when patients were positive for anticardiolipin antibodies (aCL-IgG: p = 0.01; aCL-IgM: p = 0.002), but AmtRNA-IgM were not associated with any of the clinical manifestations assessed. These findings identify mtRNA as a novel mitochondrial antigen target in SLE, and support the concept that mitochondria may provide an important source of circulating autoantigens in SLE.
Assuntos
Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , DNA/imunologia , Lúpus Eritematoso Sistêmico/imunologia , RNA Mitocondrial/imunologia , Animais , Anticorpos Anticardiolipina/sangue , Anticorpos Anticardiolipina/imunologia , Anticorpos Antinucleares/sangue , Autoanticorpos/sangue , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/imunologiaRESUMO
Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE.
Assuntos
Autoanticorpos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Mitocôndrias/imunologia , Adulto , Idoso , Animais , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/imunologia , Autoanticorpos/sangue , DNA Mitocondrial/imunologia , Modelos Animais de Doenças , Feminino , Células Hep G2 , Humanos , Lúpus Eritematoso Sistêmico/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/imunologia , Razão de Chances , Adulto JovemRESUMO
In obesity, platelets are described as hyperactive, mainly based on increased platelet size and presence of pro-thrombotic plasmatic molecules. We explored platelet functions, including calcium signalling in obesity, and the effect of weight loss. We included 40 obese patients (women with body mass index [BMI] of ≥ 35 kg/m2) who were to undergo gastric bypass surgery and 40 healthy lean subjects (women with BMI of < 25 kg/m2) as a control group. Approximately 1 year after surgery, the obese patients lost weight (75% had a BMI < 35 kg/m2). They were explored a second time with the same healthy control for the same platelet experiments. Compared with controls, obese patients' platelets displayed reduced sensitivity to thrombin (aggregation EC50 increased by 1.9 ± 0.3-fold, p = 0.005) and a lower Ca2+ response (70 ± 7% decrease, p < 10-4). In 17 pairs of patients, we performed additional experiments: in obese patients' platelets, thrombin-induced αIIbß3 activation was significantly lower (p = 0.003) and sarco-endoplasmic reticulum Ca2+ATPase (SERCA3) expression was decreased (48 ± 6% decrease, p < 10-4). These differences were abolished after weight loss. Interestingly, pharmacological inhibition of SERCA3 activity in control group's platelets mimicked similar alterations than in obese patients' platelets and was associated with defective adenosine diphosphate (ADP) secretion. Addition of ADP to agonist restored platelet functions in obese patients and in SERCA3-inhibited control platelets (five experiments) confirming the direct involvement of the SERCA3-dependent ADP secretion pathway. This is the first study demonstrating that platelets from obese patients are hypo-reactive, due to a deficiency of SERCA3-dependent ADP secretion. Weight loss restores SERCA3 activity and subsequent calcium signalling, αIIbß3 activation, platelet aggregation and ADP secretion.
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Difosfato de Adenosina/sangue , Plaquetas/metabolismo , Derivação Gástrica , Obesidade/cirurgia , Ativação Plaquetária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/sangue , Redução de Peso , Adulto , Sinalização do Cálcio , Feminino , Humanos , Obesidade/sangue , Obesidade/diagnóstico , Obesidade/fisiopatologia , Paris , Agregação Plaquetária , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Via Secretória , Fatores de Tempo , Resultado do TratamentoRESUMO
There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbß3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood-brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.