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1.
Nat Commun ; 11(1): 4837, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973183

RESUMO

ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.


Assuntos
Trifosfato de Adenosina/metabolismo , Heme/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Receptores Virais/metabolismo , Termogênese/fisiologia , Animais , Deficiência de Citocromo-c Oxidase , Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Metabolismo Energético/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Domínios Proteicos , Receptores Virais/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Células THP-1
2.
Nat Commun ; 11(1): 4498, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908142

RESUMO

The androgen receptor (AR) is the master regulator of prostate cancer (PCa) development, and inhibition of AR signalling is the most effective PCa treatment. AR is expressed in PCa cells and also in the PCa-associated stroma, including infiltrating macrophages. Macrophages have a decisive function in PCa initiation and progression, but the role of AR in macrophages remains largely unexplored. Here, we show that AR signalling in the macrophage-like THP-1 cell line supports PCa cell line migration and invasion in culture via increased Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signalling and expression of its downstream cytokines. Moreover, AR signalling in THP-1 and monocyte-derived macrophages upregulates IL-10 and markers of tissue residency. In conclusion, our data suggest that AR signalling in macrophages may support PCa invasiveness, and blocking this process may constitute one mechanism of anti-androgen therapy.


Assuntos
Macrófagos/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Idoso , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Anilidas/farmacologia , Anilidas/uso terapêutico , Biópsia , Buffy Coat/citologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Quimioterapia Adjuvante , Técnicas de Cocultura , Intervalo Livre de Doença , Humanos , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/prevenção & controle , Nitrilos/farmacologia , Nitrilos/uso terapêutico , Intervalo Livre de Progressão , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/terapia , Procedimentos Cirúrgicos Robóticos , Transdução de Sinais/imunologia , Análise de Célula Única , Células THP-1 , Compostos de Tosil/farmacologia , Compostos de Tosil/uso terapêutico
3.
Nat Commun ; 11(1): 4535, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913217

RESUMO

The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identify a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins are revealed as modulators of nanoparticle-cell association mainly through their dynamic nature. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.


Assuntos
Nanopartículas/química , Coroa de Proteína/química , Química Click , Reagentes para Ligações Cruzadas/química , Células Endoteliais , Humanos , Poliestirenos/química , Ligação Proteica , Coroa de Proteína/análise , Dióxido de Silício/química , Células THP-1
4.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 367-373, 2020 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-32935510

RESUMO

OBJECTIVE: To investigate the polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by Nippostrongylus brasiliensis proteins in vitro, so as to provide insights into the elucidation of the mechanisms underlying host immune responses to hookworm infections. METHODS: The in-vitro culture of N. brasiliensis was established and maintained in the laboratory, and the third- (L3) and fifth-stage larvae (L5) were collected under a sterile condition for preparation of L3 and L5 proteins. The in-vitro culture of THP-1 cells was established, stimulated with 500 ng/mL PMA to yield M0 macrophages that were adherent to the plate wall. The LPS + IFN-γ group, IL-4 + IL-13 group, L3 protein group and L5 protein group were given stimulation with 500 ng/mL LPS plus 100 ng/mL IFN-γ, IL-4 and IL-13 (both 100 ng/mL), L3 protein (5 mg/mL) and L5 protein (5 mg/mL), respectively, while the negative control group was given no stimulation. The cell morphology was observed using microscopy, the mRNA expression of M1/M2 macrophages-specific genes was quantified using a quantitative real-time PCR (qPCR) assay, and the surface markers of M1/M2 macrophages were detected using flow cytometry, while the levels of cytokines secreted by M1/M2 macrophages were measured using enzyme-linked immunosorbent assay (ELISA) following stimulations, so as to examine the polarization of THP-1-derived macrophages induced by N. brasiliensis proteins in vitro. RESULTS: Following stimulation with PMA, THP-1 cells appeared wall-adherent M0 macrophages, and polarized to typical M1 macrophages following stimulation with LPS + IFN-γ, and typical M2 macrophages following stimulation with IL-4 + IL-13, IL-3 protein or L5 protein. There was a significant difference in the proportion of M1 macrophages among the negative control group, the LPS + IFN-γ group, the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (χ2 = 3 721.00, P < 0.001), with the highest proportion detected in the LPS + IFN-γ group, and there was also a significant difference in the proportion of M2 macrophages among groups (χ2 = 105.43, P < 0.001). There were significant differences among groups in terms of the mRNA expression of CCL2 (F = 191.95, P < 0.001), TNF-α (F = 129.95, P < 0.001), IL-12b (F = 82.89, P < 0.001), PPARγ (F = 11.30, P < 0.001), IL-10 (F = 9.51, P < 0.001) and Mrc1 genes (F = 12.35, P < 0.001). In addition, there were significant differences in the proportion of positive CD86 and CD206 expression among groups (χ2 = 24 004.33 and 832.50, P < 0.001). Higher IL-1ß and TNF-α levels were measured in the LPS + IFN-γ group than in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (P < 0.001), and greater TGF-ß1 and IL-10 levels were seen in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group than in the negative control group and the LPS + IFN-γ group (P < 0.05). CONCLUSIONS: Both L3 and L5 proteins of N. brasiliensis may induce the polarization of THP-1-derived macrophages to M2 type in vitro.


