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1.
Front Cell Dev Biol ; 11: 1204160, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37497478

RESUMO

Chronic inflammation is a common feature of aging and numerous diseases such as diabetes, obesity, and autoimmune syndromes and has been linked to the development of hematological malignancy. Blood-forming hematopoietic stem cells (HSC) can contribute to these diseases via the production of tissue-damaging myeloid cells and/or the acquisition of mutations in epigenetic and transcriptional regulators that initiate evolution toward leukemogenesis. We previously showed that the myeloid "master regulator" transcription factor PU.1 is robustly induced in HSC by pro-inflammatory cytokines such as interleukin (IL)-1ß and limits their proliferative activity. Here, we used a PU.1-deficient mouse model to investigate the broader role of PU.1 in regulating hematopoietic activity in response to chronic inflammatory challenges. We found that PU.1 is critical in restraining inflammatory myelopoiesis via suppression of cell cycle and self-renewal gene programs in myeloid-biased multipotent progenitor (MPP) cells. Our data show that while PU.1 functions as a key driver of myeloid differentiation, it plays an equally critical role in tailoring hematopoietic responses to inflammatory stimuli while limiting expansion and self-renewal gene expression in MPPs. These data identify PU.1 as a key regulator of "emergency" myelopoiesis relevant to inflammatory disease and leukemogenesis.

2.
Cancer Res Commun ; 3(7): 1248-1259, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37465593

RESUMO

Siglec-15 (Sig15) has been implicated as an immune checkpoint expressed in solid tumor-infiltrating macrophages and is being targeted in clinical trials with mAbs to normalize the tumor immune microenvironment and stimulate antitumor immunity. However, the role of Sig15 in hematologic malignancies remains undefined. Sig15 mRNA and protein expression levels in hematologic malignancies were determined from publicly available databases, cell lines, and primary patient samples. Human B-cell acute lymphoblastic leukemia (B-ALL) cell lines were used to identify signaling pathways involved in the regulation of Sig15 expression. Secreted/soluble Sig15 and cytokine levels were measured from the plasma of children with leukemia and healthy controls. Knockdown and knockout of Siglec15 in a murine model of B-ALL was used to evaluate the effect of leukemia-derived Sig15 on the immune response to leukemia. We observed pathologic overexpression of Sig15 in a variety of hematologic malignancies, including primary B-ALL samples. This overexpression was driven by NFκB activation, which also increased the surface localization of Sig15. Secreted/soluble Sig15 was found to circulate at elevated levels in the plasma of children with B-ALL and correlated with an immune-suppressive cytokine milieu. Genetic inhibition of Sig15 in murine B-ALL promoted clearance of the leukemia by the immune system and a marked reversal of the immune-privileged leukemia bone marrow niche, including expanded early effector CD8+ T cells and reduction of immunosuppressive cytokines. Thus, Sig15 is a novel, potent immunosuppressive molecule active in leukemia that may be targeted therapeutically to activate T lymphocytes against leukemia cells. Significance: We demonstrate that Sig15 is overexpressed in hematologic malignancies driven by NFκB, is required for immune evasion in a mouse model of leukemia, and, for the first time, that it circulates at high levels in the plasma of children with leukemia.


Assuntos
Linfoma de Burkitt , Neoplasias Hematológicas , Leucemia , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Animais , Criança , Humanos , Camundongos , Imunidade Adaptativa , Linfócitos T CD8-Positivos , Citocinas , Imunoglobulinas , Proteínas de Membrana , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Microambiente Tumoral/genética
3.
Cells ; 11(4)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35203330

RESUMO

The transcription factor PU.1 is a critical regulator of lineage fate in blood-forming hematopoietic stem cells (HSC). In response to pro-inflammatory signals, such as the cytokine IL-1ß, PU.1 expression is increased in HSC and is associated with myeloid lineage expansion. To address potential functional heterogeneities arising in the phenotypic HSC compartment due to changes in PU.1 expression, here, we fractionated phenotypic HSC in mice using the SLAM surface marker code in conjunction with PU.1 expression levels, using the PU.1-EYFP reporter mouse strain. While PU.1lo SLAM cells contain extensive long-term repopulating activity and a molecular signature corresponding to HSC activity at steady state, following IL-1ß treatment, HSCLT induce PU.1 expression and are replaced in the PU.1lo SLAM fraction by CD41+ HSC-like megakaryocytic progenitors (SL-MkP) with limited long-term engraftment capacity. On the other hand, the PU.1hi SLAM fraction exhibits extensive myeloid lineage priming and clonogenic activity and expands rapidly in response to IL-1ß. Furthermore, we show that EPCR expression, but not CD150 expression, can distinguish HSCLT and SL-MkP under inflammatory conditions. Altogether, our data provide insights into the dynamic regulation of PU.1 and identify how PU.1 levels are linked to HSC fate in steady state and inflammatory stress conditions.


Assuntos
Células-Tronco Hematopoéticas , Animais , Células-Tronco Hematopoéticas/metabolismo , Camundongos
4.
J Exp Med ; 218(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33857288

RESUMO

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Inflamação/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Estresse Fisiológico/fisiologia , Transativadores/metabolismo , Animais , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Homeostase/fisiologia , Imunidade Inata/fisiologia , Camundongos , Camundongos Endogâmicos C57BL
5.
J Exp Med ; 218(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33914855

RESUMO

The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1ß (IL-1ß), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell-intrinsic competitive advantage; rather chronic IL-1ß exposure engendered potent selection for Cebpa loss. Chronic IL-1ß augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1ß, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Interleucina-1beta/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula/fisiologia , Expressão Gênica/fisiologia , Células HEK293 , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Inflamação/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo
6.
Haematologica ; 105(3): 585-597, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31101752

RESUMO

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.


Assuntos
Artrite Experimental , Artrite Reumatoide , Doenças Autoimunes , Animais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Citocinas , Modelos Animais de Doenças , Humanos , Camundongos
7.
Exp Hematol ; 81: 1-15.e6, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31863798

RESUMO

Hematopoiesis is dynamically regulated to maintain blood system function under nonhomeostatic conditions such as inflammation and injury. However, common surface marker and hematopoietic stem cell (HSC) reporter systems used for prospective enrichment of HSCs have been less rigorously tested in these contexts. Here, we use two surface markers, EPCR/CD201 and CD34, to re-analyze dynamic changes in the HSC-enriched phenotypic SLAM compartment in a mouse model of chronic interleukin (IL)-1 exposure. EPCR and CD34 coordinately identify four functionally and molecularly distinct compartments within the SLAM fraction, including an EPCR+/CD34- fraction whose long-term serial repopulating activity is only modestly impacted by chronic IL-1 exposure, relative to unfractionated SLAM cells. Notably, the other three fractions expand in frequency following IL-1 treatment and represent actively proliferating, lineage-primed cell states with limited long-term repopulating potential. Importantly, we find that the Fgd5-ZSGreen HSC reporter mouse enriches for molecularly and functionally intact HSCs regardless of IL-1 exposure. Together, our findings provide further evidence of dynamic heterogeneity within a commonly used HSC-enriched phenotypic compartment under stress conditions. Importantly, they also indicate that stringency of prospective isolation approaches can enhance interpretation of findings related to HSC function when studying models of hematopoietic stress.


Assuntos
Antígenos CD34/metabolismo , Proliferação de Células , Receptor de Proteína C Endotelial/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Estresse Fisiológico , Animais , Antígenos CD34/genética , Receptor de Proteína C Endotelial/genética , Células-Tronco Hematopoéticas/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1/efeitos adversos , Interleucina-1/farmacologia , Camundongos , Camundongos Transgênicos
8.
Cancer Res ; 79(14): 3702-3713, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31142509

RESUMO

Exploitation of the immune system has emerged as an important therapeutic strategy for acute lymphoblastic leukemia (ALL). However, the mechanisms of immune evasion during leukemia progression remain poorly understood. We sought to understand the role of calcineurin in ALL and observed that depletion of calcineurin B (CnB) in leukemia cells dramatically prolongs survival in immune-competent but not immune-deficient recipients. Immune-competent recipients were protected from challenge with leukemia if they were first immunized with CnB-deficient leukemia, suggesting robust adaptive immunity. In the bone marrow (BM), recipients of CnB-deficient leukemia harbored expanded T-cell populations as compared with controls. Gene expression analyses of leukemia cells extracted from the BM identified Cn-dependent significant changes in the expression of immunoregulatory genes. Increased secretion of IL12 from CnB-deficient leukemia cells was sufficient to induce T-cell activation ex vivo, an effect that was abolished when IL12 was neutralized. Strikingly, recombinant IL12 prolonged survival of mice challenged with highly aggressive B-ALL. Moreover, gene expression analyses from children with ALL showed that patients with higher expression of either IL12A or IL12B exhibited prolonged survival. These data suggest that leukemia cells are dependent upon calcineurin for immune evasion by restricting the regulation of proinflammatory genes, particularly IL12. SIGNIFICANCE: This report implicates calcineurin as an intracellular signaling molecule responsible for immune evasion during leukemia progression and raises the prospect of re-examining IL12 as a therapeutic in leukemia.


Assuntos
Calcineurina/imunologia , Interleucina-12/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Calcineurina/deficiência , Calcineurina/genética , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/imunologia , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Interleucina-12/biossíntese , Interleucina-12/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Evasão Tumoral
10.
Mol Cell Biol ; 38(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29915154

RESUMO

Zinc finger protein 521 (ZFP521), a DNA-binding protein containing 30 Krüppel-like zinc fingers, has been implicated in the differentiation of multiple cell types, including hematopoietic stem and progenitor cells (HSPC) and B lymphocytes. Here, we report a novel role for ZFP521 in regulating the earliest stages of hematopoiesis and lymphoid cell development via a cell-extrinsic mechanism. Mice with inactivated Zfp521 genes (Zfp521-/-) possess reduced frequencies and numbers of hematopoietic stem and progenitor cells, common lymphoid progenitors, and B and T cell precursors. Notably, ZFP521 deficiency changes bone marrow microenvironment cytokine levels and gene expression within resident HSPC, consistent with a skewing of hematopoiesis away from lymphopoiesis. These results advance our understanding of ZFP521's role in normal hematopoiesis, justifying further research to assess its potential as a target for cancer therapies.


Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Nicho de Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Citocinas/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Linfopoese/genética , Linfopoese/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mielopoese/genética , Mielopoese/fisiologia , Ligação Proteica , Nicho de Células-Tronco/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
11.
RNA Biol ; 12(11): 1169-77, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26399159

RESUMO

Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3'UTR that interact with Xrn1 to halt processive 5' to 3' decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3' UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3' UTR architecture, sfRNA production, and host adaptation.


Assuntos
Exorribonucleases/metabolismo , Flavivirus/genética , Interações Hospedeiro-Patógeno , RNA Viral/química , RNA Viral/genética , Regiões 3' não Traduzidas , Adaptação Biológica , Sequência de Bases , Evolução Molecular , Exorribonucleases/química , Exorribonucleases/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Modelos Biológicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Dobramento de RNA , Estabilidade de RNA , Alinhamento de Sequência
12.
Science ; 344(6181): 307-10, 2014 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-24744377

RESUMO

Flaviviruses are emerging human pathogens and worldwide health threats. During infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) are produced by resisting degradation by the 5'→3' host cell exonuclease Xrn1 through an unknown RNA structure-based mechanism. Here, we present the crystal structure of a complete Xrn1-resistant flaviviral RNA, which contains interwoven pseudoknots within a compact structure that depends on highly conserved nucleotides. The RNA's three-dimensional topology creates a ringlike conformation, with the 5' end of the resistant structure passing through the ring from one side of the fold to the other. Disruption of this structure prevents formation of sfRNA during flaviviral infection. Thus, sfRNA formation results from an RNA fold that interacts directly with Xrn1, presenting the enzyme with a structure that confounds its helicase activity.


Assuntos
Vírus da Encefalite do Vale de Murray/genética , Conformação de Ácido Nucleico , RNA Viral/química , Pareamento de Bases , Sequência de Bases , Cristalografia por Raios X , Vírus da Encefalite do Vale de Murray/patogenicidade , Exorribonucleases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , RNA Viral/genética , RNA Viral/metabolismo
13.
Nucleic Acids Res ; 41(1): 575-85, 2013 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-23161687

RESUMO

Long INterspersed Element one (LINE-1, or L1), is a widely distributed, autonomous retrotransposon in mammalian genomes. During retrotransposition, L1 RNA functions first as a dicistronic mRNA and then as a template for cDNA synthesis. Previously, we defined internal ribosome entry sequences (IRESs) upstream of both ORFs (ORF1 and ORF2) in the dicistronic mRNA encoded by mouse L1. Here, RNA affinity chromatography was used to isolate cellular proteins that bind these regions of L1 RNA. Four proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) R, Q and L, and nucleolin (NCL), appeared to interact specifically with the ORF2 IRES. These were depleted from HeLa cells to examine their effects on L1 IRES-mediated translation and L1 retrotransposition. NCL knockdown specifically reduced the ORF2 IRES activity, L1 and L1-assisted Alu retrotransposition without altering L1 RNA or protein abundance. These findings are consistent with NCL acting as an IRES trans-acting factor (ITAF) for ORF2 translation and hence a positive host factor for L1 retrotransposition. In contrast, hnRNPL knockdown dramatically increased L1 retrotransposition as well as L1 RNA and ORF1 protein, indicating that this cellular protein normally interferes with retrotransposition. Thus, hnRNPL joins a small, but growing list of cellular proteins that are potent negative regulators of L1 retrotransposition.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Endonucleases/genética , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Humanos , Camundongos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Biossíntese de Proteínas , RNA/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , DNA Polimerase Dirigida por RNA/genética , Ribonucleoproteínas/genética , Nucleolina
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