RESUMEN
MLL rearrangement (MLLr) is responsible for the development of acute leukemias with poor outcomes. Therefore, new therapeutic approaches are urgently needed. The NOTCH1 pathway plays a critical role in the pathogenesis of many cancers including acute leukemia. Using a CRISPR/Cas9 MLL-AF4/-AF9 translocation model, the newly developed NOTCH1 inhibitor CAD204520 with less toxic side effects allowed us to unravel the impact of NOTCH1 as a pathogenic driver and potential therapeutic target in MLLr leukemia. RNA sequencing (RNA-seq) and RT-qPCR of our MLLr model and MLLr cell lines showed the NOTCH1 pathway was overexpressed and activated. Strikingly, we confirmed this elevated expression level in leukemia patients. We also demonstrated that CAD204520 treatment of MLLr cells significantly reduces NOTCH1 and its target genes as well as NOTCH1 receptor expression. This was not observed with a comparable cytarabine treatment, indicating the specificity of the small molecule. Accordingly, treatment with CAD204520 resulted in dose-dependent reduced proliferation and viability, increased apoptosis, and the induction of cell cycle arrest via the downregulation of MLL and NOTCH1 target genes. In conclusion, our findings uncover the oncogenic relevance of the NOTCH1 pathway in MLLr leukemia. Its inhibition leads to specific anti-leukemic effects and paves the way for further evaluation in clinical settings.
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Leucemia Mieloide Aguda , Receptor Notch1 , Humanos , Puntos de Control del Ciclo Celular/genética , Citarabina/uso terapéutico , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/genética , Receptor Notch1/genéticaRESUMEN
Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. We recently showed in murine studies and in vitro human models that adoptively transferred invariant natural killer T (iNKT) cells protect from GvHD and promote graft-versus-leukemia effects. The cellular mechanisms underlying GvHD prevention by iNKT cells in humans, however, remain unknown. In order to study relevant cellular interactions, dendritic cells (DC) were either generated from monocytes or isolated directly from blood of healthy donors or GvHD patients and co-cultured in a mixed lymphocyte reaction (MLR) with T cells obtained from healthy donors or transplantation bags. Addition of culture-expanded iNKT cells to the MLR-induced DC apoptosis in a cell contact-dependent manner, thereby preventing T-cell activation and proliferation. Annexin V/propidium iodide staining and image stream assays showed that CD4+CD8-, CD4-CD8+ and double negative iNKT cells are similarly able to induce DC apoptosis. Further MLR assays revealed that conventional DC (cDC) but not plasmacytoid DC (pDC) could induce alloreactive T-cell activation and proliferation. Interestingly, cDC were also more susceptible to apoptosis induced by iNKT cells, which correlates with their higher CD1d expression, leading to a bias in favor of pDC. Remarkably, these results could also be observed in GvHD patients. We propose a new mechanism how ex vivo expanded human iNKT cells prevent alloreactivity of T cells. iNKT cells modulate T-cell responses by selective apoptosis of DC subsets, resulting in suppression of T-cell activation and proliferation while enabling beneficial immune responses through pDC.
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Enfermedad Injerto contra Huésped , Células T Asesinas Naturales , Animales , Apoptosis , Células Dendríticas , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Activación de Linfocitos , RatonesRESUMEN
OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease with poorly understood pathogenesis and limited treatment options. Patient mortality is rooted predominantly in the development of pulmonary and cardiac complications. The overactivated immune system is assumed to sustain the inflammatory signature of this autoimmune disease. Here, we investigate the potential of immunoregulatory invariant natural killer T (iNKT) cells to inhibit proinflammatory B cell responses in an in vitro model of inflammation. METHODS: B cells from healthy volunteers (n = 17) and patients with SSc (n = 15) were used for functional testing upon lipopolysaccharide (LPS) stimulation in a co-culture system with third-party iNKT cells. Cytokine production was measured with antibody-based immunoassays (ELISA) and intracellular cytokine staining. RESULTS: iNKT cells strongly inhibited the production of proinflammatory interleukin-6 by B cells upon stimulation with LPS in both healthy volunteers and patients with SSc. In a Transwell assay, cell contact between B cells and iNKT cells proved necessary for this inhibitory effect. Similarly, blocking of CD1d on the surface of B cells abolished the immunoregulatory effect of iNKT cells on B cells. B cell subsets with higher expression of CD1d, namely unswitched memory B cells, were more susceptible to iNKT cell inhibition. CONCLUSION: Our in vitro data underline the potential of iNKT cells in the control of SSc and provide a rationale for the use of novel iNKT cell-based therapeutic strategies in the context of autoimmune diseases.