Assuntos
Leucemia Monocítica Aguda , Animais , Antígenos de Helmintos/farmacologia , Criança , Humanos , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Nippostrongylus/química , Células THP-1/citologia , Células THP-1/efeitos dos fármacos
5.
PLoS Comput Biol ; 16(9): e1008179, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898132

RESUMO

Detection and segmentation of macrophage cells in fluorescence microscopy images is a challenging problem, mainly due to crowded cells, variation in shapes, and morphological complexity. We present a new deep learning approach for cell detection and segmentation that incorporates previously learned nucleus features. A novel fusion of feature pyramids for nucleus detection and segmentation with feature pyramids for cell detection and segmentation is used to improve performance on a microscopic image dataset created by us and provided for public use, containing both nucleus and cell signals. Our experimental results indicate that cell detection and segmentation performance significantly benefit from the fusion of previously learned nucleus features. The proposed feature pyramid fusion architecture clearly outperforms a state-of-the-art Mask R-CNN approach for cell detection and segmentation with relative mean average precision improvements of up to 23.88% and 23.17%, respectively.


Assuntos
Células Eucarióticas/citologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Redes Neurais de Computação , Biologia Computacional , Aprendizado Profundo , Humanos , Macrófagos/citologia , Células THP-1
6.
PLoS One ; 15(8): e0236212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797100

RESUMO

Although an impact of processing on immunogenicity of food proteins has clearly been demonstrated, the underlying mechanisms are still unclear. We applied 3 different processing methods: wet heating (60 °C) and low- or high-temperature (50 °C or 130 °C, respectively) dry-heating in absence or presence of reducing sugars, to ß-lactoglobulin (BLG), lysozyme and thyroglobulin, which represent dietary proteins with different pI or molecular weight. Uptake of the soluble fraction of the samples was tested in two types of, genetically homogeneous, antigen-presenting cells (macrophages and dendritic cells derived from THP-1 monocytes). This revealed a strong correlation between the uptake of the different protein samples by macrophages and dendritic cells, and confirmed the key role of hydrophobicity, over aggregation, in determining the uptake. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but not in cytokine production, by THP-1 macrophages.


Assuntos
Citocinas/metabolismo , Células Dendríticas/imunologia , Proteínas na Dieta/imunologia , Macrófagos/imunologia , Receptores de Superfície Celular/metabolismo , Culinária , Células Dendríticas/metabolismo , Proteínas na Dieta/química , Proteínas na Dieta/metabolismo , Temperatura Alta , Humanos , Interações Hidrofóbicas e Hidrofílicas , Macrófagos/metabolismo , Peso Molecular , Células THP-1
7.
Nat Commun ; 11(1): 4243, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843625

RESUMO

Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca2+]ex. Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1ß release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1ß release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca2+] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA.


Assuntos
Artrite Reumatoide/imunologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Calcinose , Cálcio/metabolismo , Células Cultivadas , Humanos , Inflamação , Interleucina-1beta/metabolismo , Camundongos , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Fosfatos/metabolismo , Pinocitose , Receptores de Detecção de Cálcio/deficiência , Transdução de Sinais , Células THP-1 , alfa-2-Glicoproteína-HS/metabolismo
8.
Ecotoxicol Environ Saf ; 204: 110948, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32739672

RESUMO

Exposure to arsenic is epidemiologically associated with increased lung disease. In detailing the mechanism by which arsenic exposure leads to disease, studies have emphasized that metabolic reprogramming and immune dysfunction are related to arsenic-induced lung injury. However, the association between the mechanisms listed above is not well understood. Thus, the current study aimed to investigate the interaction of energy metabolism and macrophage polarization, by which arsenic exposure adversely induced lung injury in both in vitro and human studies. First, we confirmed a shift to glycolytic metabolism resulting from mitochondrial dysfunction. This shift was accompanied by an increase in the levels of phosphorylated PDHE1α (S293) and PDK1 and a concomitant marked increase in several key markers of the HIF-1α signaling pathway (HIF-1α, p-PKM2, GLUT1 and HK-2). In addition, utilizing an in vitro model in which lung epithelial cells are cultured with macrophages, we determined that arsenic treatment polarizes macrophages towards the M2 phenotype through lactate. In the human study, the serum lactate and TGF-ß levels were higher in arsenic-exposed subjects than that in reference subjects (t= 4.50, 6.24, both p < 0.05), while FVC and FEV1 were both lower (t= 5.47, 7.59, both p < 0.05). Pearson correlation analyses showed a significant negative correlation between the serum TGF-ß and lactate levels and the lung function parameters (pcorrelation<0.05). In mediation analyses, lactate and TGF-ß significantly mediated 24.3% and 9.0%, respectively, of the association between arsenic and FVC (pmediation<0.05), while lactate and TGF-ß significantly mediated 22.2% and 12.5%, respectively, of the association between arsenic and FEV1 (pmediation<0.05). Together, the results of the in vitro and human studies indicated that there is complex communication between metabolic reprogramming and immune dysfunction, resulting in exacerbated effects in a feedback loop with increased arsenic-induced lung damage.


Assuntos
Arsênico/toxicidade , Metabolismo Energético/efeitos dos fármacos , Lesão Pulmonar/metabolismo , Macrófagos/efeitos dos fármacos , Adulto , Células Epiteliais , Feminino , Humanos , Lesão Pulmonar/etiologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Células THP-1
9.
Anticancer Res ; 40(8): 4707-4710, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727796

RESUMO

BACKGROUND/AIM: Serum-derived macrophage activating factor (serum-MAF) can rapidly activate macrophage phagocytic activity by inducing characteristic membrane ruffles designated as Frill-like structures. Serum-MAF contains γ-globulin, an activator of phagocytosis. This study examined whether serum-MAF and γ-globulin activate macrophages similarly. MATERIALS AND METHODS: Morphological changes in macrophages were observed by time-lapse imaging and the efficiency of engulfment was analysed quantitatively. Immunological staining of talin-1 and a calpain inhibitor were performed. RESULTS: The engulfment efficiency of serum-MAF- and γ-globulin-activated macrophages was significantly different. Talin-1 showed weak co-localisation with the Frill-like structures. Treatment with a calpain inhibitor similarly down-regulated phagocytosis irrespective of the activation factor. CONCLUSION: There was a difference between macrophage activation mechanisms by γ-globulin and serum-MAF. Talin may slightly contribute to serum-MAF activation. It is possible to distinguish between the calpain-dependent fundamental 'mechanism of phagocytosis' and the activating factor-dependent rapid 'activation mechanism'.


Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Fatores Ativadores de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , gama-Globulinas/farmacologia , Calpaína/farmacologia , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Humanos , Fagocitose/efeitos dos fármacos , Células THP-1
10.
Nat Commun ; 11(1): 3484, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661253

RESUMO

Sustainable developments of nanotechnology necessitate the exploration of structure-activity relationships (SARs) at nano-bio interfaces. While ferroptosis may contribute in the developments of some severe diseases (e.g., Parkinson's disease, stroke and tumors), the cellular pathways and nano-SARs are rarely explored in diseases elicited by nano-sized ferroptosis inducers. Here we find that WS2 and MoS2 nanosheets induce an iron-dependent cell death, ferroptosis in epithelial (BEAS-2B) and macrophage (THP-1) cells, evidenced by the suppression of glutathione peroxidase 4 (GPX4), oxygen radical generation and lipid peroxidation. Notably, nano-SAR analysis of 20 transition metal dichalcogenides (TMDs) disclosures the decisive role of surface vacancy in ferroptosis. We therefore develop methanol and sulfide passivation as safe design approaches for TMD nanosheets. These findings are validated in animal lungs by oropharyngeal aspiration of TMD nanosheets. Overall, our study highlights the key cellular events as well as nano-SARs in TMD-induced ferroptosis, which may facilitate the safe design of nanoproducts.


