RESUMO
Tumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults (n = 4) and patients with precursor (n = 8) and full-blown MM (n = 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27- and CD27+ T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations.
Assuntos
Mieloma Múltiplo , Adulto , Humanos , Animais , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Linfócitos T , Receptor de Morte Celular Programada 1/genética , Lenalidomida , Células ClonaisRESUMO
Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n = 12 of 21), branching (n = 4 of 21), and clonal evolution (n = 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in â¼80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.
Assuntos
Leucemia Mieloide Aguda , Humanos , Idoso , Citometria de Fluxo/métodos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/complicações , Prognóstico , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients.
Assuntos
Síndromes Mielodisplásicas , Fatores de Transcrição , Adulto , Humanos , Idoso , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Síndromes Mielodisplásicas/patologia , Eritropoese/genética , Células-Tronco Hematopoéticas/metabolismo , Regulação da Expressão Gênica , Fator de Transcrição CHOP/genéticaRESUMO
Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88L265P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88L265P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.
Assuntos
Linfoma de Células B , Linfoma , Macroglobulinemia de Waldenstrom , Idoso , Animais , Humanos , Linfoma de Células B/metabolismo , Camundongos , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Macroglobulinemia de Waldenstrom/diagnóstico , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/patologiaRESUMO
Large-scale immune monitoring is becoming routinely used in clinical trials to identify determinants of treatment responsiveness, particularly to immunotherapies. Flow cytometry remains one of the most versatile and high throughput approaches for single-cell analysis; however, manual interpretation of multidimensional data poses a challenge when attempting to capture full cellular diversity and provide reproducible results. We present FlowCT, a semi-automated workspace empowered to analyze large data sets. It includes pre-processing, normalization, multiple dimensionality reduction techniques, automated clustering, and predictive modeling tools. As a proof of concept, we used FlowCT to compare the T-cell compartment in bone marrow (BM) with peripheral blood (PB) from patients with smoldering multiple myeloma (SMM), identify minimally invasive immune biomarkers of progression from smoldering to active MM, define prognostic T-cell subsets in the BM of patients with active MM after treatment intensification, and assess the longitudinal effect of maintenance therapy in BM T cells. A total of 354 samples were analyzed and immune signatures predictive of malignant transformation were identified in 150 patients with SMM (hazard ratio [HR], 1.7; P < .001). We also determined progression-free survival (HR, 4.09; P < .0001) and overall survival (HR, 3.12; P = .047) in 100 patients with active MM. New data also emerged about stem cell memory T cells, the concordance between immune profiles in BM and PB, and the immunomodulatory effect of maintenance therapy. FlowCT is a new open-source computational approach that can be readily implemented by research laboratories to perform quality control, analyze high-dimensional data, unveil cellular diversity, and objectively identify biomarkers in large immune monitoring studies. These trials were registered at www.clinicaltrials.gov as #NCT01916252 and #NCT02406144.
Assuntos
Mieloma Múltiplo Latente , Biomarcadores , Medula Óssea , Citometria de Fluxo/métodos , Humanos , ImunofenotipagemRESUMO
Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation-related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Transcriptoma , Adulto , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Mieloma Múltiplo/genética , Plasmócitos/metabolismo , Células Tumorais CultivadasRESUMO
Information on the immunopathobiology of coronavirus disease 2019 (COVID-19) is rapidly increasing; however, there remains a need to identify immune features predictive of fatal outcome. This large-scale study characterized immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection using multidimensional flow cytometry, with the aim of identifying high-risk immune biomarkers. Holistic and unbiased analyses of 17 immune cell-types were conducted on 1,075 peripheral blood samples obtained from 868 COVID-19 patients and on samples from 24 patients presenting with non-SARS-CoV-2 infections and 36 healthy donors. Immune profiles of COVID-19 patients were significantly different from those of age-matched healthy donors but generally similar to those of patients with non-SARS-CoV-2 infections. Unsupervised clustering analysis revealed three immunotypes during SARS-CoV-2 infection; immunotype 1 (14% of patients) was characterized by significantly lower percentages of all immune cell-types except neutrophils and circulating plasma cells, and was significantly associated with severe disease. Reduced B-cell percentage was most strongly associated with risk of death. On multivariate analysis incorporating age and comorbidities, B-cell and non-classical monocyte percentages were independent prognostic factors for survival in training (n=513) and validation (n=355) cohorts. Therefore, reduced percentages of B-cells and non-classical monocytes are high-risk immune biomarkers for risk-stratification of COVID-19 patients.