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Células T Asesinas Naturales , Esclerodermia Sistémica , Citocinas/metabolismo , Humanos , Interleucina-6/metabolismo , Lipopolisacáridos , Activación de Linfocitos , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/terapiaRESUMEN
Annexin A1 is an intracellular calcium/phospholipid-binding protein that is involved in membrane organization and the regulation of the immune system. It has been attributed an anti-inflammatory role at various control levels, and recently we could show that annexin A1 externalization during secondary necrosis provides an important fail-safe mechanism counteracting inflammatory responses when the timely clearance of apoptotic cells has failed. As such, annexin A1 promotes the engulfment of dying cells and dampens the postphagocytic production of proinflammatory cytokines. In our current follow-up study, we report that exposure of annexin A1 during secondary necrosis coincided with proteolytic processing within its unique N-terminal domain by ADAM10. Most importantly, we demonstrate that the released peptide and culture supernatants of secondary necrotic, annexin A1-externalizing cells induced chemoattraction of monocytes, which was clearly reduced in annexin A1- or ADAM10-knockdown cells. Thus, altogether our findings indicate that annexin A1 externalization and its proteolytic processing into a chemotactic peptide represent final events during apoptosis, which after the transition to secondary necrosis contribute to the recruitment of monocytes and the prevention of inflammation.
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Proteínas ADAM/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Anexina A1/inmunología , Factores Quimiotácticos/inmunología , Quimiotaxis/inmunología , Proteínas de la Membrana/inmunología , Monocitos/inmunología , Proteolisis , Transducción de Señal/inmunología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Anexina A1/genética , Anexina A1/metabolismo , Factores Quimiotácticos/genética , Factores Quimiotácticos/metabolismo , Quimiotaxis/genética , Técnicas de Silenciamiento del Gen , Células HL-60 , Humanos , Células Jurkat , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Monocitos/metabolismo , Monocitos/patología , Necrosis/genética , Necrosis/inmunología , Necrosis/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal/genética , Células U937RESUMEN
The elimination of apoptotic cells, called efferocytosis, is fundamentally important for tissue homeostasis and prevents the onset of inflammation and autoimmunity. Serum proteins are known to assist in this complex process. In the current study, we performed a multistep chromatographic fractionation of human serum and identified plasminogen, a protein involved in fibrinolysis, wound healing, and tissue remodeling, as a novel serum-derived factor promoting apoptotic cell removal. Even at levels significantly lower than its serum concentration, purified plasminogen strongly enhanced apoptotic prey cell internalization by macrophages. Plasminogen acted mainly on prey cells, whereas on macrophages no enhancement of the engulfment process was observed. We further demonstrate that the efferocytosis-promoting activity essentially required the proteolytic activation of plasminogen and was completely abrogated by the urokinase plasminogen activator inhibitor-1 and serine protease inhibitor aprotinin. Thus, our study assigns a new function to plasminogen and plasmin in apoptotic cell clearance.
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Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/inmunología , Fagocitosis/inmunología , Plasminógeno/metabolismo , Sistema del Grupo Sanguíneo ABO/sangre , Proteínas Reguladoras de la Apoptosis/sangre , Línea Celular Tumoral , Cromatografía de Afinidad/métodos , Humanos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Plasminógeno/deficiencia , Plasminógeno/fisiología , Cultivo Primario de Células , Suero/inmunologíaRESUMEN
Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative trait locus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis with recombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small molecule inhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human gene C5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to the identification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal role in fibrogenesis across species.
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Complemento C5/genética , Cirrosis Hepática/genética , Sitios de Carácter Cuantitativo , Animales , Mapeo Cromosómico , Complemento C5/metabolismo , Genotipo , Humanos , Ratones , Ratones Endogámicos , Polimorfismo GenéticoRESUMEN
Dysregulated cholesterol homeostasis promotes tumorigenesis and progression. Therefore, metabolic reprogramming constitutes a new hallmark of cancer. However, until today, only few therapeutic approaches exist to target this pathway due to the often-observed negative feedback induced by agents like statins leading to controversially increased cholesterol synthesis upon inhibition. Sterol regulatory element-binding proteins (SREBPs) are key transcription factors regulating the synthesis of cholesterol and fatty acids. Since SREBP2 is difficult to target, we performed pharmacological inhibition of retinoic acid receptor (RAR)-related orphan receptor gamma (RORγ), which acts upstream of SREBP2 and serves as master regulator of the cholesterol metabolism. This resulted in an inactivated cholesterol-related gene program with significant downregulation of cholesterol biosynthesis. Strikingly, these effects were more pronounced than the effects of fatostatin, a direct SREBP2 inhibitor. Upon RORγ inhibition, RNA sequencing showed strongly increased cholesterol efflux genes leading to leukemic cell death and cell cycle changes in a dose- and time-dependent manner. Combinatorial treatment of t(4;11) cells with the RORγ inhibitor showed additive effects with cytarabine and even strong anti-leukemia synergism with atorvastatin by circumventing the statin-induced feedback. Our results suggest a novel therapeutic strategy to inhibit tumor-specific cholesterol metabolism for the treatment of t(4;11) leukemia.
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Colesterol , Leucemia , Neoplasias , Humanos , Colesterol/metabolismo , Homeostasis , Leucemia/tratamiento farmacológico , Leucemia/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
Locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis. The introduction of PD-1 inhibitors has led to a significant improvement in survival, but only a subpopulation of patients responds to therapy. Current biomarkers cannot reliably identify these patients. The identification of biomarkers for the prediction and monitoring of immunotherapy is therefore of great importance. In this study, we characterized lymphocyte subsets in the peripheral blood of HNSCC patients under PD-1 inhibition. Patients with primary response (n=11) to PD-1 inhibition showed an increase of the CD3+ effector memory (CD3/EM) population and an elevated expression of the activation marker CD69 in CD3+ T cells, particularly in the CD3/EM subpopulation at 3 months when treatment response was assessed. In contrast, patients with primary treatment failure and progressive disease (n=9) despite PD-1 inhibition had lower absolute lymphocyte counts and an increased expression of CTLA-4 in CD3+ T cells at the time of treatment failure compared with baseline, particularly in CD4+ and CD8+ effector memory populations. Our results demonstrate that HNSCC patients' response to immune checkpoint inhibition shows a distinct immune signature in peripheral blood, which could help identify refractory patients earlier. Furthermore, strategies to overcome primary therapy failure by inducing a beneficial T cell phenotype or adding alternative immune checkpoint inhibitors could improve response rates and survival of HNSCC patients.
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BACKGROUND: Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties. METHODS: iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings. RESULTS: In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses. CONCLUSION: In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT.
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Enfermedad Injerto contra Huésped , Células T Asesinas Naturales , Receptores Quiméricos de Antígenos , Humanos , Ratones , Animales , Receptor de Muerte Celular Programada 1 , Antígenos CD19 , Enfermedad Injerto contra Huésped/etiología , RecurrenciaRESUMEN
Peritoneal carcinomatosis is an advanced form of metastatic disease characterized by cancer cell dissemination onto the peritoneum. It is commonly observed in ovarian and colorectal cancers and is associated with poor patient survival. Novel therapies consist of cytoreductive surgery in combination with intraperitoneal chemotherapy, aiming at tumor cell death induction. The resulting dying tumor cells are considered to be eliminated by professional as well as semi-professional phagocytes. In the present study, we have identified a hitherto unknown type of 'amateur' phagocyte in this environment: human peritoneal mesothelial cells (HMCs). We demonstrate that HMCs engulf corpses of dying ovarian and colorectal cancer cells, as well as other types of apoptotic cells. Flow cytometric, confocal and electron microscopical analyses revealed that HMCs ingest dying cell fragments in a dose- and time-dependent manner and the internalized material subsequently traffics into late phagolysosomes. Regarding the mechanisms of prey cell recognition, our results show that HMCs engulf apoptotic corpses in a serum-dependent and -independent fashion and quantitative real-time PCR (qRT-PCR) analyses revealed that diverse opsonin receptor systems orchestrating dying cell clearance are expressed in HMCs at high levels. Our data strongly suggest that HMCs contribute to dying cell removal in the peritoneum, and future studies will elucidate in what manner this influences tumor cell dissemination and the antitumor immune response.