Assuntos
Morte Celular/fisiologia , Endocitose/fisiologia , Ferroptose/fisiologia , Animais , Biomarcadores/metabolismo , Western Blotting , Morte Celular/genética , Linhagem Celular , Sobrevivência Celular/fisiologia , Feminino , Humanos , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Confocal , Nanotecnologia , Relação Estrutura-Atividade , Células THP-1
11.
PLoS One ; 15(7): e0235857, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730263

RESUMO

OBJECTIVE: The risk loci for juvenile idiopathic arthritis (JIA) consist of extended haplotypes that include functional elements in addition to canonical coding genes. As with most autoimmune diseases, the risk haplotypes for JIA are highly enriched for H3K4me1/H3K27ac histone marks, epigenetic signatures that typically identify poised or active enhancers. In this study, we test the hypothesis that genetic risk for JIA is exerted through altered enhancer-mediated gene regulation. METHODS: We mined publically available HiC and other chromatin conformation data to determine whether H3K27ac-marked regions in 25 JIA risk loci showed physical evidence of contact with gene promoters. We also used in vitro reporter assays to establish as proof-of-concept the idea that genetic variants in linkage disequilibrium with GWAS-identified tag SNPs alter enhancer function. RESULTS: All 25 loci examined showed multiple contact sites in the 4 different cell lines that we queried. These regions were characterized by HiC-defined loop structures that included 237 immune-related genes. Using in vitro assays, we found that a 657 bp, H3K4me1/H3K27-marked region within the first intron of IL2RA shows enhancer activity in reporter assays, and this activity is attenuated by SNPs on the IL2RA haplotype that we identified using whole genome sequencing of children with JIA. Similarly, we identified a 1,669 bp sequence in an intergenic region of the IL6R locus where SNPs identified in children with JIA increase enhancer function in reporter assays. CONCLUSIONS: These studies provide evidence that altered enhancer function contributes to genetic risk in JIA. Further studies to identify the specific target genes of genetically altered enhancers are warranted.


Assuntos
Artrite Juvenil/genética , Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Histonas/genética , Polimorfismo de Nucleotídeo Único , Humanos , Células K562 , Locos de Características Quantitativas , Células THP-1
12.
Life Sci ; 258: 118093, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32673666

RESUMO

BACKGROUND: Nuclear factor-kappa B (NF-κB) is an important nuclear transcription factor in cells, involving in a series of processes such as cell proliferation, apoptosis, and differentiation. In this study, we explored the specific mechanism of NF-κB on the differentiation of osteoclasts. METHODS: MicroRNAs (miRNAs) expression microarray data GSE105027 related to osteoarthritis was obtained to screen out the differentially expressed miRNA. Phorbol-12-myristate-13-acetate (PMA) was used to induce THP-1 cells to differentiate into macrophages, followed by induction to osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). ELISA and RT-qPCR were conducted to examine IL-6 and IL-1ß expression. The binding of NF-κB to the miR-1276 promoter region was demonstrated by ChIP assay, and targeting relationship between miR-1276 and MITF was verified by dual luciferase reporter assay. KK, iKBα, NF-kB, p-IKK, p-iKBα, p-NF-kB expression was analyzed by western blot. NF-κB and miR-1276 expression in osteoclasts was examined later. After gain- and less-of-function study, the effects on osteoclast differentiation were detected by TRAP-positive osteoclasts, TRAP activity, TRAP-5b content, F-Actin expression, as well as osteoclast differentiation marker genes expression. RESULTS: NF-κB was activated in osteoclasts, and down-regulation of NF-κB inhibited osteoclast differentiation. Next, miR-1276 was downregulated in osteoclasts after differentiation from monocytes. Meanwhile, NF-κB decreased the expression of miR-1276 by binding to the miR-1276 promoter, thereby elevating MITF expression, thereby promoting osteoclast differentiation. CONCLUSION: In summary, NF-κB promoted osteoclast differentiation through downregulating miR-1276 to upregulate MITF.


Assuntos
Diferenciação Celular , Regulação para Baixo/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , NF-kappa B/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Inativação Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Células THP-1
13.
Arch Virol ; 165(8): 1827-1835, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32507978

RESUMO

Human cytomegalovirus (HCMV) infection causes high morbidity and mortality among immunocompromised patients and can remain in a latent state in host cells. Expression of the immediate-early (IE) genes sustains HCMV replication and reactivation. As a novel genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been extensively utilized to modify and edit genomic DNA. In the present study, the CRISPR/Cas9 system was used to target the IE region of the HCMV genome via specific single-guide RNAs (sgRNAs). Infection with CRISPR/Cas9/sgRNA lentiviral constructs significantly reduced viral gene expression and virion production in HFF primary fibroblasts and inhibited viral DNA production and reactivation in the THP-1 monocytic cell line. Thus, the CRISPR/Cas9/sgRNA system can accurately and efficiently target HCMV replication and reactivation and represents a novel therapeutic strategy against latent HCMV infection.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citomegalovirus/genética , Endonucleases/genética , Genes Virais/genética , Replicação Viral/genética , Linhagem Celular , Infecções por Citomegalovirus/virologia , DNA Viral/genética , Fibroblastos/virologia , Edição de Genes/métodos , Expressão Gênica/genética , Células HEK293 , Humanos , RNA Guia/genética , Células THP-1
14.
Life Sci ; 256: 117925, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522570

RESUMO

AIMS: This study aims to explore the effect and underlying mechanism of zoledronic acid (ZA) on the incidence of thyroid cancer (TC) tumorigenesis. MATERIALS AND METHODS: Human mononuclear cells THP-1 were differentiated into M2-like tumor associated macrophages (TAMs) by incubation with PMA followed by additional incubation of IL-4 and IL-13. TC cells TPC-1 and IHH4 were co-cultured with M2-like TAMs. Identification of M2-like TAMs markers were determined by immunohistochemistry or flow cytometry. Cell proliferation, stemness and migration/invasion ability were measured by colony, sphere formation assay and transwell assay, respectively. The expression levels of cell stemness, EMT and Wnt/ß-catenin pathway-related factors were verified by qRT-PCR, Western blotting, and immunofluorescence. A subcutaneous tumor model was established in nude mice to examine the in vivo effects of ZA. KEY FINDINGS: M2-like TAMs were enriched in TC tissues, and they promoted the colony/sphere formation, accompanied with a down-regulated expression in E-cadherin and an up-regulated expression in N-cadherin, Vimentin and other stemness-associated markers (CD133, Oct4, c-Myc) in TC cells. The effects were suppressed when ZA co-treatment was given, because ZA inhibited the polarization of M2-like TAMs and ß-catenin entry into the nucleus. Moreover, in agreement with in vitro data, ZA also limited subcutaneous tumor formation and macrophage enrichment in nude mice. SIGNIFICANCE: ZA suppressed M2-like TAMs induced TC cell proliferation, stemness and metastasis through inhibiting M2-like TAMs polarization and Wnt/ß-catenin pathway, which sheds light on the mechanisms of TC and provides avenues for the development of clinical therapy to TC.


Assuntos
Macrófagos/metabolismo , Neoplasias da Glândula Tireoide/tratamento farmacológico , Ácido Zoledrônico/farmacologia , beta Catenina/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Carcinogênese/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Transdução de Sinais , Células THP-1/metabolismo , Microambiente Tumoral , Via de Sinalização Wnt
15.
Nanotoxicology ; 14(7): 929-946, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32538272

RESUMO

Multi-walled carbon nanotubes (MWCNTs) are known to induce pulmonary inflammatory effects through stimulating pro-inflammatory cytokine secretion from alveolar macrophages. Despite extensive studies on MWCNTs' pro-inflammatory reactivity, the understanding of molecular mechanisms involved is still incomplete. In this study, we investigated hemichannel's involvement in MWCNTs-induced macrophage IL-1ß release. Our results showed that the unmodified and COOH MWCNTs could induce ATP release and ATP-P2X7R axis-dependent IL-1ß secretion from THP-1 macrophages. By using various inhibitors, we confirmed that the MWCNTs-induced ATP release was primarily through hemichannels. EtBr dye uptake assay detected significant hemichannels opening in MWCNTs exposed THP-1 macrophages. Inhibition of hemichannels by CBX, 43Gap27, or 10Panx1 pretreatment results in decreased ATP and IL-1ß release. The addition of ATP restored the reduced IL-1ß secretion level from hemichannel inhibition. We also confirmed with five other types of MWCNTs that the induction of hemichannels by MWCNTs strongly correlates with their capacity to induce IL-1ß secretion. Taken together, we conclude that hemichannels-mediated ATP release and subsequent NLRP3 inflammasome activation through P2X7R may be one mechanism by which MWCNTs induce macrophage IL-1ß secretion. Our findings may provide a novel molecular mechanism for MWCNTs induced IL-1ß secretion.


Assuntos
Trifosfato de Adenosina/metabolismo , Conexinas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Proteínas do Tecido Nervoso/metabolismo , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nanotubos de Carbono/química , Células THP-1
16.
Nat Immunol ; 21(7): 727-735, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541831

RESUMO

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.


Assuntos
Herpes Simples/imunologia , Proteínas de Membrana/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Animais , Carbolinas/farmacologia , Células Cultivadas , DNA Viral/imunologia , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Feminino , Fibroblastos , Complexo de Golgi/metabolismo , Células HEK293 , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Homeostase/imunologia , Humanos , Imunidade Inata , Peroxidação de Lipídeos/genética , Peroxidação de Lipídeos/imunologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Nucleotidiltransferases/metabolismo , Oxirredução , Oximas/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Cultura Primária de Células , Carbonilação Proteica/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sulfonamidas/farmacologia , Células THP-1 , Replicação Viral/imunologia
17.
Nanotoxicology ; 14(7): 947-967, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32574520

RESUMO

Multi-walled carbon nanotubes (MWCNTs) are one of the most widely used types of novel nano-fiber materials. The aim of this study was to establish an experimental system based on actual exposure dosage and environments and explore the roles and mechanisms of inflammation in the malignant transformation of pleural mesothelial cells induced by MWCNTs after low doses and long-term exposure. Here, we established an in vitro system by co-culturing macrophages and mesothelial cells and exposing these cells to high aspect ratio MWCNTs (0.1 µg/mL) for three months. Results indicated that IL-1ß, secreted by macrophages stimulated by MWCNTs, may significantly enhance the release of inflammatory cytokines, such as IL-8, TNF-α, and IL-6, from mesothelial cells. Results obtained from proliferation, migration, invasion, colony formation, and chromosomal aberration studies indicated that MWCNTs may promote malignant transformation of mesothelial cells after long-term and low-dose exposure via inflammation. Furthermore, the obtained results demonstrated that the NF-κB/IL-6/STAT3 pathway was active in the malignant transformation of Met 5A cells, induced by MWCNTs, and played an important role in the process. In conclusion, our results showed that the NF-κB (p65)/IL-6/STAT3 molecular pathway, which was mediated by inflammation, played an important role in the malignant transformation of pleural mesothelial cells induced by MWCNTs. These findings also provide novel ideas and references for the treatment of mesothelioma and offers options for the occupational safety of nanomaterial practitioners.


Assuntos
Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Neoplasias Pulmonares/imunologia , Macrófagos/efeitos dos fármacos , Mesotelioma/imunologia , Nanotubos de Carbono/toxicidade , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Células Epiteliais/imunologia , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Humanos , Inflamação , Interleucina-1beta/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/imunologia , Macrófagos/patologia , Mesotelioma/metabolismo , Mesotelioma/patologia , Células THP-1 , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
18.
Food Chem ; 330: 127257, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32535321

RESUMO

Huangshui (HS), the by-product of Chinese Baijiu, has attracted considerable attention due to its nutrient and microbial composition; however, none of the studies has explored the polysaccharides in HS yet. Here, from HS, we isolated a novel polysaccharide, HSP-3, with an average molecular weight of 26.40 kDa. The structure was elucidated based on monosaccharide composition and methylation analysis, NMR, FT-IR, and AFM analysis. It is mainly composed of mannose (46.6%), galactose (17.3%), arabinose (11.2%), glucose (10.5%), xylose (8.2%), fucose (5.2%), and rhamnose (1.0%). The backbone of HSP-3 was made up of â†’ 2)-ß-d-Manp-(1 â†’ 2,6)-ß-d-Manp-(1 â†’ 6)-ß-d-Galp-(1 â†’ 3,6)-ß-d-Galp-(1 â†’ 4)-α-l-Rhap-(1 â†’ 3,4)-α-l-Rhap-(1 â†’ . Moreover, stimulation of the production of ROS, NO, TNF-α and IL-6, upregulation of the mRNA and protein expression levels of TNF-α and IL-6 in THP-1 cells, and enhanced the pinocytic and phagocytic capacities of THP-1 cells exhibited significant immunomodulatory properties of HSP-3. Altogether, this study suggests that HSP-3 could be used as an active component in functional foods.


Assuntos
Imunossupressores/farmacologia , Interleucina-6/metabolismo , Óxido Nítrico/biossíntese , Polissacarídeos/farmacologia , Rios/química , Fator de Necrose Tumoral alfa/metabolismo , Humanos , Imunossupressores/química , Peso Molecular , Polissacarídeos/química , Células THP-1
19.
PLoS Pathog ; 16(6): e1008611, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511263

RESUMO

Human infection with avian influenza A (H5N1) and (H7N9) viruses causes severe respiratory diseases. PB1-F2 protein is a critical virulence factor that suppresses early type I interferon response, but the mechanism of its action in relation to high pathogenicity is not well understood. Here we show that PB1-F2 protein of H7N9 virus is a particularly potent suppressor of antiviral signaling through formation of protein aggregates on mitochondria and inhibition of TRIM31-MAVS interaction, leading to prevention of K63-polyubiquitination and aggregation of MAVS. Unaggregated MAVS accumulated on fragmented mitochondria is prone to degradation by both proteasomal and lysosomal pathways. These properties are proprietary to PB1-F2 of H7N9 virus but not shared by its counterpart in WSN virus. A recombinant virus deficient of PB1-F2 of H7N9 induces more interferon ß in infected cells. Our findings reveal a subtype-specific mechanism for destabilization of MAVS and suppression of interferon response by PB1-F2 of H7N9 virus.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Subtipo H7N9 do Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Agregação Patológica de Proteínas/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cães , Células HEK293 , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/patologia , Interferon beta/genética , Interferon beta/metabolismo , Células Madin Darby de Rim Canino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Agregação Patológica de Proteínas/genética , Células THP-1 , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética
20.
Life Sci ; 256: 117894, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502544

RESUMO

AIMS: Pathological alterations in the brain can cause microglial activation (MA). Thus, inhibiting MA could provide a new approach for treating neurodegenerative disorders. MAIN METHODS: To investigate the effect of C16 peptide and angiopoietin-1 (Ang1) on inflammation following MA, we stimulated microglial BV-2 cells with lipopolysaccharide (LPS) and used dexmedetomidine (DEX) as a positive control. Specific inhibitors of Tie2, αvß3 and α5ß1 integrins, and PI3K/Akt were applied to investigate the neuron-protective and anti-inflammatory effects and signaling pathway of C16 + Ang1 treatment in the LPS-induced BV-2 cells. KEY FINDINGS: Our results showed that C16 + Ang1 treatment reduced the microglia M1 phenotype but promoted the microglia M2 phenotype. In addition, C16 + Ang1 treatment suppressed leukocyte migration across human pulmonary microvascular endothelial cells, reduced the levels of pro-inflammatory factors [inducible nitric oxide synthase (iNOS), interleukin (IL)-1ß, tumor necrosis factor (TNF-α)], and cellular apoptosis factors (caspase-3 and p53), and decreased lactate dehydrogenase (LDH) release, but promoted anti-inflammatory cytokine (IL-10) expression and cell proliferation in the LPS-activated BV-2 cells. The signaling pathways underlying the neuron-protective and anti-inflammatory effects of C16 + Ang1 may be mediated by Tie2-PI3K/Akt, Tie2-integrin and integrin-PI3K/Akt. SIGNIFICANCE: The neuron-protective and anti-inflammatory effects of C16 + Ang1 treatment included M1 to M2 microglia phenotype switching, blocking leukocyte transmigration, decreasing apoptotic and inflammatory factors, and promoting cellular viability.


Assuntos
Angiopoietina-1/farmacologia , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Microglia/patologia , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Pulmão/irrigação sanguínea , Camundongos , Microglia/efeitos dos fármacos , Microvasos/patologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor TIE-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1
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