Assuntos
COVID-19/imunologia , COVID-19/mortalidade , Imunidade Adaptativa , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Biomarcadores , COVID-19/patologia , Feminino , Humanos , Imunidade Inata , Linfopenia/imunologia , Linfopenia/mortalidade , Linfopenia/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Prognóstico , SARS-CoV-2 , Análise de Sobrevida , Adulto JovemRESUMO
Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ósseas/secundário , MicroRNAs/genética , Mieloma Múltiplo/patologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Apoptose , Neoplasias Ósseas/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Prognóstico , Células Tumorais Cultivadas , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of â¼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species-mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Neoplasia Residual/patologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos de Boro/uso terapêutico , Bortezomib/uso terapêutico , Aberrações Cromossômicas , Dexametasona/uso terapêutico , Feminino , Citometria de Fluxo , Glicina/análogos & derivados , Glicina/uso terapêutico , Humanos , Lenalidomida/uso terapêutico , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Resultado do TratamentoRESUMO
Not available.
Assuntos
COVID-19/imunologia , Neoplasias Hematológicas/imunologia , SARS-CoV-2/imunologia , COVID-19/epidemiologia , Neoplasias Hematológicas/epidemiologia , HumanosRESUMO
Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14-CD15+CD33+HLADR- cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P < .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC-related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.
Assuntos
Antígenos CD , Mieloma Múltiplo , Células Supressoras Mieloides , Proteínas de Neoplasias , Antígenos CD/sangue , Antígenos CD/genética , Antígenos CD/imunologia , Feminino , Seguimentos , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Transcrição Gênica/imunologiaRESUMO
Risk of developing myelodysplastic syndrome (MDS) is significantly increased in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance, suggesting that it is therapy independent. However, the incidence and sequelae of dysplastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for the presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 patients with MM enrolled in the PETHEMA/GEM2012MENOS65 trial (#NCT01916252). We investigated the clinical significance of monocytic MDS-PA in a larger series of 1252 patients enrolled in 4 PETHEMA/GEM protocols. At diagnosis, 33 (11.6%) of 285 cases displayed MDS-PA. Bulk and single-cell-targeted sequencing of MDS recurrently mutated genes in CD34+ progenitors (and dysplastic lineages) from 67 patients revealed clonal hematopoiesis in 13 (50%) of 26 cases with MDS-PA vs 9 (22%) of 41 without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86 of 285 patients and showed that in most cases (69 of 86 [80%]), MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations infrequently emerged after high-dose therapy. Based on MFC profiling, patients with MDS-PA have altered hematopoiesis and T regulatory cell distribution in the tumor microenvironment. Importantly, the presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematologic toxicity and was independently associated with inferior progression-free survival (hazard ratio, 1.5; P = .02) and overall survival (hazard ratio, 1.7; P = .01). This study reveals the biological and clinical significance of dysplastic hematopoiesis in newly diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC.
Assuntos
Hematopoiese Clonal , Mieloma Múltiplo/patologia , Síndromes Mielodisplásicas/etiologia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos Fase III como Assunto , Terapia Combinada , Feminino , Citometria de Fluxo/métodos , Transplante de Células-Tronco Hematopoéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/terapia , Mutação , Prognóstico , Intervalo Livre de Progressão , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Transplante Autólogo , Microambiente TumoralRESUMO
UNLABELLED: Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl-/HCO3- anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2a,b(-/-) mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2a,b(-/-) mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥ 10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8(+) T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2'-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8(+) T cells from aged knockouts. CONCLUSION: Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8+ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.