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Carcinoma/patología , Neoplasias Colorrectales/patología , Células Epiteliales/patología , Neoplasias Ováricas/patología , Neoplasias Peritoneales/patología , Peritoneo/patología , Fagocitosis/fisiología , Apoptosis/fisiología , Células CACO-2 , Línea Celular Tumoral , Células Cultivadas , Femenino , Células HT29 , Humanos , Microscopía Confocal , Neoplasias Peritoneales/secundario , Microambiente TumoralRESUMEN
MLL rearranged (MLLr) leukemias are associated with a poor prognosis and a limited response to conventional therapies. Moreover, chemotherapies result in severe side effects with significant impairment of the immune system. Therefore, the identification of novel treatment strategies is mandatory. Recently, we developed a human MLLr leukemia model by inducing chromosomal rearrangements in CD34+ cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This MLLr model authentically mimics patient leukemic cells and can be used as a platform for novel treatment strategies. RNA sequencing of our model revealed MYC as one of the most important key drivers to promote oncogenesis. However, in clinical trials the BRD4 inhibitor JQ-1 leading to indirect blocking of the MYC pathway shows only modest activity. We and others previously reported that epigenetic drugs targeting MAT2A or PRMT5 promote cell death in MLLr cells. Therefore, we use these drugs in combination with JQ-1 leading to augmented anti-leukemic effects. Moreover, we found activation of T, NK and iNKT cells, release of immunomodulatory cytokines and downregulation of the PD-1/PD-L1 axis upon inhibitor treatment leading to improved cytotoxicity. In summary, the inhibition of MYC and MAT2A or PRMT5 drives robust synergistic anti-leukemic activity in MLLr leukemia. Moreover, the immune system is concomitantly activated upon combinatorial inhibitor treatment, hereby further augmenting the therapeutic efficiency.
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Leucemia , Proteína de la Leucemia Mieloide-Linfoide , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Leucemia/tratamiento farmacológico , Leucemia/genética , Epigénesis Genética , Proteínas de Ciclo Celular/genética , Proteína-Arginina N-Metiltransferasas/genética , Metionina Adenosiltransferasa/genéticaRESUMEN
The NALP3 inflammasome is activated by low intracellular potassium concentrations [K(+)](i), leading to the secretion of the proinflammatory cytokine IL-1ß. However, the mechanism of [K(+)](i) lowering after phagocytosis of monosodium urate crystals is still elusive. Here, we propose that endosomes containing monosodium urate crystals fuse with acidic lysosomes. The low pH in the phagolysosome causes a massive release of sodium and raises the intracellular osmolarity. This process is balanced by passive water influx through aquaporins leading to cell swelling. This process dilutes [K(+)](i) to values below the threshold of 90 mm known to activate NALP3 inflammasomes without net loss of cytoplasmic potassium ions. In vitro, the inhibitors of lysosomal acidification (ammonium chloride, chloroquine) and of aquaporins (mercury chloride, phloretin) all significantly decreased the production of IL-1ß. In vivo, only the pharmacological inhibitor of lysosome acidification chloroquine could be used which again significantly reduced the IL-1ß production. As a translational aspect one may consider the use of chloroquine for the anti-inflammatory treatment of refractory gout.
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Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Sodio/metabolismo , Agua/metabolismo , Animales , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Fagocitos/citología , Fagocitos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Potasio/metabolismo , Ácido Úrico/química , Ácido Úrico/metabolismoRESUMEN
Cytomegalovirus (CMV) reactivation is common after allogeneic hematopoietic cell transplantation (HCT) and may result in fatal CMV disease. Invariant natural killer T (iNKT) cells are potent modulators of the immune system preventing graft-versus-host disease while promoting graft-versus-leukemia effects. It is thought that iNKT cells selectively influence mediators of both innate and adaptive immunity. Here, we investigated the impact of graft iNKT cells on CMV reactivation in patients undergoing allogeneic HCT. We found a significantly decreased cumulative incidence of CMV reactivation in patients with higher numbers of iNKT cells in the allograft. Therefore iNKT-cell-enriched grafts or adoptive transfer of iNKT cells are compelling cytotherapeutic strategies to improve outcomes after allogeneic HCT.
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Infecciones por Citomegalovirus , Trasplante de Células Madre Hematopoyéticas , Células T Asesinas Naturales , Citomegalovirus , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Factores de RiesgoRESUMEN
The engulfment of apoptotic cells is of crucial importance for tissue homeostasis in multicellular organisms. A failure of this process results in secondary necrosis triggering proinflammatory cytokine production and autoimmune disease. In the present study, we investigated the role of annexin A1, an intracellular protein that has been implicated in the efficient removal of apoptotic cells. Consistent with its function as bridging protein in the phagocyte synapse, opsonization of apoptotic cells with purified annexin A1 strongly enhanced their phagocytic uptake. A detailed analysis, however, surprisingly revealed that annexin A1 was hardly exposed to the cell surface of primary apoptotic cells, but was strongly externalized only on secondary necrotic cells. Interestingly, while the exposure of annexin A1 failed to promote the uptake of these late secondary necrotic cells, it efficiently prevented induction of cytokine production in macrophages during engulfment of secondary necrotic cells. Our results therefore suggest that annexin A1 exposure during secondary necrosis provides an important failsafe mechanism counteracting inflammatory responses, even when the timely clearance of apoptotic cells has failed.
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Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/fisiología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Apoptosis/inmunología , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/patología , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Citocinas/metabolismo , Humanos , Células Jurkat , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Proteínas de la Membrana/fisiología , Necrosis/inmunología , Necrosis/prevención & control , Fagocitosis/inmunologíaRESUMEN
BACKGROUND & AIMS: Fibrosis is the hallmark of chronic liver diseases, yet many aspects of its mechanism remain to be defined. Chemokines are ubiquitous chemotactic molecules that mediate many acute and chronic inflammatory conditions, and CXC chemokine genes colocalize with a locus previously shown to include fibrogenic genes. We investigated the roles of the chemokine CXCL9 and its receptor CXCR3 in liver fibrosis. METHODS: The effects of CXCL variants on fibrogenesis were analyzed using samples from patients with hepatitis C virus infection and by induction of fibrosis in CXCR3(-/-) and wild-type mice. In mice, intrahepatic immune cell subsets were investigated and interferon gamma messenger RNA levels were measured at baseline and after injury. Human serum CXCL9 levels were measured and correlated with CXCL9 variant and fibrosis severity. The effects of stimulation with CXCL9 were investigated on human hepatic stellate cells (LX-2). RESULTS: Specific CXCL9 variants were associated with liver fibrosis in mice and humans; CXCL9 serum concentrations correlated with genotypes and levels of fibrosis in patients. In contrast to other chemokines, CXCL9 exerted antifibrotic effects in vitro, suppressing collagen production in LX-2 cells. CXCR3(-/-) mice had increased liver fibrosis; progression was associated with decreased numbers of intrahepatic interferon gamma-positive T cells and reduced interferon gamma messenger RNA, indicating that CXCL9-CXCR3 regulates Th1-associated immune pathways. CONCLUSIONS: This is the first description of a chemokine-based antifibrotic pathway in the liver; antifibrotic therapies might be developed to modulate CXC chemokine levels.
