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1.
J Enzyme Inhib Med Chem ; 35(1): 311-324, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31809612

RESUMO

Hybridization of reported weakly active antiproliferative hit 5-amino-4-pyrimidinol derivative with 2-anilino-4-phenoxypyrimidines suggests a series of 2,5-diamino-4-pyrimidinol derivatives as potential antiproliferative agents. Few compounds belonging to the proposed series were reported as CSF1R/DAPK1 inhibitors as anti-tauopathies. However, the correlation between CSF1R/DAPK1 signalling pathways and cancer progression provides motives to reprofile them against cancer therapy. The compounds were synthesised, characterized, and evaluated against M-NFS-60 cells and a kinase panel which bolstered predictions of their antiproliferative activity and suggested the involvement of diverse molecular targets. Compound 6e, the most potent in the series, showed prominent broad-spectrum antiproliferative activity inhibiting the growth of hematological, NSCLC, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers by 84.1, 52.79, 72.15, 66.34, 66.48, 51.55, 55.95, 61.85, and 60.87%, respectively. Additionally, it elicited an IC50 value of 1.97 µM against M-NFS-60 cells and good GIT absorption with Pe value of 19.0 ± 1.1 × 10-6 cm/s (PAMPA-GIT). Molecular docking study for 6e with CSF1R and DAPK1 was done to help to understand the binding mode with both kinases. Collectively, compound 6e could be a potential lead compound for further development of anticancer therapies.


Assuntos
Antineoplásicos/farmacologia , Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas Quinases Associadas com Morte Celular/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Relação Estrutura-Atividade
2.
Radiat Res ; 192(6): 612-620, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31560640

RESUMO

Radiation-induced acute myeloid leukemia (rAML) in C3H mice is commonly developed through inactivation of PU.1 transcription factor encoded in Sfpi1 on chromosome 2. PU.1 inactivation involves two steps: hemizygous deletion of the Sfpi1 gene (DSG) and point mutation of the allele Sfpi1 gene (PMASG). In this study, we investigated the dose-rate dependence of the frequency of both DSG and PMASG in hematopoietic stem cells (HSCs) of C3H mice that received a total of 3 Gy gamma-ray exposure at dose rates of 20 mGy/day, 200 mGy/day or 1,000 mGy/min. All mice were followed for 250 days from start of irradiation. Fluorescent in situ hybridization of the Sfpi1 gene site indicated that frequency of HSCs with DSG was proportional to dose rate. In cell surface profiles, PU.1-inactivated HSCs by both DSG and PMASG were still positive for PU.1, but negative for GM-CSF receptor-α (GMCSFRα), which is transcriptionally regulated by PU.1. Immunofluorescent staining analysis of both PU.1 and GM-CSFRα also showed dose-rate-dependent levels of PU.1-inactivated HSCs. This study provides evidence that both DSG and PMASG are dose-rate dependent; these experimental data offer new insights into the dose-rate effects in HSCs that can lead to radiation-induced leukemogenesis.


Assuntos
Células-Tronco Hematopoéticas/efeitos da radiação , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Induzida por Radiação/tratamento farmacológico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/genética , Transativadores/fisiologia , Alelos , Animais , Carcinogênese , Membrana Celular/metabolismo , Proliferação de Células , Relação Dose-Resposta a Droga , Raios gama , Deleção de Genes , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Hibridização In Situ , Leucemia Mieloide Aguda/genética , Leucemia Induzida por Radiação/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Mutação Puntual , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo
3.
Nat Commun ; 10(1): 3758, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434879

RESUMO

Many risk genes for the development of Alzheimer's disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aß deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis.