Assuntos
Doenças Autoimunes/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Colangite/metabolismo , Fígado/metabolismo , Ativação Linfocitária , Receptor de Morte Celular Programada 1/metabolismo , Fatores Etários , Animais , Apoptose , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/deficiência , Antiportadores de Cloreto-Bicarbonato/genética , Colangite/genética , Colangite/imunologia , Colangite/patologia , Deleção Clonal , Metilação de DNA , Modelos Animais de Doenças , Epigênese Genética , Feminino , Predisposição Genética para Doença , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fenótipo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , TransfecçãoRESUMO
BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive biliary cystogenesis. Current therapies show short-term and/or modest beneficial effects. Cystic cholangiocytes hyperproliferate as a consequence of diminished intracellular calcium levels ([Ca(2+)]i). Here, the therapeutic value of ursodeoxycholic acid (UDCA) was investigated. METHODS: Effect of UDCA was examined in vitro and in polycystic (PCK) rats. Hepatic cystogenesis and fibrosis, and the bile acid (BA) content were evaluated from the liver, bile, serum, and kidneys by HPLC-MS/MS. RESULTS: Chronic treatment of PCK rats with UDCA inhibits hepatic cystogenesis and fibrosis, and improves their motor behaviour. As compared to wild-type animals, PCK rats show increased BA concentration ([BA]) in liver, similar hepatic Cyp7a1 mRNA levels, and diminished [BA] in bile. Likewise, [BA] is increased in cystic fluid of PLD patients compared to their matched serum levels. In PCK rats, UDCA decreases the intrahepatic accumulation of cytotoxic BA, normalizes their diminished [BA] in bile, increases the BA secretion in bile and diminishes the increased [BA] in kidneys. In vitro, UDCA inhibits the hyperproliferation of polycystic human cholangiocytes via a PI3K/AKT/MEK/ERK1/2-dependent mechanism without affecting apoptosis. Finally, the presence of glycodeoxycholic acid promotes the proliferation of polycystic human cholangiocytes, which is inhibited by both UDCA and tauro-UDCA. CONCLUSIONS: UDCA was able to halt the liver disease of a rat model of PLD through inhibiting cystic cholangiocyte hyperproliferation and decreasing the levels of cytotoxic BA species in the liver, which suggests the use of UDCA as a potential therapeutic tool for PLD patients.
Assuntos
Apoptose , Cistos/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Fígado/patologia , Ácido Ursodesoxicólico/farmacologia , Animais , Ácidos e Sais Biliares/metabolismo , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colagogos e Coleréticos/farmacologia , Cistos/metabolismo , Cistos/patologia , Modelos Animais de Doenças , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias/metabolismo , Hepatopatias/patologia , Ratos , Espectrometria de Massas em TandemRESUMO
Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi ). Subsequent pHi regulation may involve HCO3 (-) extrusion through Cl(-) /HCO3 (-) exchangers and/or Na(+) -HCO3 (-) co-transporters with acid-loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8(+) T-cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na(+) -independent Cl(-) /HCO3 (-) anion-exchange activity). Here we report that CD8(+) T cells but not CD4(+) T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4(+) T cells) exhibit Ae1 expression and Na(+) -HCO3 (-) co-transport with acidifying potential, CD8(+) T cells lack these acid-loading mechanisms. In Ae2-KO mice, CD4(+) but not CD8(+) T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2-KO CD8(+) T cells exhibit alkalinized pHi , and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2-deficient CD8(+) T cells show enhanced intracellular production of IL-2 and membrane expression of its receptor IL-2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8(+) T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8(+) T-cell responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Homeostase/fisiologia , Ativação Linfocitária/fisiologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/imunologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Concentração de Íons de Hidrogênio , Interleucina-2/biossíntese , Interleucina-2/genética , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Transporte de Íons/fisiologia , Camundongos , Camundongos KnockoutRESUMO
UNLABELLED: Cl(-) /HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3'untranslated region (3'UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. CONCLUSION: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3'UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Antiporters/genética , Antiporters/metabolismo , Ductos Biliares Intra-Hepáticos/fisiopatologia , Cirrose Hepática Biliar , MicroRNAs/metabolismo , Bicarbonatos/metabolismo , Ductos Biliares Intra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/metabolismo , Linhagem Celular Tumoral , Antiportadores de Cloreto-Bicarbonato , Cloretos/metabolismo , Simulação por Computador , Epitélio/fisiologia , Humanos , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/metabolismo , Cirrose Hepática Biliar/fisiopatologia , MicroRNAs/genética , Cultura Primária de Células , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Proteínas SLC4A , Regulação para Cima/genéticaRESUMO
BACKGROUND & AIMS: Cl(-)/HCO(3)(-) anion exchanger 2 (AE2) is involved in intracellular pH (pH(i)) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2(a.b)-deficient mice. METHODS: In this murine model, splenocyte pH(i) and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. RESULTS: Ae2(a,b)(-/-) mice exhibit splenomegaly, elevated pH(i) in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8(+) T-cell population, and under represented CD4(+)FoxP3(+)/regulatory T cells. Most Ae2(a,b)(-/-) mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2(a,b)(-/-) mice had extensive portal inflammation with CD8(+) and CD4(+) T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2(a,b)(-/-) mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. CONCLUSIONS: Ae2 deficiency alters pH(i) homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC.