RESUMO
Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.
Assuntos
Compostos de Anilina , Antibacterianos , Sobrevivência Celular , Macrófagos , Fagocitose , Animais , Humanos , Camundongos , Compostos de Anilina/farmacologia , Antibacterianos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Nitrilas/farmacologia , Fagocitose/efeitos dos fármacos , Quinolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Kupffer cells are liver resident macrophages and play critical role in fatty liver disease, yet the underlying mechanisms remain unclear. Here, we show that activation of G-protein coupled receptor 3 (GPR3) in Kupffer cells stimulates glycolysis and protects mice from obesity and fatty liver disease. GPR3 activation induces a rapid increase in glycolysis via formation of complexes between ß-arrestin2 and key glycolytic enzymes as well as sustained increase in glycolysis through transcription of glycolytic genes. In mice, GPR3 activation in Kupffer cells results in enhanced glycolysis, reduced inflammation and inhibition of high-fat diet induced obesity and liver pathogenesis. In human fatty liver biopsies, GPR3 activation increases expression of glycolytic genes and reduces expression of inflammatory genes in a population of disease-associated macrophages. These findings identify GPR3 activation as a pivotal mechanism for metabolic reprogramming of Kupffer cells and as a potential approach for treating fatty liver disease.
Assuntos
Células de Kupffer , Hepatopatia Gordurosa não Alcoólica , Humanos , Animais , Camundongos , Células de Kupffer/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Glicólise , Obesidade/metabolismo , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Microglia shift toward an inflammatory phenotype during aging that is thought to exacerbate age-related neurodegeneration. The molecular and cellular signals that resolve neuroinflammation post-injury are largely undefined. Here, we exploit systems genetics methods based on the extended BXD murine reference family and identify IGFBPL1 as an upstream cis-regulator of microglia-specific genes to switch off inflammation. IGFBPL1 is expressed by mouse and human microglia, and higher levels of its expression resolve lipopolysaccharide-induced neuroinflammation by resetting the transcriptome signature back to a homeostatic state via IGF1R signaling. Conversely, IGFBPL1 deficiency or selective deletion of IGF1R in microglia shifts these cells to an inflammatory landscape and induces early manifestation of brain tauopathy and retinal neurodegeneration. Therapeutic administration of IGFBPL1 drives pro-homeostatic microglia and prevents glaucomatous neurodegeneration and vision loss in mice. These results identify IGFBPL1 as a master driver of the counter-inflammatory microglial modulator that presents an endogenous resolution of neuroinflammation to prevent neurodegeneration in eye and brain.
Assuntos
Microglia , Tauopatias , Camundongos , Animais , Humanos , Microglia/metabolismo , Doenças Neuroinflamatórias , Tauopatias/metabolismo , Inflamação/metabolismo , Encéfalo/metabolismo , Homeostase , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Antibiotic resistance critically limits treatment options for infection caused by opportunistic pathogens such as enterococci. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX) in vitro and in vivo against vancomycin-resistant Enterococcus faecalis (VRE). We show that, in vitro, MTX is a potent antibiotic against Gram-positive bacteria through induction of reactive oxygen species and DNA damage. MTX also synergizes with vancomycin against VRE, rendering the resistant strains more permeable to MTX. In a murine wound infection model, single-dose MTX treatment effectively reduces VRE numbers, with further reduction when combined with vancomycin. Multiple MTX treatments accelerate wound closure. MTX also promotes macrophage recruitment and proinflammatory cytokine induction at the wound site and augments intracellular bacterial killing in macrophages by up-regulating the expression of lysosomal enzymes. These results show that MTX represents a promising bacterium- and host-targeted therapeutic for overcoming vancomycin resistance.
Assuntos
Enterococcus faecalis , Enterococos Resistentes à Vancomicina , Animais , Camundongos , Enterococcus faecalis/genética , Resistência a Vancomicina/genética , Vancomicina/farmacologia , Mitoxantrona/farmacologia , Antibacterianos/farmacologia , Enterococos Resistentes à Vancomicina/genéticaRESUMO
Acute myeloid leukemia (AML) remains a therapeutic challenge, and a paucity of tumor-specific targets has significantly hampered the development of effective immune-based therapies. Recent paradigm-changing studies have shown that natural killer (NK) cells exhibit innate memory upon brief activation with IL-12 and IL-18, leading to cytokine-induced memory-like (CIML) NK cell differentiation. CIML NK cells have enhanced antitumor activity and have shown promising results in early phase clinical trials in patients with relapsed/refractory AML. Here, we show that arming CIML NK cells with a neoepitope-specific chimeric antigen receptor (CAR) significantly enhances their antitumor responses to nucleophosphmin-1 (NPM1)-mutated AML while avoiding off-target toxicity. CIML NK cells differentiated from peripheral blood NK cells were efficiently transduced to express a TCR-like CAR that specifically recognizes a neoepitope derived from the cytosolic oncogenic NPM1-mutated protein presented by HLA-A2. These CAR CIML NK cells displayed enhanced activity against NPM1-mutated AML cell lines and patient-derived leukemic blast cells. CAR CIML NK cells persisted in vivo and significantly improved AML outcomes in xenograft models. Single-cell RNA sequencing and mass cytometry analyses identified up-regulation of cell proliferation, protein folding, immune responses, and major metabolic pathways in CAR-transduced CIML NK cells, resulting in tumor-specific, CAR-dependent activation and function in response to AML target cells. Thus, efficient arming of CIML NK cells with an NPM1-mutation-specific TCR-like CAR substantially improves their innate antitumor responses against an otherwise intracellular mutant protein. These preclinical findings justify evaluating this approach in clinical trials in HLA-A2+ AML patients with NPM1c mutations.
Assuntos
Memória Imunológica , Células de Memória Imunológica , Imunoterapia Adotiva , Células Matadoras Naturais , Leucemia Mieloide Aguda , Nucleofosmina , Receptores de Antígenos Quiméricos , Antígeno HLA-A2/imunologia , Humanos , Células de Memória Imunológica/imunologia , Células de Memória Imunológica/transplante , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutação , Nucleofosmina/genética , Nucleofosmina/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologiaRESUMO
Malignant ascites is a common clinical problem in ovarian cancer. NK cells are present in the ascites, but their antitumor activity is inhibited. The underlying mechanisms of the inhibition have yet to be fully elucidated. Using an Fcγ receptor-mediated NK cell activation assay, we show that ascites from ovarian cancer patients potently inhibits NK cell activation. Part of the inhibitory activity is mediated by CA125, a mucin 16 fragment shed from ovarian cancer tumors. Moreover, transcriptional analyses by RNA sequencing reveal upregulation of genes involved in multiple metabolic pathways but downregulation of genes involved in cytotoxicity and signaling pathways in NK cells purified from ovarian cancer patient ascites. Transcription of genes involved in cytotoxicity pathways are also downregulated in NK cells from healthy donors after in vitro treatment with ascites or with a CA125-enriched protein fraction. These results show that ascites and CA125 inhibit antitumor activity of NK cells at transcriptional levels by suppressing expression of genes involved in NK cell activation and cytotoxicity. Our findings shed light on the molecular mechanisms by which ascites inhibits the activity of NK cells and suggest possible approaches to reactivate NK cells for ovarian cancer immunotherapy.
Assuntos
Ascite , Antígeno Ca-125 , Células Matadoras Naturais , Neoplasias Ovarianas , Ascite/metabolismo , Antígeno Ca-125/genética , Antígeno Ca-125/metabolismo , Feminino , Humanos , Células Matadoras Naturais/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Ativação TranscricionalRESUMO
BackgroundResponses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse.MethodsWe initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion.ResultsIn the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires.ConclusionGiven their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.Trial RegistrationClinicalTrials.gov NCT04024761.FundingDunkin' Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Interleucina-2 , Humanos , Células Matadoras Naturais , Recidiva , Transplante HomólogoRESUMO
Macrophages are plastic and, in response to different local stimuli, can polarize toward multi-dimensional spectrum of phenotypes, including the pro-inflammatory M1-like and the anti-inflammatory M2-like states. Using a high-throughput phenotypic screen in a library of ~4000 FDA-approved drugs, bioactive compounds and natural products, we find ~300 compounds that potently activate primary human macrophages toward either M1-like or M2-like state, of which ~30 are capable of reprogramming M1-like macrophages toward M2-like state and another ~20 for the reverse repolarization. Transcriptional analyses of macrophages treated with 34 non-redundant compounds identify both shared and unique targets and pathways through which the tested compounds modulate macrophage activation. One M1-activating compound, thiostrepton, is able to reprogram tumor-associated macrophages toward M1-like state in mice, and exhibit potent anti-tumor activity. Our compound-screening results thus help to provide a valuable resource not only for studying the macrophage biology but also for developing therapeutics through modulating macrophage activation.
Assuntos
Anti-Inflamatórios/farmacologia , Produtos Biológicos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Produtos Biológicos/química , Linhagem Celular Tumoral , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Humanos , Macrófagos/classificação , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Fenótipo , Células THP-1 , Tioestreptona/química , Tioestreptona/farmacologiaRESUMO
ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.
Assuntos
Trifosfato de Adenosina/metabolismo , Heme/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Receptores Virais/metabolismo , Termogênese/fisiologia , Animais , Deficiência de Citocromo-c Oxidase , Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Metabolismo Energético/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Domínios Proteicos , Receptores Virais/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Células THP-1RESUMO
Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.
Assuntos
Antígenos CD/genética , Epitopos/imunologia , Proteínas de Checkpoint Imunológico/genética , Tolerância Imunológica , Linfoma/genética , Monócitos/imunologia , Receptores Imunológicos/genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/metabolismo , Antígenos CD/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Mapeamento de Epitopos , Epitopos/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Xenoenxertos , Humanos , Proteínas de Checkpoint Imunológico/imunologia , Linfoma/imunologia , Linfoma/mortalidade , Linfoma/patologia , Camundongos , Monócitos/citologia , Biblioteca de Peptídeos , Cultura Primária de Células , Receptores Imunológicos/agonistas , Receptores Imunológicos/imunologia , Análise de Sobrevida , Linfócitos T/citologia , Linfócitos T/imunologia , Transplante HomólogoRESUMO
Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated cancer. Many hematologic and solid tumors are resistant to therapeutic antibodies in the bone marrow (BM), but not in the periphery (e.g., spleen). We previously showed that cyclophosphamide (CTX) sensitizes the BM niche to antibody therapeutics. Here, we show that (i) BM resistance was induced not only by the tumor but also by the intrinsic BM microenvironment; (ii) CTX treatment overcame both intrinsic and extrinsic resistance mechanisms by augmenting macrophage activation and phagocytosis, including significant upregulation of activating Fcγ receptors (FcγRIII and FcγRIV) and downregulation of the inhibitory receptor, FcγRIIB; and (iii) CTX synergized with cetuximab (anti-EGFR) and trastuzumab (anti-Her2) in eliminating metastatic breast cancer in the BM of humanized mice. These findings provide insights into the mechanisms by which CTX synergizes with antibody therapeutics in resistant niche-specific organs and its applicability in treating BM-resident tumors.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Medula Óssea/imunologia , Ciclofosfamida/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Receptores de IgG/genética , Animais , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Ciclofosfamida/imunologia , Ciclofosfamida/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoterapia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Receptores de IgG/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by emphysema and/or obstructive bronchiolitis. Deficiency in vitamin D3 (VD3), which regulates gene expression through binding to vitamin D receptor (VDR), is associated with high risks of COPD susceptibility. Alveolar macrophages (AM), which are generated during early ontogeny and maintained in alveoli by self-renewal in response to cytokine GM-CSF, are positively correlated with severity of emphysema. However, whether and how VD3, VDR and AM interact to contribute to COPD pathogenesis at the molecular and cellular levels are largely unknown. METHODS: We used systems biology approaches to analyze gene expression in mouse macrophages from different tissues to identify key transcription factors (TF) for AM and infer COPD disease genes. We used RNA-seq and ChIP-seq to identify genes that are regulated by VD3 in AM. We used VDR-deficient (Vdr-/-) mice to investigate the role of VD3-VDR axis in the pathogenesis of COPD and characterized the transcriptional and functional alterations of Vdr-/- AM. FINDINGS: We find that VDR is a key TF for AM and a COPD disease gene. VDR is highly expressed in AM and in response to VD3 inhibits GM-CSF-induced AM proliferation. In Vdr-/- AM, genes involved in proliferation and immune response are upregulated. Consistently, Vdr-/- mice progressively accumulate AM and concomitantly develop emphysema without apparent infiltration of immune cells into the lung tissue. Intratracheal transfer of Vdr-/- AM into wildtype mice readily induces emphysema. The production of reactive oxygen species at basal level and in response to heme or lipopolysaccharide is elevated in Vdr-/- AM and suppressed by VD3 in wildtype AM. INTERPRETATION: These results show that the VD3-VDR axis is critical to counteract GM-CSF-induced AM proliferation and defect in this regulation leads to altered AM homeostasis and function. Our findings identify that VD3 deficiency contributes to emphysema by altering AM function without contributing to bronchiolitis. Our findings also suggest possibilities of modulating the VD3-VDR axis for inhibiting emphysema in COPD patients.
Assuntos
Colecalciferol/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Doença Pulmonar Obstrutiva Crônica/genética , Enfisema Pulmonar/genética , Receptores de Calcitriol/genética , Animais , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Homeostase/genética , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Camundongos , Ligação Proteica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Transdução de SinaisRESUMO
Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer, early events of tumorigenesis have already taken place. Similarly, development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article, we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model, human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1), referred to as NPM1c. Following engraftment into immunodeficient mice, transduced HSPCs give rise to human myeloid leukemia, whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML, with CD123+ leukemic stem or initiating cells (LSC), resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics.
Assuntos
Carcinogênese , Leucemia Mieloide , Proteínas Nucleares , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NucleofosminaRESUMO
Pleural macrophages play critical roles in pathogenesis of tuberculous pleuritis, but very little is known about their response to anti-tuberculosis antibiotics treatment. Here, we examined whether and how pleural macrophages change in phenotype, transcription and function following antibiotics treatment in patients with tuberculous pleuritis. Results show pro-inflammatory cytokines were down-regulated significantly post antibiotic treatment in the pleural effusions and pleural macrophages up-regulated markers characteristic of M2 macrophages such as CD163 and CD206. Differential expression analysis of transcriptomes from four paired samples before and after treatment identified 230 treatment-specific responsive genes in pleural macrophages. Functional analysis identified interferon-related pathway to be the most responsive genes and further confirmed macrophage polarization to M2-like phenotype. We further demonstrate that expression of a significant fraction of responsive genes was modulated directly by antibiotics in pleural macrophages in vitro. Our results conclude that pleural macrophages polarize from M1-like to M2-like phenotype within a mean of 3.5 days post antibiotics treatment, which is dependent on both pleural cytokine environment and direct modulatory effects of antibiotics. The treatment-specific genes could be used to study the roles of pleural macrophages in the pathogenesis of tuberculous pleuritis and to monitor the response to antibiotics treatment.
Assuntos
Antibacterianos/farmacologia , Polaridade Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Pleura/efeitos dos fármacos , Tuberculose Pleural/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Biomarcadores/metabolismo , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Pleura/patologia , Tuberculose Pleural/tratamento farmacológico , Adulto JovemRESUMO
Club cells are known to function as regional progenitor cells to repair the bronchiolar epithelium in response to lung damage. By lineage tracing in mice, we have shown recently that club cells also give rise to alveolar type 2 cells (AT2s) and alveolar type 1 cells (AT1s) during the repair of the damaged alveolar epithelium. Here, we show that when highly purified, anatomically and phenotypically confirmed club cells are seeded in 3-dimensional culture either in bulk or individually, they proliferate and differentiate into both AT2- and AT1-like cells and form alveolar-like structures. This differentiation was further confirmed by transcriptomic analysis of freshly isolated club cells and their cultured progeny. Freshly isolated club cells express Sca-1 and integrin α6, markers commonly used to characterize lung stem/progenitor cells. Together, current study for the first time isolated highly purified club cells for in vitro study and demonstrated club cells' capacity to differentiate into alveolar epithelial cells at the single-cell level.
Assuntos
Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Diferenciação Celular , Mucosa Respiratória/citologia , Células-Tronco/citologia , Animais , Ataxina-1/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/genética , Células Cultivadas , Imunofluorescência , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Imunofenotipagem , Integrina alfa6/metabolismo , Camundongos , Fenótipo , TranscriptomaRESUMO
The genome sequence available for different Plasmodium species is a valuable resource for understanding malaria parasite biology. However, comparative genomics on its own cannot fully explain all the species-specific differences which suggests that other genomic aspects such as regulation of gene expression play an important role in defining species-specific characteristics. Here, we developed a comprehensive approach to measure transcriptional changes of the evolutionary conserved syntenic orthologs during the intraerythrocytic developmental cycle across six Plasmodium species. We show significant transcriptional constraint at the mid-developmental stage of Plasmodium species while the earliest stages of parasite development display the greatest transcriptional variation associated with critical functional processes. Modeling of the evolutionary relationship based on changes in transcriptional profile reveal a phylogeny pattern of the Plasmodium species that strictly follows its mammalian hosts. In addition, the work shows that transcriptional conserved orthologs represent potential future targets for anti-malaria intervention as they would be expected to carry out key essential functions within the parasites. This work provides an integrated analysis of orthologous transcriptome, which aims to provide insights into the Plasmodium evolution thereby establishing a framework to explore complex pathways and drug discovery in Plasmodium species with broad host range.
Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Malária/veterinária , Plasmodium/genética , Animais , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Especificidade de Hospedeiro , Malária/parasitologia , Camundongos , Filogenia , Plasmodium/classificação , Plasmodium/fisiologia , Especificidade da Espécie , SinteniaRESUMO
Memory CD8⺠T-cell development is defined by the expression of a specific set of memory signature genes. Despite recent progress, many components of the transcriptional control of memory CD8⺠T-cell development are still unknown. To identify transcription factors and their interactions in memory CD8⺠T-cell development, we construct a genome-wide regulatory network and apply it to identify key transcription factors that regulate memory signature genes. Most of the known transcription factors having a role in memory CD8⺠T-cell development are rediscovered and about a dozen new ones are also identified. Sox4, Bhlhe40, Bach2 and Runx2 are experimentally verified, and Bach2 is further shown to promote both development and recall proliferation of memory CD8⺠T cells through Prdm1 and Id3. Gene perturbation study identifies the interactions between the transcription factors, with Sox4 positioned as a hub. The identified transcription factors and insights into their interactions should facilitate further dissection of molecular mechanisms underlying memory CD8⺠T-cell development.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Redes Reguladoras de Genes/imunologia , Estudo de Associação Genômica Ampla , Memória Imunológica/genética , Fatores de Transcrição/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Cães , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição SOXC/genéticaRESUMO
BACKGROUND: In recent years a number of genome sequences for different plasmodium species have become available. This has allowed the identification of numerous conserved genes across the different species and has significantly enhanced our understanding of parasite biology. In contrast little is known about species specific differences between the different genomes partly due to the lower sequence coverage and therefore relatively poor annotation of some of the draft genomes particularly the rodent malarias parasite species. RESULTS: To improve the current annotation and gene identification status of the draft genomes of P. berghei, P. chabaudi and P. yoelii, we performed genome-wide comparisons between these three species. Through analyses via comparative genome hybridizations using a newly designed pan-rodent array as well as in depth bioinformatics analysis, we were able to improve on the coverage of the draft rodent parasite genomes by detecting orthologous genes between these related rodent parasite species. More than 1,000 orthologs for P. yoelii were now newly associated with a P. falciparum gene. In addition to extending the current core gene set for all plasmodium species this analysis also for the first time identifies a relatively small number of genes that are unique to the primate malaria parasites while a larger gene set is uniquely conserved amongst the rodent malaria parasites. CONCLUSIONS: These findings allow a more thorough investigation of the genes that are important for host specificity in malaria.
Assuntos
Hibridização Genômica Comparativa/métodos , Genoma de Protozoário , Plasmodium/genética , Animais , Biologia Computacional , DNA de Protozoário/genética , Genes de Protozoários , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Functions have yet to be defined for the majority of genes of Plasmodium falciparum, the agent responsible for the most serious form of human malaria. Here we report changes in P. falciparum gene expression induced by 20 compounds that inhibit growth of the schizont stage of the intraerythrocytic development cycle. In contrast with previous studies, which reported only minimal changes in response to chemically induced perturbations of P. falciparum growth, we find that approximately 59% of its coding genes display over three-fold changes in expression in response to at least one of the chemicals we tested. We use this compendium for guilt-by-association prediction of protein function using an interaction network constructed from gene co-expression, sequence homology, domain-domain and yeast two-hybrid data. The subcellular localizations of 31 of 42 proteins linked with merozoite invasion is consistent with their role in this process, a key target for malaria control. Our network may facilitate identification of novel antimalarial drugs and vaccines.