Assuntos
Doença de Alzheimer/metabolismo , Microglia/metabolismo , Compostos Orgânicos/farmacologia , Placa Amiloide/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Doença de Alzheimer/genética , Animais , Comportamento Animal , Encéfalo/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hipocampo/metabolismo , Humanos , Memória , Camundongos , Camundongos Transgênicos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Transcriptoma
4.
BMC Pulm Med ; 19(1): 158, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438916

RESUMO

BACKGROUND: Severe non-allergic eosinophilic asthma (SNEA) is a rare asthma phenotype associated with severe clinical course, frequent exacerbations, and resistance to therapy, including high steroid doses. The key feature is type 2 inflammation with predominant airway eosinophilia. Eosinophil maturation, activation, survivability, and recruitment are mainly induced by interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) through their receptors on eosinophil surface and related with integrins activation states. The aim of the study was to estimate the expression of eosinophil ß chain-signaling cytokines receptors, outer-membrane integrins, and serum-derived type 2 inflammation biomarkers in SNEA. METHODS: We examined 8 stable SNEA patients with high inhaled steroid doses, 12 steroid-free patients with non-severe allergic asthma (AA), 12 healthy subjects (HS). Blood eosinophils were isolated using Ficol gradient centrifugation and magnetic separation. Eosinophils were lysed, and mRNA was isolated. Gene expressions of IL-5Rα, IL-3Rα, GM-CSFRα, and α4ß1, αMß2 integrins were analyzed using quantitative real-time reverse transcription polymerase chain reaction. Type 2 inflammation activity was evaluated measuring exhaled nitric oxide concentration (FeNO) collected with the electrochemical sensing device. Serum IL-5, IL-3, GM-CSF, periostin, chemokine ligand (CCL) 17 and eotaxin concentrations were assessed by enzyme-linked immunosorbent assay. RESULTS: Eosinophils from SNEA patients demonstrated significantly increased gene expression of IL-3Rα, IL-5Rα and GM-CSFRα as well as α4, ß1 and αM integrin subunits compared with the AA group. The highest IL-5 serum concentration was in the SNEA group; it significantly differed compared with AA and HS. GM-CSF serum levels were similar in the SNEA and AA groups and were significantly lower in the HS group. No differences in serum IL-3 concentration were found among all groups. Furthermore, serum levels of eotaxin, CCL17 and FeNO, but not periostin, differed in all groups, with the highest levels in SNEA patients. CONCLUSIONS: Eosinophil demonstrated higher expression of IL-3, IL-5, GM-CSF α-chain receptors and α4, ß1, αM integrins subunits in SNEA compared with the AA group. Additionally, SNEA patients had increased serum levels of IL-5, eotaxin and CCL-17. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03388359.


Assuntos
Asma/sangue , Eosinófilos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Integrinas/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Adulto , Asma/fisiopatologia , Biomarcadores/sangue , Quimiocina CCL17/sangue , Feminino , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Inflamação/metabolismo , Interleucina-3/sangue , Interleucina-3/genética , Interleucina-5/sangue , Interleucina-5/genética , Contagem de Leucócitos , Lituânia , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória , Adulto Jovem
5.
Nat Commun ; 10(1): 3215, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324781

RESUMO

The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1rΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peritoneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r-/- rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals.


Assuntos
Genes fms/genética , Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Deleção de Sequência , Animais , Sequência de Bases , Diferenciação Celular , Proliferação de Células , Modelos Animais de Doenças , Células-Tronco Embrionárias/patologia , Fator de Crescimento Epidérmico , Feminino , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Monócitos/metabolismo , Fagocitose , Células RAW 264.7 , Sequências Reguladoras de Ácido Nucleico/genética
6.
Comput Biol Chem ; 80: 374-383, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31103918

RESUMO

Colony-stimulating factor 1 receptor is a type III receptor protein tyrosine kinase belonging to PDGFR family. CSF1R signaling is essential for differentiation, proliferation and survival of macrophages. Aberrant expression of CSF1R appears to be an attractive target in several cancer types. Higher expression of CSF1R ligands correlates to tumor progression. CSF1R inhibitors have been shown to suppress cancers. We have attempted an in silico fragment derived drug discovery approach by screening ˜25,000 in-house compounds as potential CSF1R inhibitors. Using FBDD approach we have identified six diverse fragments that exhibit affinity towards hinge region of CSF1R. Some of the fragments 5-nitroindole and 7-azaindole and their derivatives were synthesized for further evaluation. The in silico and in vitro enzyme activity studies reveal moderate inhibition of CSF1R kinase activity by 5-nitroindole and good inhibition by 7-azaindole fragments. Bio and chemiinformatics studies have shown that 7-azaindole compounds have better membrane permeability and enzyme inhibition properties. Molecular docking studies show that the amino acid residues 664-666 in the hinge region of the cytosolic domain of CSF1R to be the preferred region of binding for nitroindole and azaindole derivatives. Further optimization and biological analysis would identify these fragments as potential and promising leads as CSF1R inhibitors.


Assuntos
Indóis/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Biologia Computacional , Desenho de Drogas , Humanos , Indóis/síntese química , Indóis/química , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
7.
PLoS One ; 14(5): e0216275, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31042769

RESUMO

INTRODUCTION: Murine Kupffer cells (KCs) comprise CD11bhi and F4/80hi subsets. Tissue-resident macrophages are known to express the tyrosine kinase receptors colony-stimulating factor 1 receptor (Csf1r) and Mer. However, the expression of Csf1r and Mer on KC subsets and the importance of these tyrosine kinases during liver regeneration (LR) are unknown. METHODS: KCs from wild-type and Csf1r-GFP mice were characterized by flow cytometry. Partial hepatectomy (PH) was performed in mice treated with clodronate liposomes, a Csf1r small molecule inhibitor or depleting antibody, or a small molecule Mer inhibitor. Sera and livers were analyzed. The function of sorted KC subsets was tested in vitro. RESULTS: Mer was specifically expressed on tissue-resident F4/80hi KCs, 55% of which also expressed Csf1r. Mer+Csf1r+ and Mer+Csf1r- KCs had distinct expression of macrophage markers. Csf1r inhibition in mice reduced F4/80hi KCs by approximately 50%, but did not affect CD11bhi KCs. Clodronate liposomes depleted F4/80hi KCs, but also altered levels of other intrahepatic leukocytes. Csf1r inhibition delayed LR, as demonstrated by a 20% reduction in liver-to-body weight ratios 7 days after PH. At 36h after PH, Csf1r inhibition increased serum ALT and histological liver injury, and decreased liver cell proliferation. A small molecule inhibitor of Mer did not alter the percentage of KCs or their proliferation and just modestly delayed LR. In vitro, Csf1r or Mer inhibition did not decrease KC viability, but did attenuate their cytokine response to stimulation. CONCLUSIONS: F4/80hi KCs are Mer+ and can be subdivided based on Csf1r expression. Csf1r or Mer inhibition each reduces KC cytokine production and delays LR.


Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Macrófagos do Fígado/metabolismo , Regeneração Hepática/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Antígenos de Diferenciação/análise , Hepatectomia , Macrófagos do Fígado/citologia , Macrófagos do Fígado/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores Imunológicos/antagonistas & inibidores
8.
Nat Commun ; 10(1): 1935, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028249

RESUMO

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.


Assuntos
Regulação da Expressão Gênica , Leucemia Mielomonocítica Crônica/genética , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Sistemas CRISPR-Cas , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Corantes Fluorescentes/química , Edição de Genes , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Maleimidas/química , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais , Células THP-1 , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo
9.
Leukemia ; 33(10): 2442-2453, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30940906

RESUMO

The microenvironment strongly influences mantle cell lymphoma (MCL) survival, proliferation, and chemoresistance. However, little is known regarding the molecular characterization of lymphoma niches. Here, we focused on the interplay between MCL cells and the associated monocytes/macrophages. Using circulating MCL cells (n = 58), we showed that, through the secretion of CSF1 and, to a lesser extent, IL-10, MCL polarized monocytes into specific CD163+ M2-like macrophages (MϕMCL). In turn, MϕMCL favored lymphoma survival and proliferation ex vivo. We next demonstrated that BTK inhibition abrogated CSF1 and IL-10 production in MCL cells, leading to the inhibition of macrophage polarization and consequently resulting in the suppression of microenvironment-dependent MCL expansion. In vivo, we showed that CSF1 and IL-10 plasma concentrations were higher in MCL patients than in healthy donors, and that monocytes from MCL patients overexpressed CD163. Further analyses of serial samples from ibrutinib-treated patients (n = 8) highlighted a rapid decrease of CSF1, IL-10, and CD163 in responsive patients. Finally, we showed that targeting the CSF1R abrogated MϕMCL-dependent MCL survival, irrespective of their sensitivity to ibrutinib. These data reinforced the role of the microenvironment in lymphoma and suggested that macrophages are a potential target for developing novel therapeutic strategies in MCL.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Linfoma de Célula do Manto/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Interleucina-10/metabolismo , Linfoma de Célula do Manto/metabolismo , Macrófagos/metabolismo , Masculino , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Receptores de Superfície Celular/metabolismo , Microambiente Tumoral/efeitos dos fármacos
10.
Int Immunopharmacol ; 72: 112-123, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30974282

RESUMO

The tyrosine kinase inhibitor, Nintedanib (NTD), has been approved for the treatment of idiopathic pulmonary fibrosis (IPF). In cell-free systems, NTD was recently shown to inhibit kinase activity of the human recombinant colony-stimulating factor 1 (CSF1) receptor (CSF1R) which mediates major functions of pulmonary macrophages. In the present study, we have investigated the effects of NTD on the phenotype of human monocyte-derived macrophages controlled by CSF1 in order to identify its anti-inflammatory properties via CSF1R inhibition. NTD (0.01 to 1 µM) prevented the CSF1-induced phosphorylation of CSF1R and activation of the downstream signaling pathways. NTD, like the CSF1R inhibitor GW2580, significantly decreased the adhesion of macrophages and production of the chemokine ligand (CCL) 2. NTD also altered the polarization of macrophages to classical M1 and alternative M2a macrophages. It reduced the secretion of several pro-inflammatory and/or pro-fibrotic cytokines (IL-1ß, IL-8, IL-10 and CXCL13) by M1 macrophages but did not prevent the expression of M1 markers. While NTD (50-200 nM) partially blocked the synthesis of M2a markers (CD11b, CD200R, CD206, and CD209), it did not reduce synthesis of the M2a pro-fibrotic cytokines CCL22 and PDGF-BB, and increased CCL18 release when used at its highest concentration (1 µM). The effects of NTD on macrophage polarization only was partially mimicked by GW2580, suggesting that the drug inhibits other molecules in addition to CSF1R. In conclusion, NTD alters the CSF1-controlled phenotype of human macrophages mainly by blocking the activation of CSF1R that thus constitutes a new molecular target of NTD, at least in vitro.


Assuntos
Indóis/farmacologia , Macrófagos/efeitos dos fármacos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Humanos , Fibrose Pulmonar Idiopática , Macrófagos/metabolismo , Fenótipo
11.
Mol Genet Genomic Med ; 7(4): e00595, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30729751

RESUMO

BACKGROUND: Colony-stimulating factor 1 receptor is a tyrosine kinase transmembrane protein that mediates proliferation, differentiation, and survival of monocytes/macrophages and microglia. CSF1R gene mutations cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an autosomal-dominantly inherited microgliopathy, leading to early onset dementia with high lethality. METHODS: By interdisciplinary assessment of a complex neuropsychiatric condition in a 44-year old female patient, we narrowed down the genetic diagnostic to CSF1R gene sequencing. Flow cytometric analyses of uncultivated peripheral blood monocytes were conducted sequentially to measure the cell surface CSF1 receptor and autophosphorylation levels. Monocyte subpopulations were monitored during disease progression. RESULTS: We identified a novel heterozygous deletion-insertion mutation c.2527_2530delinsGGCA, p.(Ile843_Leu844delinsGlyIle) in our patient with initial signs of HDLS. Marginally elevated cell surface CSF1 receptor levels with increased Tyr723 autophosphorylation suggest an enhanced receptor activity. Furthermore, we observed a shift in monocyte subpopulations during disease course. CONCLUSION: Our data indicate a mutation-related CSF1R gain-of-function, accompanied by an altered composition of the peripheral innate immune cells in our patient with HDLS. Since pharmacological targeting of CSF1R with tyrosine kinase inhibitors prevents disease progression in mouse models of neurodegenerative disorders, a potential pharmacological benefit of CSF1R inhibition remains to be elucidated for patients with HDLS.


Assuntos
Mutação com Ganho de Função , Leucoencefalopatias/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Adulto , Feminino , Heterozigoto , Humanos , Leucoencefalopatias/patologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo
12.
PLoS Biol ; 17(2): e3000134, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735499

RESUMO

Microglia are resident immune cells that play critical roles in maintaining the normal physiology of the central nervous system (CNS). Remarkably, microglia have an intrinsic capacity to repopulate themselves after acute ablation. However, the underlying mechanisms that drive such restoration remain elusive. Here, we characterized microglial repopulation both spatially and temporally following removal via treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. We show that microglia were replenished via self-renewal, with no contribution from nonmicroglial lineages, including Nestin+ progenitors and the circulating myeloid population. Interestingly, spatial analyses with dual-color labeling revealed that newborn microglia recolonized the parenchyma by forming distinctive clusters that maintained stable territorial boundaries over time, indicating the proximal expansive nature of adult microgliogenesis and the stability of microglia tiling. Temporal transcriptome profiling at different repopulation stages revealed that adult newborn microglia gradually regain steady-state maturity from an immature state that is reminiscent of the neonatal stage and follow a series of maturation programs, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, interferon immune activation, and apoptosis. Importantly, we show that the restoration of microglial homeostatic density requires NF-κB signaling as well as apoptotic egress of excessive cells. In summary, our study reports key events that take place from microgliogenesis to homeostasis reestablishment.


Assuntos
Envelhecimento/genética , Encéfalo/metabolismo , Homeostase/genética , Microglia/metabolismo , NF-kappa B/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/genética , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Interferons/genética , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Nestina/genética , Nestina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Compostos Orgânicos/toxicidade , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Regeneração/genética , Transdução de Sinais , Transcriptoma
13.
Proc Natl Acad Sci U S A ; 116(5): 1686-1691, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30635412

RESUMO

While neuroinflammation is an evolving concept and the cells involved and their functions are being defined, microglia are understood to be a key cellular mediator of brain injury and repair. The ability to measure microglial activity specifically and noninvasively would be a boon to the study of neuroinflammation, which is involved in a wide variety of neuropsychiatric disorders including traumatic brain injury, demyelinating disease, Alzheimer's disease (AD), and Parkinson's disease, among others. We have developed [11C]CPPC [5-cyano-N-(4-(4-[11C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide], a positron-emitting, high-affinity ligand that is specific for the macrophage colony-stimulating factor 1 receptor (CSF1R), the expression of which is essentially restricted to microglia within brain. [11C]CPPC demonstrates high and specific brain uptake in a murine and nonhuman primate lipopolysaccharide model of neuroinflammation. It also shows specific and elevated uptake in a murine model of AD, experimental allergic encephalomyelitis murine model of demyelination and in postmortem brain tissue of patients with AD. Radiation dosimetry in mice indicated [11C]CPPC to be safe for future human studies. [11C]CPPC can be synthesized in sufficient radiochemical yield, purity, and specific radioactivity and possesses binding specificity in relevant models that indicate potential for human PET imaging of CSF1R and the microglial component of neuroinflammation.


Assuntos
Fator Estimulador de Colônias de Macrófagos/metabolismo , Microglia/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Placa Amiloide/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Primatas , Compostos Radiofarmacêuticos/metabolismo
14.
Gen Comp Endocrinol ; 273: 163-171, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29966660

RESUMO

Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male's life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.


Assuntos
Escorpiões/citologia , Espermatogônias/citologia , Nicho de Células-Tronco , Tartarugas/metabolismo , Animais , Biomarcadores/metabolismo , Forma Celular , Tamanho Celular , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Escorpiões/metabolismo , América do Sul , Espermatogônias/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
15.
Bioorg Med Chem Lett ; 29(1): 115-118, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30442420

RESUMO

We report the discovery of a novel azetidine scaffold for colony stimulating factor-1 receptor (CSF-1R) Type II inhibitors by using a structure-based drug design (SBDD) based on a docking model. The work leads to the representative compound 4a with high CSF-1R inhibitory activity (IC50 = 9.1 nM). The obtained crystal structure of an azetidine compound with CSF-1R, which matched our predicted docking model, demonstrates that the azetidine compounds bind to the DFG-out conformation of the protein as a Type II inhibitor.


Assuntos
Azetidinas/farmacologia , Descoberta de Drogas , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Azetidinas/síntese química , Azetidinas/química , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Relação Estrutura-Atividade
16.
Lung ; 197(1): 89-94, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30474709

RESUMO

PURPOSE: Diabetes mellitus (DBM) reduces immunological activity and increases susceptibility to various infections, including tuberculosis (TB). Human alveolar macrophage (hAM) functions are altered in DBM. METHODS: To mimic hyperglycemic conditions in the lung alveolus, we co-cultured a hAM cell line (Daisy cell line) with human umbilical vein endothelial cells for 48 h in the presence of culture media alone, normal glucose (5 mM), and high glucose (22 mM). Using flow cytometry, immunophenotype characterization included cell surface markers CD 11c, CD14, CD16, CD86, CD163, CD169, CD206, CX3CR-1, CSF-1R, and matrix metalloproteinase-9 (MMP9). Phagocytic function was measured by immunofluorescence microscopy at 24 h after inoculation of cells with GFP-expressing Mycobacterium smegmatis. RESULTS: Direct exposure of AMs to high glucose and exposure in the co-culture system yield different results for the same phenotypic markers. MMP9 expression was increased under both conditions. CD169 and CX3CR1 expressions were decreased when AMs were exposed directly to high glucose but increased under co-culture. Immunofluorescence assay revealed that phagocytosis decreased in AMs when directly exposed to increased glucose levels from 2.5 mM to normal glucose (5 mM), yet AMs under co-culture did not show decreased phagocytosis until concentrations were raised to 25 mM. CONCLUSION: Alteration in the expression of certain receptors may contribute to defective sentinel function of AMs, promoting susceptibility to TB in a diabetic host. Variability in cell surface marker expression under direct glucose exposure compared to exposure via co-culture reveals that cell signaling between endothelial cells and AMs may play a crucial role in the phenotypic expression of AMs.


Assuntos
Glucose/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Mycobacterium smegmatis/fisiologia , Fagocitose/efeitos dos fármacos , Antígenos CD/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Comunicação Celular , Linhagem Celular , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Metaloproteinase 9 da Matriz/metabolismo , Fenótipo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo
17.
J Innate Immun ; 11(1): 99-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30408777

RESUMO

Early exposure to inflammatory signals may have a lasting impact on immune function. Present throughout embryogenesis, macrophages are key cells providing innate immune protection to the developing fetus and newborn. Here, we have used an established model of macrophage development to test how early inflammatory signals can impact cellular differentiation and function. Bone marrow-derived macrophages were treated with Escherichia coli lipopolysaccharide (LPS) 2 days after initial isolation and culture. LPS treatment during this early stage of differentiation decreased the expression of CSF1R and increased that of the mature macrophage marker F4/80. These early changes in macrophage differentiation were also measured in cells from mice lacking IKKß, but the change in CSF1R expression after LPS treatment was blocked with MAPK inhibition. LPS-induced changes in macrophage marker expression persisted following LPS removal, suggesting that early inflammatory activation could induce a lasting developmental impact. Early LPS exposure inhibited macrophage phagocytosis of labeled E. coli while LPS had no effect on fully differentiated macrophages. Our data demonstrate that early inflammatory exposure to a microbial stimulus induce lasting phenotypic changes in macrophages.


Assuntos
Diferenciação Celular , Ativação de Macrófagos , Macrófagos , Receptores Toll-Like/metabolismo , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Escherichia coli , Quinase I-kappa B/metabolismo , Imunidade Inata , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagocitose/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais
18.
Nat Commun ; 9(1): 5279, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538245

RESUMO

Regulatory mechanisms controlling the pool size of spleen dendritic cells (DC) remain incompletely understood. DCs are continuously replenished from hematopoietic stem cells, and FLT3-mediated signals cell-intrinsically regulate homeostatic expansion of spleen DCs. Here we show that combining FLT3 and CSF1R-deficiencies results in specific and complete abrogation of spleen DCs in vivo. Spatiotemporally controlled CSF1R depletion reveals a cell-extrinsic and non-hematopoietic mechanism for DC pool size regulation. Lack of CSF1R-mediated signals impedes the differentiation of spleen macrophages of embryonic origin, and the resulted macrophage depletion during development or in adult mice results in loss of DCs. Moreover, embryo-derived macrophages are important for the physiologic regeneration of DC after activation-induced depletion in situ. In summary, we show that the differentiation of DC and their regeneration relies on ontogenetically distinct spleen macrophages, thereby providing a novel regulatory principle that may also be important for the differentiation of other hematopoietic cell types.


Assuntos
Células Dendríticas/citologia , Macrófagos/citologia , Camundongos/embriologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Diferenciação Celular , Células Dendríticas/metabolismo , Feminino , Macrófagos/metabolismo , Masculino , Camundongos/metabolismo , Camundongos Knockout , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Baço/citologia , Baço/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
19.
J Neuroinflammation ; 15(1): 311, 2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-30413160

RESUMO

BACKGROUND: Activation of inflammation pathways in the brain occurs in Alzheimer's disease and may contribute to the accumulation and spread of pathological proteins including tau. The goal of this study was to identify how changes in microglia, a key inflammatory cell type, may contribute to tau protein accumulation and pathology-associated changes in immune and non-immune cell processes such as neuronal degeneration, astrocyte physiology, cytokine expression, and blood vessel morphology. METHODS: We used PLX3397 (290 mg/kg), a colony-stimulating factor receptor 1 (CSF1R) inhibitor, to reduce the number of microglia in the brains of a tau-overexpressing mouse model. Mice were fed PLX3397 in chow or a control diet for 3 months beginning at 12 months of age and then were subsequently analyzed for changes in blood vessel morphology by in vivo two-photon microscopy and tissues were collected for biochemistry and histology. RESULTS: PLX3397 reduced microglial numbers by 30% regardless of genotype compared to control diet-treated mice. No change in tau burden, cortical atrophy, blood vessels, or astrocyte activation was detected. All Tg4510 mice were observed to have an increased in "disease-associated" microglial gene expression, but PLX3397 treatment did not reduce expression of these genes. Surprisingly, PLX3397 treatment resulted in upregulation of CD68 and Tgf1ß. CONCLUSIONS: Manipulating microglial activity may not be an effective strategy to combat tau pathological lesions. Higher doses of PLX3397 may be required or earlier intervention in the disease course. Overall, this indicates a need for a better understanding of specific microglial changes and their relation to the disease process.


Assuntos
Envelhecimento , Microglia/patologia , Tauopatias/patologia , Proteínas tau/genética , Proteínas tau/metabolismo , Aminopiridinas/farmacologia , Animais , Vasos Sanguíneos/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Mutação/genética , Pirróis/farmacologia , RNA Mensageiro/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Tauopatias/genética
20.
Glia ; 66(11): 2385-2396, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30370589

RESUMO

Microglia are the resident immune cell of the central nervous system (CNS), and serve to protect and maintain the local brain environment. Microglia are critically dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R); administration of CSF1R inhibitors that cross the blood brain barrier (BBB) lead to the elimination of up to 99% of microglia, depending on CNS exposure and treatment duration. Once microglia are depleted, withdrawal of inhibitor stimulates repopulation of the entire CNS with new cells, conceivably enabling a therapeutic strategy for beneficial renewal of the entire microglial tissue. We have explored the kinetics and limits of this repopulation event and show that the rate of microglial repopulation is proportional to the extent of microglial depletion - greater depletion of microglia results in more rapid repopulation. Using a CSF1R inhibitor formulation that eliminates approximately 99% of microglia within 7 days, we subjected mice to multiple rounds of elimination (7 days' treatment) and repopulation (7 days' recovery) and found that the brain only has the capacity for a single complete repopulation event; subsequent elimination and CSF1R inhibitor withdrawal fail to repopulate the brain. However, if the recovery time between, or after, cycles is extended sufficiently then the brain can ultimately repopulate. These kinetic studies define the opportunities and possible limits of the remarkable renewal capacities of microglia.


Assuntos
Encéfalo/citologia , Microglia/fisiologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Aminopiridinas/farmacologia , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Ontologia Genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Pirróis/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
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