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Quimiocina CXCL9/metabolismo , Cirrosis Hepática/prevención & control , Hígado/inmunología , Receptores CXCR3/metabolismo , Animales , Tetracloruro de Carbono , Células Cultivadas , Quimiocina CXCL9/sangre , Quimiocina CXCL9/genética , Estudios de Cohortes , Colágeno/metabolismo , Modelos Animales de Enfermedad , Genotipo , Células Estrelladas Hepáticas/inmunología , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/inmunología , Humanos , Interferón gamma/metabolismo , Hígado/metabolismo , Hígado/patología , Hígado/virología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/inmunología , Cirrosis Hepática/patología , Cirrosis Hepática/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Polimorfismo de Nucleótido Simple , ARN Mensajero/metabolismo , Receptores CXCR3/deficiencia , Receptores CXCR3/genética , Índice de Severidad de la Enfermedad , Células TH1/inmunologíaRESUMEN
OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease with a significant morbidity and reduced survival of patients. Effective treatment and clinical control of the disease remain challenging. In particular, the development of pulmonary and cardiac fibrosis and pulmonary hypertension are severe complications responsible for excessive mortality. Currently available treatment strategies only alleviate symptoms and slow disease progression. Here, we investigated the therapeutic potential of ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor used in B cell malignancies, to alter B cell pathology in SSc in an in vitro model of autoimmunity. METHODS: PBMCs and sorted B cells of 24 patients with SSc were used for functional testing after stimulation with hypomethylated DNA fragments (CpG) to induce an innate immune response. The effects of ibrutinib on cytokine production, autoantibody release, and activation of the transcription factor NFκB were evaluated. RESULTS: Ibrutinib was able to reduce the production of the profibrotic hallmark cytokines IL-6 and TNF-α mainly from the effector B cell population in patients with SSc. Importantly, small doses of ibrutinib (0.1 µM) preserved the production of immunoregulatory IL-10 while effectively inhibiting hyperactivated, profibrotic effector B cells. In a flow cytometry analysis of phosphorylated NFκB, an important transcription factor in the induction of innate immune responses in B cells, significantly less activation was observed with ibrutinib treatment. CONCLUSION: Our data could pave the avenue for a clinical application of ibrutinib for patients with SSc as a novel treatment option for the underlying pathogenetic immune imbalance contributing to disease onset and progression.
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Adenina/análogos & derivados , Linfocitos B/efectos de los fármacos , Piperidinas/farmacología , Adenina/farmacología , Adulto , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Anciano , Anciano de 80 o más Años , Linfocitos B/citología , Linfocitos B/metabolismo , Células Cultivadas , Citocinas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patologíaRESUMEN
Mixed lineage leukemia (MLL) (KMT2A) rearrangements (KMT2Ar) play a crucial role in leukemogenesis. Dependent on age, major differences exist regarding disease frequency, main fusion partners and prognosis. In infants, up to 80% of acute lymphoid leukemia (ALL) bear a MLL translocation and half of them are t(4;11), resulting in a poor prognosis. In contrast, in adults only 10% of acute myeloid leukemia (AML) bear t(9;11) with an intermediate prognosis. The reasons for these differences are poorly understood. Recently, we established an efficient CRISPR/Cas9-based KMT2Ar model in hematopoietic stem and progenitor cells (HSPCs) derived from human cord blood (huCB) and faithfully mimicked the underlying biology of the disease. Here, we applied this model to HSPCs from adult bone marrow (huBM) to investigate the impact of the cell of origin and fusion partner on disease development. Both genome-edited infant and adult KMT2Ar cells showed monoclonal outgrowth with an immature morphology, myelomonocytic phenotype and elevated KMT2Ar target gene expression comparable to patient cells. Strikingly, all KMT2Ar cells presented with indefinite growth potential except for MLL-AF4 huBM cells ceasing proliferation after 80 days. We uncovered FFAR2, an epigenetic tumor suppressor, as potentially responsible for the inability of MLL-AF4 to immortalize adult cells under myeloid conditions.
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Epigenetic dysregulation plays a pivotal role in mixed-lineage leukemia (MLL) pathogenesis, therefore serving as a suitable therapeutic target. S-adenosylmethionine (SAM) is the universal methyl donor in human cells and is synthesized by methionine adenosyltransferase 2A (MAT2A), which is deregulated in different cancer types. Here, we used our human CRISPR/Cas9-MLL-rearranged (CRISPR/Cas9-MLLr) leukemia model, faithfully mimicking MLLr patients' pathology with indefinite growth potential in vitro, to evaluate the unknown role of MAT2A. Comparable to publicly available patient data, we detected MAT2A to be significantly overexpressed in our CRISPR/Cas9-MLLr model compared to healthy controls. By using non-MLLr and MLLr cell lines and our model, we detected an MLLr-specific enhanced response to PF-9366, a new MAT2A inhibitor, and small interfering (si) RNA-mediated knockdown of MAT2A, by alteration of the proliferation, viability, differentiation, apoptosis, cell cycling, and histone methylation. Moreover, the combinational treatment of PF-9366 with chemotherapy or targeted therapies against the SAM-dependent methyltransferases, disruptor of telomeric silencing 1 like (DOT1L) and protein arginine methyltransferase 5 (PRMT5), revealed even more pronounced effects. In summary, we uncovered MAT2A as a key regulator in MLL leukemogenesis and its inhibition led to significant anti-leukemic effects. Therefore, our study paves the avenue for clinical application of PF-9366 to improve the treatment of poor prognosis MLLr leukemia.
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Phagocytosis of apoptotic cells, e.g., neutrophils, by monocytes is essential for resolution of inflammation. Delayed removal leads to secondary necrosis, perpetuating inflammation, and tissue destruction. Common histologic features in neonatal chronic inflammatory disorders are an accumulation of apoptotic cells in inflamed tissues. We hypothesized that apoptotic cell removal by monocytes is compromised in newborns. PKH-26 labeled autologous or allogeneic apoptotic neutrophils were fed to monocytes of adult donors (PBMO) and cord blood (CBMO), and phagocytic activity was analyzed by flow cytometry and confocal microscopy. Relative mRNA-expression levels of 21 surface receptors and bridging molecules relevant for apoptotic cell removal were measured, as was postphagocytic IL-8 production upon LPS-stimulation. Compared with PBMO, CBMO exhibited a significantly diminished phagocytotic competence for autologous and allogeneic apoptotic neutrophils. mRNA-expression levels of milk fat globule-EGF factor 8 and T cell immunoglobulin- and mucin-domain-containing molecule, two crucial members of the phagocytic synapse of apoptotic cell removal, were reduced in CBMO. In PBMO, interaction with autologous apoptotic neutrophils reduced LPS-induced IL-8 production whereas it was enhanced in CBMO. Our data suggest a specific defect in CBMO during clearance of apoptotic neutrophils resulting in impaired anti-inflammatory capacity.
Asunto(s)
Apoptosis , Sangre Fetal/citología , Interleucina-8/metabolismo , Monocitos/citología , Neutrófilos/citología , Células Cultivadas , Femenino , Humanos , Recién Nacido , Leucocitos Mononucleares/citología , Lipopolisacáridos/metabolismo , Microscopía Confocal/métodos , Neutrófilos/metabolismo , Fagocitosis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting graft-vs.-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients and occurrence of graft-vs.-host disease (GVHD) prevents further dose escalation. Previous data indicate that invariant natural killer T (iNKT) cells promote anti-tumor immunity without exacerbating GVHD. In the present study we investigated lysis of leukemic blasts through iNKT cells from donor-derived lymphocytes for leukemia control and found that iNKT cells constitute about 0.12% of cryopreserved donor T cells. Therefore, we established a 2-week cell culture protocol allowing for a robust expansion of iNKT cells from cryopreserved DLIs (DLI-iNKTs) that can be used for further preclinical and clinical applications. Such DLI-iNKTs efficiently lysed leukemia cell lines and primary patient AML blasts ex vivo in a dose- and CD1d-dependent manner. Furthermore, expression of CD1d on target cells was required to release proinflammatory cytokines and proapoptotic effector molecules. Our results suggest that iNKT cells from donor-derived lymphocytes are involved in anti-tumor immunity after allo-HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival.