RESUMO
OBJECTIVE: Mucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory immune cell function, we here focus on the enzyme ATP citrate lyase (ACLY) in mucosal T cell immunometabolism and its relevance for IBD. DESIGN: ACLY expression and its immunometabolic impact on colitogenic T cell function were analysed in mucosal T cells from patients with IBD and in two experimental colitis models. RESULTS: ACLY was markedly expressed in colon tissue under steady-state conditions but was significantly downregulated in lamina propria mononuclear cells in experimental dextran sodium sulfate-induced colitis and in CD4+ and to a lesser extent in CD8+ T cells infiltrating the inflamed gut in patients with IBD. ACLY-deficient CD4+ T cells showed an impaired capacity to induce intestinal inflammation in a transfer colitis model as compared with wild-type T cells. Assessment of T cell immunometabolism revealed that ACLY deficiency dampened the production of IBD-relevant cytokines and impaired glycolytic ATP production but enriched metabolites involved in the biosynthesis of phospholipids and phosphatidylcholine. Interestingly, the short-chain fatty acid butyrate was identified as a potent suppressor of ACLY expression in T cells, while IL-36α and resolvin E1 induced ACLY levels. In a translational approach, in vivo administration of the butyrate prodrug tributyrin downregulated mucosal infiltration of ACLYhigh CD4+ T cells and ameliorated chronic colitis. CONCLUSION: ACLY controls mucosal T cell immunometabolism and experimental colitis. Therapeutic modulation of ACLY expression in T cells emerges as a novel strategy to promote the resolution of intestinal inflammation.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Linfócitos Intraepiteliais , Humanos , Animais , Linfócitos Intraepiteliais/metabolismo , ATP Citrato (pro-S)-Liase/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Colite/metabolismo , Inflamação/metabolismo , Butiratos , Mucosa Intestinal/metabolismo , Sulfato de Dextrana , Modelos Animais de DoençasRESUMO
Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH4Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.
Assuntos
Autofagia , Linfócitos B , Glicerofosfolipídeos , Lisossomos , Animais , Camundongos , Linfócitos B/metabolismo , Glicerofosfolipídeos/metabolismo , Metabolismo dos Lipídeos , Lipidômica/métodos , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Proteômica/métodosRESUMO
Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.
Assuntos
Adenilato Quinase/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Células HEK293 , Células HeLa , Humanos , Transporte ProteicoRESUMO
B cells have a pivotal function in the pathogenesis of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus. In autoimmune disease, B cells orchestrate antigen presentation, cytokine production and autoantibody production, the latter via their differentiation into antibody-secreting plasmablasts and plasma cells. This article addresses the current therapeutic strategies to deplete B cells in order to ameliorate or potentially even cure autoimmune disease. It addresses the main target antigens in the B-cell lineage that are used for therapeutic approaches. Furthermore, it summarises the current evidence for successful treatment of autoimmune disease with monoclonal antibodies targeting B cells and the limitations and challenges of these approaches. Finally, the concept of deep B-cell depletion and immunological reset by chimeric antigen receptor T cells is discussed, as well as the lessons from this approach for better understanding the role of B cells in autoimmune disease.
RESUMO
Effective vaccines and monoclonal antibodies have been developed against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the appearance of virus variants with higher transmissibility and pathogenicity is a major concern because of their potential to escape vaccines and clinically approved SARS-CoV-2- antibodies. Here, we use flow cytometry-based binding and pseudotyped SARS-CoV-2 neutralization assays to determine the efficacy of boost immunization and therapeutic antibodies to neutralize the dominant Omicron variant. We provide compelling evidence that the third vaccination with BNT162b2 increases the amount of neutralizing serum antibodies against Delta and Omicron variants, albeit to a lower degree when compared to the parental Wuhan strain. Therefore, a third vaccination is warranted to increase titers of protective serum antibodies, especially in the case of the Omicron variant. We also found that most clinically approved and otherwise potent therapeutic antibodies against the Delta variant failed to recognize and neutralize the Omicron variant. In contrast, some antibodies under preclinical development potentially neutralized the Omicron variant. Our studies also support using a flow cytometry-based antibody binding assay to rapidly monitor therapeutic candidates and serum titers against emerging SARS-CoV-2 variants.
Assuntos
Antineoplásicos Imunológicos , COVID-19 , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , Vacinas contra COVID-19 , Humanos , SARS-CoV-2 , VacinaçãoRESUMO
TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Camundongos , SARS-CoV-2RESUMO
To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and successful vaccination against coronavirus disease 2019 (COVID-19), the kinetics of neutralizing or blocking anti-SARS-CoV-2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS-CoV-2 blocking assay (SUBA) using immobilized recombinant human angiotensin-converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS-CoV-2 spike protein. Spike protein-expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell-associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2- or 3-approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell-bound SARS-CoV-2 spike protein and can be rapidly adjusted to quickly pre-screen already approved therapeutic antibodies or sera from vaccinated individuals for their ACE2 blocking activities against any emerging SARS-CoV-2 variants.
Assuntos
Anticorpos Bloqueadores/sangue , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/análise , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Citometria de Fluxo/métodos , Anticorpos Bloqueadores/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Long-lived antibody-secreting plasma cells are essential to establish humoral memory against pathogens. While a regulatory transcription factor network has been established in plasma cell differentiation, the regulatory role of miRNAs remains enigmatic. We have recently identified miR-148a as the most abundant miRNA in primary mouse and human plasma cells. To determine whether this plasma cell signature miRNA controls the in vivo development of B cells into long-lived plasma cells, we established mice with genomic, conditional, and inducible deletions of miR-148a. The analysis of miR-148a-deficient mice revealed reduced serum Ig, decreased numbers of newly formed plasmablasts and reduced CD19-negative, CD93-positive long-lived plasma cells. Transcriptome and metabolic analysis revealed an impaired glucose uptake, a reduced oxidative phosphorylation-based energy metabolism, and an altered abundance of homing receptors CXCR3 (increase) and CXCR4 (reduction) in miR-148a-deficient plasma cells. These findings support the role of miR-148a as a positive regulator of the maintenance of long-lived plasma cells.
Assuntos
Diferenciação Celular/genética , Metabolismo Energético , Regulação da Expressão Gênica , MicroRNAs/genética , Plasmócitos/metabolismo , Animais , Antígenos CD19/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Medula Óssea/imunologia , Medula Óssea/metabolismo , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Epitopos de Linfócito B/imunologia , Técnicas de Silenciamento de Genes , Imunofenotipagem , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Plasmócitos/citologia , Plasmócitos/imunologia , Interferência de RNARESUMO
Psychostimulants are widely abused drugs that may cause addiction in vulnerable individuals. While the reward circuitry of the brain is involved in addiction establishment, various pathways in the brain may provide protection at the molecular level that limits the acute and chronic effects of drugs. These targets may be used for strategies designed to prevent and treat addiction. Swiprosin-1/EF hand domain 2 (EFhd2) is a Ca2+ -binding cytoskeletal adaptor protein involved in sensation-seeking behaviour, anxiety and alcohol addiction. Here, we tested how EFhd2 contributes to the physiological and behavioural effects of the psychostimulant drugs methamphetamine (METH) and cocaine. An in vivo microdialysis study in EFhd2 knockout mice revealed that EFhd2 controls METH- and cocaine-induced changes in extracellular dopamine, serotonin and noradrenaline levels through different mechanisms in the nucleus accumbens and prefrontal cortex. Electrophysiological recordings in a slice preparation showed that a lack of EFhd2 increases dopaminergic neuronal activity in the ventral tegmental area and increases the sensitivity of neurons to stimulation. We report a role of EFhd2 in METH-induced locomotor activation and in the conditioned locomotor effects. No role, however, was observed in the establishment of METH- or cocaine-induced conditioned place preference. These findings may suggest that EFhd2 modulates the activity of the dopaminergic system and the neurochemical effects of METH and cocaine, which translate into a modulation of the behavioural effects of these drugs at the level of the acute and conditioned locomotor activity.
Assuntos
Encéfalo/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Locomoção/efeitos dos fármacos , Metanfetamina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND/AIMS: Swiprosin-1/ EF-hand domain 2 (EFhd2) is a Ca2+ sensor protein that plays an important role in the immune system. Its abundant expression in the brain, however, suggested also a role in neuronal circuits and behavior. METHODS: Here we review recent discoveries on the structure and molecular function, its role in immunity and its function in the brain regarding behavioral control and pathologies. RESULTS: While EFhd2 did not emerge as a vital protein for brain development, changes in its expression may nevertheless shape the adult behavioral repertoire significantly and contribute to adult personality traits. A defective function of EFhd2 may also render individuals more prone to the development of psychiatric disorders. Most prominently, EFhd2 proved to be a resilience factor protecting from fast establishment of drug addiction. Moreover, EFhd2 is critical for adult neurogenesis and as a modulator of monoaminergic systems. CONCLUSION: Dysregulated activity of EFhd2 is increasingly considered as a contributing factor for the development of numerous neurodegenerative disorders. Whether EFhd2 can be used as biomarker or in therapeutic approaches has to be addressed in future research.
Assuntos
Encefalopatias/imunologia , Encefalopatias/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/imunologia , Transtornos da Personalidade/imunologia , Transtornos da Personalidade/metabolismo , Animais , Encefalopatias/genética , Proteínas de Ligação ao Cálcio/genética , Humanos , Transtornos da Personalidade/genéticaRESUMO
The prevalence of neurodegenerative disease and arthritis increases with age. Despite both processes being associated with immune activation and inflammation, little is known about the mechanistic interactions between neurodegenerative disease and arthritis. In this article, we show that tau-transgenic (tau-tg) mice that develop neurodegenerative disease characterized by deposition of tau tangles in the brain are highly susceptible to developing arthritis. Already at steady-state conditions, tau-tg mice exhibit peripheral immune activation that is manifested by higher numbers of granulocytes, plasmablasts, and inflammatory Ly6Chi CCR2+ monocytes, as well as increased levels of proinflammatory cytokines, such as TNF-α and IL-17. Upon induction of collagen-induced arthritis (CIA), tau-tg mice displayed an increased incidence and an earlier onset of CIA that was associated with a more pronounced inflammatory cytokine response. Furthermore, induction of CIA led to significantly elevated numbers of Iba-1-expressing cells in the brain, indicative of microglia activation, and the formation of anti-tau Abs in tau-tg mice. These changes were accompanied by the resolution of tau tangles and significantly decreased neurodegenerative pathology. In summary, these data show that neurodegenerative disease enhances the development of arthritis. In addition, arthritis, once induced, triggers innate immune responses in the brain, leading to resolution of neurodegenerative changes.
Assuntos
Encéfalo/imunologia , Microglia/imunologia , Proteínas tau/metabolismo , Animais , Artrite Experimental , Autoanticorpos/sangue , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Doenças Neurodegenerativas , Emaranhados Neurofibrilares/imunologia , Proteínas tau/genética , Proteínas tau/imunologiaRESUMO
The critical size for strong interaction of hydrophobic particles with phospholipid bilayers has been predicted to be 10 nm. Because of the wide spreading of nonpolar nanoparticles (NPs) in the environment, we aimed to reveal the ability of living organisms to entrap NPs via formation of neutrophil extracellular traps (NETs). Upon interaction with various cell types and tissues, 10- to 40-nm-sized NPs induce fast (<20 min) damage of plasma membranes and instability of the lysosomal compartment, leading to the immediate formation of NETs. In contrast, particles sized 100-1,000 nm behaved rather inertly. Resulting NET formation (NETosis) was accompanied by an inflammatory reaction intrinsically endowed with its own resolution, demonstrated in lungs and air pouches of mice. Persistence of small NPs in joints caused unremitting arthritis and bone remodeling. Small NPs coinjected with antigen exerted adjuvant-like activity. This report demonstrates a cellular mechanism that explains how small NPs activate the NETosis pathway and drive their entrapping and resolution of the initial inflammatory response.
Assuntos
Armadilhas Extracelulares/metabolismo , Inflamação/patologia , Nanopartículas/química , Tamanho da Partícula , Animais , Membrana Celular/metabolismo , Eritrócitos/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunidade , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Nanodiamantes/química , Nanodiamantes/ultraestrutura , Nanopartículas/ultraestrutura , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Espécies Reativas de Oxigênio/metabolismoRESUMO
We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.
Assuntos
Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Plasmócitos/classificação , Plasmócitos/imunologia , Coloração e Rotulagem/métodos , Animais , Antígenos CD19/análise , Células da Medula Óssea/imunologia , Diferenciação Celular , Proteínas de Fluorescência Verde , Imunofenotipagem/instrumentação , Antígenos Comuns de Leucócito/análise , Camundongos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Baço/imunologia , Sindecana-1/análise , Fatores de Transcrição/análiseRESUMO
The IL-1 family member IL-36α has proinflammatory and pathogenic properties in psoriasis. IL-36α binds to the IL-36 receptor leading to nuclear factor kappa B/mitogen activated protein kinase mediated cytokine release. The IL-36R antagonist prevents recruitment of IL-1 receptor accessory protein and therefore IL-36-dependent cell activation. In inflamed human tissue, we previously could show that resident B cells and plasma cells (PC) express IL-36α. Further, fibroblast-like synoviocytes (FLS) produced proinflammatory cytokines upon IL-36α-stimulation. We hypothesize an IL-36-specific crosstalk between B cells/PCs and FLS permitting a proinflammatory B cell niche. Here, we firstly demonstrated that B cell lines and B cells from healthy donors express IL-36α and stimulation increased IL-36α in B cells and primary plasmablasts/PCs. Moreover, FLS respond specifically to IL-36α by proliferation and production of matrix metalloproteinases via p38/HSP27 signaling. Importantly, IL-36R-deficiency abrogated IL-36α-induced production of inflammatory mediators in FLS and changed the intrinsic FLS-phenotype. Using an in vitro co-culture system, we could show that IL-36R-deficient FLS had a limited capacity to support PC survival compared to wild-type FLS. Hence, we demonstrated an IL-36R-dependent crosstalk between B cells/PCs and FLS. Our data support the concept of initiation and maintenance of a proinflammatory niche by B cells in the joints.
Assuntos
Fibroblastos/imunologia , Plasmócitos/imunologia , Receptores de Interleucina-1/imunologia , Membrana Sinovial/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica/imunologia , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-1/farmacologia , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Plasmócitos/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/metabolismoRESUMO
The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body's own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca2+-mediated mitoflashes. Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Metabolismo Energético/imunologia , Animais , Cálcio/metabolismo , Glicólise/imunologia , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fosforilação OxidativaRESUMO
The CC-chemokine receptor 6 (CCR6) can be detected on naive and activated B cells. Counterintuitively, its absence accelerates the appearance of germinal centres (GCs) and increases the production of low-affinity antibodies. The detailed mechanism of CCR6 function during the humoral response has remained elusive, but previously we identified a distinct CCR6high B-cell population in vivo early after antigenic challenge. In this study, we defined this population specifically as early, activated pre-GC B cells. In accordance, we show that CCR6 is upregulated rapidly within hours on the protein or mRNA level after activation in vitro. In addition, only activated B cells migrated specifically towards CCL20, the specific ligand for CCR6. Lack of CCR6 increased the dark zone/light zone ratio of GC and led to decreased antigen-specific IgG1 and IgG2a antibody generation in a B-cell intrinsic manner in mixed bone marrow chimeras. In contrast, antigen-specific IgM responses were normal. Hence, CCR6 negatively regulates entry of activated, antigen-specific pre-GC B cells into the GC reaction.
Assuntos
Formação de Anticorpos/imunologia , Linfócitos B/metabolismo , Centro Germinativo/metabolismo , Receptores CCR6/metabolismo , Animais , Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CCL20/farmacologia , Citometria de Fluxo , Centro Germinativo/efeitos dos fármacos , Cinética , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR6/genética , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVES: Janus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-ß. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts. METHODS: The antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance. RESULTS: The antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis. CONCLUSION: Fibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.
Assuntos
Fibroblastos/efeitos dos fármacos , Janus Quinase 1/efeitos dos fármacos , Janus Quinase 2/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fibrose Pulmonar/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Escleroderma Sistêmico , Sulfonamidas/farmacologia , Adulto , Animais , Antibióticos Antineoplásicos/toxicidade , Benzoquinonas/farmacologia , Bleomicina/toxicidade , Western Blotting , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Imuno-Histoquímica , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Lactamas Macrocíclicas/farmacologia , Pulmão/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Nitrilas , Fosforilação/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FL-integral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry.
Assuntos
Linfócitos B/metabolismo , Citometria de Fluxo/métodos , Antígeno de Macrófago 1/química , Neutrófilos/metabolismo , Receptores de Antígenos de Linfócitos B/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/ultraestrutura , Carbocianinas/química , Separação Celular , Fluorescência , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Antígeno de Macrófago 1/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Cultura Primária de Células , Transporte Proteico , Receptores de Antígenos de Linfócitos B/imunologia , Coloração e Rotulagem/métodosRESUMO
Synaptic dysfunction and dysregulation of Ca(2+) are linked to neurodegenerative processes and behavioural disorders. Our understanding of the causes and factors involved in behavioural disorders and neurodegeneration, especially Alzheimer's disease (AD), a tau-related disease, is on the one hand limited and on the other hand controversial. Here, we review recent data about the links between the Ca(2+)-binding EF-hand-containing cytoskeletal protein Swiprosin-1/EFhd2 and neurodegeneration. Specifically, we summarize the functional biochemical data obtained in vitro with the use of recombinant EFhd2 protein, and integrated them with in vivo data in order to interpret the emerging role of EFhd2 in synaptic plasticity and in the pathophysiology of neurodegenerative disorders, particularly involving the tauopathies. We also discuss its functions in actin remodelling through cofilin and small GTPases, thereby linking EFhd2, synapses and the actin cytoskeleton. Expression data and functional experiments in mice and in humans have led to the hypothesis that down-regulation of EFhd2, especially in the cortex, is involved in dementia.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Sistema Nervoso/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Neurônios/metabolismo , Transporte Proteico , Sinapses/metabolismoRESUMO
The bone marrow provides niches for early B cell differentiation and long-lived plasma cells. Therefore, it has been hypothesized that perturbing bone homeostasis might impact B cell function and Ab production. This hypothesis is highly relevant for patients receiving long-term treatment with antiresorptive drugs. We therefore analyzed the humoral immune response of mice chronically treated with ibandronate, a commonly used bisphosphonate. We confirmed the increased bone mass caused by inhibition of osteoclast activity and also the strongly reduced bone formation because of decreased osteoblast numbers in response to ibandronate. Thus, bisphosphonate drastically inhibited bone remodeling. When ibandronate was injected into mice after a primary immunization to mimic common antiosteoporotic treatments, the generation of the various B cell populations, the response to booster immunization, and the generation of plasma cells were surprisingly normal. Mice also responded normally to immunization when ibandronate was applied to naive mice. However, there, ibandronate shunted the homing of bone marrow plasma cells. Interestingly, ibandronate reduced the numbers of megakaryocytes, a known component of the bone marrow plasma cell niche. In line with normal Ab responses, increased plasma cell populations associated with increased megakaryocyte numbers were then observed in the spleens of the ibandronate-treated mice. Thus, although inhibition of bone remodeling disturbed the bone marrow plasma cell niche, a compensatory niche may have been created by relocating the megakaryocytes into the spleen, thereby allowing normal B cell responses. Therefore, megakaryocytes may act as a key regulator of plasma cell niche plasticity.