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1.
Eur J Immunol ; 51(9): 2237-2250, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34107067

RESUMO

Early embryonic hematopoiesis in mammals is defined by three successive waves of hematopoietic progenitors which exhibit a distinct hematopoietic potential and provide continuous support for the development of the embryo and adult organism. Although the functional importance of each of these waves has been analyzed, their spatio-temporal overlap and the lack of wave-specific markers hinders the accurate separation and assessment of their functional roles during early embryogenesis. We have recently shown that TLR2, in combination with c-kit, represents the earliest signature of emerging precursors of the second hematopoietic wave, erythro-myeloid precursors (EMPs). Since the onset of Tlr2 expression distinguishes EMPs from primitive progenitors which coexist in the yolk sac from E7.5, we generated a novel transgenic "knock in" mouse model, Tlr2Dtr , suitable for inducible targeted depletion of TLR2+ EMPs. In this model, the red fluorescent protein and diphtheria toxin receptor sequences are linked via a P2A sequence and inserted into the Tlr2 locus before its stop codon. We show that a timely controlled deletion of TLR2+ EMPs in Tlr2Dtr embryos results in a marked decrease in both erythroid as well as myeloid lineages and, consequently, in embryonic lethality peaking before E13.5. These findings validate the importance of EMPs in embryonic development.


Assuntos
Embrião de Mamíferos/patologia , Desenvolvimento Embrionário/genética , Hematopoese/genética , Células Progenitoras Mieloides/citologia , Receptor 2 Toll-Like/genética , Animais , Embrião de Mamíferos/embriologia , Eritrócitos/citologia , Hematopoese/fisiologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
2.
Sci Rep ; 11(1): 10736, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031489

RESUMO

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell-cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.


Assuntos
Reabsorção Óssea/genética , Antígenos CD13/genética , Antígenos CD13/metabolismo , Osteoclastos/patologia , Animais , Densidade Óssea , Reabsorção Óssea/patologia , Diferenciação Celular , Fusão Celular , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Osteoclastos/metabolismo , Células U937
3.
Front Immunol ; 12: 621665, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815375

RESUMO

Toll-like receptor 5 (TLR5) is the receptor of bacterial Flagellin. Reportedly, TLR5 engagement helps to combat infections, especially at mucosal sites, by evoking responses from epithelial cells and immune cells. Here we report that TLR5 is expressed on a previously defined bipotent progenitor of macrophages (MΦs) and osteoclasts (OCs) that resides in the mouse bone marrow (BM) and circulates at low frequency in the blood. In vitro, Flagellin promoted the generation of MΦs, but not OCs from this progenitor. In vivo, MΦ/OC progenitors were recruited from the blood into the lung upon intranasal inoculation of Flagellin, where they rapidly differentiated into MΦs. Recruitment of the MΦ/OC progenitors into the lung was likely promoted by the CCL2/CCR2 axis, since the progenitors expressed CCR2 and type 2 alveolar epithelial cells (AECs) produced CCL2 upon stimulation by Flagellin. Moreover, CCR2 blockade reduced migration of the MΦ/OC progenitors toward lung lavage fluid (LLF) from Flagellin-inoculated mice. Our study points to a novel role of the Flagellin/TLR5 axis in recruiting circulating MΦ/OC progenitors into infected tissue and stimulating these progenitors to locally differentiate into MΦs. The progenitor pathway to produce MΦs may act, next to monocyte recruitment, to fortify host protection against bacterial infection at mucosal sites.


Assuntos
Flagelina/metabolismo , Pulmão/imunologia , Macrófagos/fisiologia , Células Progenitoras Mieloides/fisiologia , Osteoclastos/fisiologia , Receptor 5 Toll-Like/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR2/metabolismo , Transdução de Sinais
4.
Elife ; 102021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33830019

RESUMO

Innate immune cellular effectors are actively consumed during systemic inflammation, but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here, we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to regional lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NF-κB independent, Traf6/IκB-kinase/SNAP23 activation dependent, and is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205+/CD172a+ conventional dendritic cells type 2 and upregulation of BM myeloid progenitor Ccr7 signaling. Mature myeloid Traf6 signaling is anti-inflammatory and necessary for lymph node myeloid cell development. This report unveils the existence and the mechanistic basis of a very early direct traffic of myeloid progenitors from BM to lymphatics during inflammation.


Assuntos
Medula Óssea/metabolismo , Movimento Celular , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Linfadenopatia/metabolismo , Sistema Linfático/metabolismo , Células Progenitoras Mieloides/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Medula Óssea/imunologia , Medula Óssea/patologia , Linhagem da Célula , Células Cultivadas , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Células Progenitoras de Granulócitos e Macrófagos/imunologia , Células Progenitoras de Granulócitos e Macrófagos/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Linfadenopatia/imunologia , Linfadenopatia/patologia , Sistema Linfático/imunologia , Sistema Linfático/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/patologia , Fenótipo , Transdução de Sinais , Fatores de Tempo , Adulto Jovem
5.
Immunol Cell Biol ; 99(7): 749-766, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33866598

RESUMO

Brown adipose tissue (BAT) may be an important metabolic regulator of whole-body glucose. While important roles have been ascribed to macrophages in regulating metabolic functions in BAT, little is known of the roles of other immune cells subsets, particularly dendritic cells (DCs). Eating a high-fat diet may compromise the development of hematopoietic stem and progenitor cells (HSPCs)-which give rise to DCs-in bone marrow, with less known of its effects in BAT. We have previously demonstrated that ongoing exposure to low-dose ultraviolet radiation (UVR) significantly reduced the 'whitening' effect of eating a high-fat diet upon interscapular (i) BAT of mice. Here, we examined whether this observation may be linked to changes in the phenotype of HSPCs and myeloid-derived immune cells in iBAT and bone marrow of mice using 12-colour flow cytometry. Many HSPC subsets declined in both iBAT and bone marrow with increasing metabolic dysfunction. Conversely, with rising adiposity and metabolic dysfunction, conventional DCs (cDCs) increased in both of these tissues. When compared with a low-fat diet, consumption of a high-fat diet significantly reduced proportions of myeloid, common myeloid and megakaryocyte-erythrocyte progenitors in iBAT, and short-term hematopoietic stem cells in bone marrow. In mice fed the high-fat diet, exposure to low-dose UVR significantly reduced proportions of cDCs in iBAT, independently of nitric oxide release from irradiated skin [blocked using the scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)], but did not significantly modify HSPC subsets in either tissue. Further studies are needed to determine whether changes in these cell populations contribute towards metabolic dysfunction .


Assuntos
Tecido Adiposo Marrom , Células-Tronco Hematopoéticas , Tecido Adiposo Marrom/fisiologia , Animais , Dieta Hiperlipídica/efeitos adversos , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Células Progenitoras Mieloides , Raios Ultravioleta
6.
Nat Commun ; 12(1): 2455, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33911081

RESUMO

The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.


Assuntos
Hematopoiese Clonal/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Polimerase Dirigida por DNA/genética , Leucemia Mieloide/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Afidicolina/farmacologia , Calreticulina/genética , Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Células Progenitoras Mieloides , Proteínas Repressoras/genética , Deleção de Sequência/genética , Fatores de Processamento de Serina-Arginina/genética
7.
Nat Immunol ; 22(5): 595-606, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33903766

RESUMO

Although the pathological significance of tumor-associated macrophage (TAM) heterogeneity is still poorly understood, TAM reprogramming is viewed as a promising anticancer therapy. Here we show that a distinct subset of TAMs (F4/80hiCD115hiC3aRhiCD88hi), endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), plays a critical role in shaping a prometastatic tumor microenvironment favoring immunosuppression, angiogenesis and epithelial-to-mesenchymal transition. This population originates from F4/80+HO-1+ bone marrow (BM) precursors, accumulates in the blood of tumor bearers and preferentially localizes at the invasive margin through a mechanism dependent on the activation of Nrf2 and coordinated by the NF-κB1-CSF1R-C3aR axis. Inhibition of F4/80+HO-1+ TAM recruitment or myeloid-specific deletion of HO-1 blocks metastasis formation and improves anticancer immunotherapy. Relative expression of HO-1 in peripheral monocyte subsets, as well as in tumor lesions, discriminates survival among metastatic melanoma patients. Overall, these results identify a distinct cancer-induced HO-1+ myeloid subgroup as a new antimetastatic target and prognostic blood marker.


Assuntos
Biomarcadores Tumorais/metabolismo , Heme Oxigenase-1/metabolismo , Neoplasias Pulmonares/imunologia , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Linhagem Celular Tumoral/transplante , Quimioterapia Adjuvante/métodos , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/imunologia , Feminino , Heme/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/sangue , Heme Oxigenase-1/genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Masculino , Melanoma/mortalidade , Melanoma/secundário , Melanoma/terapia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/metabolismo , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/metabolismo
8.
Nat Chem Biol ; 17(5): 567-575, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33664520

RESUMO

The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.


Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células Progenitoras Mieloides/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Triazóis/farmacologia , Antineoplásicos/síntese química , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Concentração Inibidora 50 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Células Progenitoras Mieloides/enzimologia , Células Progenitoras Mieloides/patologia , Piperazinas/farmacologia , Cultura Primária de Células , Piridinas/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Transdução de Sinais , Relação Estrutura-Atividade , Transcriptoma , Triazóis/síntese química
10.
Br J Haematol ; 192(6): 1054-1063, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33618432

RESUMO

Clonal haematopoiesis (CH) in patients with acute myeloid leukaemia (AML) may persist beyond attaining complete remission. From a consecutive cohort of 67 patients with nucleophosmin 1-mutated (NPM1mut ) AML, we identified 50 who achieved NPM1mut clearance and had parallel multicolour flow cytometry (MFC) and next generation sequencing (NGS). In total, 13 (26%) cleared all mutations, 37 (74%) had persistent CH frequently involving DNA methyltransferase 3α (DNMT3A,70%), tet methylcytosine dioxygenase 2 (TET2, 27%), isocitrate dehydrogenase 2 (IDH2, 19%) and IDH1 (11%). A small number (<1%) of aberrant CD34+ myeloblasts, but immunophenotypically different from original AML blasts [herein referred to as a pre-leukaemic (PL) phenotype], was detected in 17 (49%) patients with CH, but not in any patients with complete clearance of all mutations (P = 0·0037). A PL phenotype was associated with higher mutation burden (P = 0·005). Persistent IDH2 and serine and arginine-rich splicing factor 2 (SRSF2) mutations were exclusively observed in PL+ CH+ cases (P = 0·016). Persistent dysplasia was seen exclusively in cases with a PL+ phenotype (29% vs. none; P = 0·04). The PL+ phenotype did not correlate with age, intensity of induction therapy or relapse-free survival. Post-remission CH in the setting of NPM1mut clearance is common and may result in immunophenotypic changes in myeloid progenitors. It is important to not misinterpret these cells as AML measurable residual disease (MRD).


Assuntos
Medula Óssea , Hematopoiese Clonal , Leucemia Mieloide Aguda , Mutação , Células Progenitoras Mieloides , Proteínas de Neoplasias , Proteínas Nucleares , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/metabolismo , Medula Óssea/patologia , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Indução de Remissão
11.
Nat Immunol ; 22(3): 301-311, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33603226

RESUMO

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.


Assuntos
Linhagem da Célula , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Células Dendríticas/metabolismo , Elementos Facilitadores Genéticos , Fatores Reguladores de Interferon/metabolismo , Monócitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células da Medula Óssea , Células Cultivadas , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/genética , Células Dendríticas/imunologia , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Monócitos/imunologia , Células Progenitoras Mieloides/imunologia , Fenótipo , Transdução de Sinais
12.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33635312

RESUMO

Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier.


Assuntos
Gengiva/citologia , Gengiva/imunologia , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/imunologia , Animais , Medula Óssea/metabolismo , Feminino , Hematopoese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , RNA-Seq/métodos , Análise de Célula Única/métodos
13.
Stem Cell Reports ; 16(2): 324-336, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33482101

RESUMO

Hemorrhagic shock induces an aberrant immune response characterized by simultaneous induction of a proinflammatory state and impaired host defenses. The objective of this study was to evaluate the impact of conditionally immortalized neutrophil progenitors (NPs) on this aberrant immune response. We employed a mouse model of hemorrhagic shock, followed by the adoptive transfer of NPs and subsequent inoculation of Staphylococcus aureus to induce pneumonia. We observed that transplant of NPs decreases the proportion of host neutrophils that express programmed death ligand 1 and intercellular adhesion molecule 1 in the context of prior hemorrhage. Following hemorrhage, NP transplant decreased proinflammatory cytokines in the lungs, increased neutrophil migration into the airspaces, and enhanced bacterial clearance. Further, hemorrhagic shock improved NP engraftment in the bone marrow. These results suggest that NPs hold the potential for use as a cellular therapy in the treatment and prevention of secondary infection following hemorrhagic shock.


Assuntos
Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/metabolismo , Neutrófilos/imunologia , Pneumonia/imunologia , Choque Hemorrágico/imunologia , Choque Hemorrágico/metabolismo , Staphylococcus aureus/imunologia , Animais , Antígeno B7-H1/metabolismo , Medula Óssea/imunologia , Linhagem Celular , Movimento Celular , Terapia Baseada em Transplante de Células e Tecidos , Citocinas/metabolismo , Modelos Animais de Doenças , Imunidade , Molécula 1 de Adesão Intercelular/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/transplante , Pneumonia/microbiologia , Choque Hemorrágico/complicações
14.
Sci Rep ; 11(1): 2478, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510180

RESUMO

Despite the frequent use of ionising radiation (IR) in therapy and diagnostics and the unavoidable exposure to external radiation sources, our knowledge regarding the radiosensitivity of human blood cell populations is limited and published data, obtained under different experimental conditions, are heterogeneous. To compare the radiosensitivity of different hematopoietic cell populations, we set out to determine the responses of cells obtained from peripheral blood of healthy volunteers under identical conditions (resting, non-stimulated cells). First, we measured the radiation response of T cells (Treg, Th, CTL), B cells, NK cells, CD34+ progenitor cells and monocytes obtained from peripheral blood and monocyte-derived macrophages (Mph) and immature dendritic cells (iDC) ex vivo and show that T and B cells are highly sensitive, starting to undergo apoptosis following IR with a dose as low as 0.125 Gy. Importantly, there was no clear threshold dose and cell death/apoptosis increased up to a saturation level with a dose of 2 Gy. The sensitivity decreased in the order of T cells > NK and B cells > monocytes > macrophages and iDC. The data confirm a previous report that Mph and iDC are radiation-resistant compared to their progenitor monocytes. Although non-stimulated T and B cells were highly radiation-sensitive compared to monocytes and macrophages, they were competent in the repair of DNA double-strand breaks, as shown by a decline in γH2AX foci in the post-exposure period. CD34+ cells obtained from peripheral blood also showed γH2AX decline post-exposure, indicating they are repair competent. Granulocytes (CD15+) did not display any γH2AX staining following IR. Although peripheral blood lymphocytes, the main fraction are T cells, were significantly more radiation-sensitive than monocytes, they displayed the expression of the repair proteins XRCC1, ligase III and PARP-1, which were nearly non-expressed in monocytes. To assess whether monocytes are depleted in vivo following IR, we measured the amount of T cells and monocytes in cancer patients who received total-body radiation (TBR, 6 × 2 Gy). We observed that the number of T cells in the peripheral blood significantly declined already after the first day of TBR and remained at a low level, which was accompanied by an increase in the number of γH2AX foci in the surviving CD3+ T cell fraction. In contrast, the number of monocytes did not decline extensively, reflecting their radiation resistance compared to T cells. Monocytes also showed an accumulation of γH2AX foci in vivo, but the levels were significantly lower than in T cells. CD56+ NK cells displayed a response similar to T cells. The data support the notion that unstimulated T cell subfractions are nearly equally radiation sensitive. There are, however, remarkable differences in the radiation sensitivity between the lymphoid and the myeloid lineage, with lymphoid cells being significantly more sensitive than cells of the myeloid lineage. In the myeloid lineage, macrophages and iDCs were the most radio-resistant cell types.


Assuntos
Reparo do DNA/efeitos da radiação , Células Dendríticas/metabolismo , Raios gama , Linfócitos/metabolismo , Macrófagos/metabolismo , Células Progenitoras Mieloides/metabolismo , Tolerância a Radiação/efeitos da radiação , Humanos
15.
J Immunol ; 206(2): 432-445, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33310871

RESUMO

Although neutrophils play important roles in immunity and inflammation, their analysis is strongly hindered by their short-lived and terminally differentiated nature. Prior studies reported conditional immortalization of myeloid progenitors using retroviral expression of an estrogen-dependent fusion protein of the HoxB8 transcription factor. This approach allowed the long-term culture of mouse myeloid progenitors (HoxB8 progenitors) in estrogen-containing media, followed by differentiation toward neutrophils upon estrogen withdrawal. Although several reports confirmed the in vitro functional responsiveness of the resulting differentiated cells (HoxB8 neutrophils), little is known about their capacity to perform in vivo neutrophil functions. We have addressed this issue by an in vivo transplantation approach. In vitro-generated HoxB8 neutrophils showed a neutrophil-like phenotype and were able to perform conventional neutrophil functions, like respiratory burst, chemotaxis, and phagocytosis. The i.v. injection of HoxB8 progenitors into lethally irradiated recipients resulted in the appearance of circulating donor-derived HoxB8 neutrophils. In vivo-differentiated HoxB8 neutrophils were able to migrate to the inflamed peritoneum and to phagocytose heat-killed Candida particles. The reverse passive Arthus reaction could be induced in HoxB8 chimeras but not in irradiated, nontransplanted control animals. Repeated injection of HoxB8 progenitors also allowed us to maintain stable circulating HoxB8 neutrophil counts for several days. Injection of arthritogenic K/B×N serum triggered robust arthritis in HoxB8 chimeras, but not in irradiated, nontransplanted control mice. Taken together, our results indicate that HoxB8 progenitor-derived neutrophils are capable of performing various in vivo neutrophil functions, providing a framework for using the HoxB8 system for the in vivo analysis of neutrophil function.


Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Proteínas de Homeodomínio/metabolismo , Inflamação/imunologia , Células Progenitoras Mieloides/imunologia , Neutrófilos/imunologia , Animais , Diferenciação Celular , Linhagem Celular Transformada , Quimiotaxia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Transgenes/genética
16.
Sci Rep ; 10(1): 21804, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311522

RESUMO

Previous studies examining the role of the histone deacetylase Hdac3 within myeloid cells demonstrated that Hdac3 promotes M2 activation and tissue healing in inflammatory conditions. Since myeloid lineage cells are required for proper bone formation and regeneration, in this study we examined the functions of Hdac3 during bone healing. Conditional deletion of Hdac3 within myeloid progenitors accelerates healing of cortical bone defects. Moreover, reduced osteoclast numbers within the defect site are correlated with Hdac3 suppression. Ex vivo osteoclastogenesis assays further demonstrate that Hdac3 deficiency limits osteoclastogenesis, the number of nuclei per cell and bone resorption, suggesting a defect in cell fusion. High throughput RNA sequencing identified the transmembrane protein Pmepa1 as a differentially expressed gene within osteoclast progenitor cells. Knockdown of Pmepa1 partially restores defects in osteoclastogenesis induced by Hdac3 deficiency. These results show that Hdac3 is required for optimal bone healing and osteoclast fusion, potentially via its regulation of Pmepa1 expression.


Assuntos
Regeneração Óssea , Osso Cortical/metabolismo , Deleção de Genes , Histona Desacetilases/deficiência , Proteínas de Membrana/metabolismo , Células Progenitoras Mieloides/metabolismo , Osteoclastos/metabolismo , Animais , Fusão Celular , Osso Cortical/lesões , Osso Cortical/patologia , Feminino , Histona Desacetilases/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Células Progenitoras Mieloides/patologia , Osteoclastos/patologia
17.
PLoS Biol ; 18(11): e3000920, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33137094

RESUMO

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3' splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.


Assuntos
Ribossomos/metabolismo , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Substituição de Aminoácidos , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Inativação Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , Ribossomos/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
18.
Elife ; 92020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33168134

RESUMO

Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation of innate immune cells occurs at the level of myeloid progenitors, which adds exciting opportunities for novel treatment strategies.


Assuntos
Aterosclerose/metabolismo , Células da Medula Óssea/fisiologia , Técnicas de Reprogramação Celular , Doença da Artéria Coronariana/metabolismo , Leucócitos Mononucleares/fisiologia , Células Progenitoras Mieloides/fisiologia , Idoso , Estudos de Casos e Controles , Diferenciação Celular , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade
20.
Nucleic Acids Res ; 48(20): 11335-11346, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33119742

RESUMO

High-content imaging and single-cell genomics are two of the most prominent high-throughput technologies for studying cellular properties and functions at scale. Recent studies have demonstrated that information in large imaging datasets can be used to estimate gene mutations and to predict the cell-cycle state and the cellular decision making directly from cellular morphology. Thus, high-throughput imaging methodologies, such as imaging flow cytometry can potentially aim beyond simple sorting of cell-populations. We introduce IFC-seq, a machine learning methodology for predicting the expression profile of every cell in an imaging flow cytometry experiment. Since it is to-date unfeasible to observe single-cell gene expression and morphology in flow, we integrate uncoupled imaging data with an independent transcriptomics dataset by leveraging common surface markers. We demonstrate that IFC-seq successfully models gene expression of a moderate number of key gene-markers for two independent imaging flow cytometry datasets: (i) human blood mononuclear cells and (ii) mouse myeloid progenitor cells. In the case of mouse myeloid progenitor cells IFC-seq can predict gene expression directly from brightfield images in a label-free manner, using a convolutional neural network. The proposed method promises to add gene expression information to existing and new imaging flow cytometry datasets, at no additional cost.


Assuntos
Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Processamento de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Análise de Célula Única/métodos , Animais , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Sangue Fetal/metabolismo , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Redes Neurais de Computação , Transcriptoma
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