RESUMO
Monocytes and macrophages that originate from common myeloid progenitors perform various crucial roles in the innate immune system. Stimulation with LPS combined with TLR4 drives the production of pro-inflammatory cytokines through MAPKs and NF-κB pathway in different cells. However, the difference in LPS susceptibility between monocytes and macrophages is poorly understood. In this study, we found that pro-inflammatory cytokines-IL-1ß, IL-6 and TNFα showed greater induction in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells than in THP-1 cells. To determine the difference in cytokine expression, the surface proteins such as TLR4-related proteins and intracellular adaptor proteins were more preserved in PMA-differentiated THP-1 cells than in THP-1 cells. MyD88 is a key molecule responsible for the difference in LPS susceptibility. Moreover, MAPKs and NF-κB pathway-related molecules showed higher levels of phosphorylation in PMA-differentiated THP-1 cells than in THP-1 cells. Upon MyD88 depletion, there was no difference in the phosphorylation of MAPK pathway-related molecules. Therefore, these results demonstrate that the difference in LPS susceptibility between THP-1 cells and PMA-differentiated THP-1 cells occur as a result of gap between the activated MAPKs and NF-κB pathways via changes in the expression of LPS-related receptors and MyD88.
Assuntos
Lipopolissacarídeos , Células THP-1 , Receptor 4 Toll-Like , Citocinas/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Receptor 4 Toll-Like/metabolismoRESUMO
Adhesion of monocytes to endothelial cells is an important initiating step in atherogenesis. One of the most abundant flavonoids in the diet, quercetin has been reported to inhibit monocyte adhesion to endothelial cells. However, it is poorly absorbed in the upper gastrointestinal tract during oral intake but rather is metabolized by the intestinal microbiota into various phenolic acids. Since the biological properties of the microbial metabolites of quercetin remain largely unknown, herein, we investigated how the microbial metabolite of quercetin, 3-(3-hydroxyphenyl)propionic acid (3HPPA) impact monocyte adhesion to endothelial cells. Direct treatment with 3HPPA for 24 h was not cytotoxic to human aortic endothelial cells (HAECs). Cotreatment with 3HPPA inhibited tumor necrosis factor α (TNFα)-induced adhesion of THP-1 monocytes to HAECs, and suppressed the upregulation of cell adhesion molecule E-selectin but not intercellular adhesion molecule 1 or vascular cell adhesion molecule 1. Furthermore, 3HPPA was found to inhibit TNFα-induced nuclear translocation and phosphorylation of the p65 subunit of nuclear factor κB (NF-κB). We conclude that 3HPPA mitigates the adhesion of monocytes to endothelial cells by suppressing the expression of the cell adhesion molecule E-selectin in HAECs via inhibition of the NF-κB pathway, providing additional evidence for the health benefits of dietary flavonoids and their microbial metabolites as therapeutic agents in atherosclerosis.
Assuntos
Aterosclerose/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Propionatos/metabolismo , Quercetina/metabolismo , Células THP-1/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , HumanosRESUMO
BACKGROUND: The tyrosinase inhibitor rhododendrol (RD), used as a skin whitening agent, reportedly has the potential to induce leukoderma. OBJECTIVE: Although an immune response toward melanocytes was demonstrated to be involved in leukoderma, the molecular mechanism is not fully understood. METHODS: We hypothesized that if RD is a pro-hapten and tyrosinase-oxidized RD metabolites are melanocyte-specific sensitizers, the sensitizing process could be reproduced by the human cell line activation test (h-CLAT) cocultured with melanocytes (h-CLATw/M) composed of human DC THP-1 cells and melanoma SK-MEL-37 cells. Cell surface expression, ROS generation and ATP release, mRNA expression, and the effects of several inhibitors were examined. RESULTS: When RD was added to the h-CLATw/M, the expression of cell-surface CD86 and IL-12 mRNA was greatly enhanced in THP-1 cells compared with those in the h-CLAT. The rapid death of melanoma cells was induced, with ROS generation and ATP release subsequently being greatly enhanced, resulting in the cooperative upregulation of CD86 and IL-12. Consistent with those observations, an ROS inhibitor, ATP receptor P2X7 antagonist, or PERK inhibitor antagonized the upregulation. CD86 upregulation was similarly observed with another leukoderma-inducible tyrosinase inhibitor, raspberry ketone, but not with the leukoderma noninducible skin-whitening agents ascorbic acid and tranexamic acid. CONCLUSION: RD is a pro-hapten sensitizer dependent on tyrosinase that induces ROS generation and ATP release from melanocytes for CD86 and IL-12 upregulation in DCs, possibly leading to the generation of tyrosinase-specific cytotoxic T lymphocytes. The coculture system h-CLATw/M may be useful for predicting the sensitizing potential to induce leukoderma.
Assuntos
Antígeno B7-2 , Butanóis , Hipopigmentação , Preparações Clareadoras de Pele , Humanos , Trifosfato de Adenosina/metabolismo , Técnicas de Cocultura , Hipopigmentação/metabolismo , Interleucina-12/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Preparações Clareadoras de Pele/farmacologia , Células THP-1/efeitos dos fármacos , Regulação para Cima , Antígeno B7-2/metabolismo , Butanóis/farmacologiaRESUMO
BACKGROUND/AIM: Effective ex vivo maturation of dendritic cells (DCs) can increase the efficiency of cancer immunotherapy. We aimed to identify novel chemicals with the potential to differentiate and activate immature DCs (iDCs) to mature DCs (mDCs). MATERIALS AND METHODS: The expression of surface markers on THP-1 monocytes treated with the screened compounds was analyzed using FACS. Subsequent DC subset analysis and secreted cytokine profiling were also performed. RESULTS: FACS analysis showed that THP-1 cells treated with amsacrine hydrochloride, a DNA topoisomerase II inhibitor, exhibited the typical phenotype of conventional DCs (cDCs). The expression of DC activation markers was also increased after amsacrine treatment. The profile of cytokines produced by THP-1 cells treated with amsacrine was similar to that of mDCs. CONCLUSION: Amsacrine has an ex vivo capability of differentiating THP-1 monocytes into cDCs. As amsacrine has been used as a stable chemotherapeutic agent in humans, it can be useful for producing mDCs for cancer immunotherapy.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células THP-1/efeitos dos fármacos , Inibidores da Topoisomerase II/uso terapêutico , Diferenciação Celular , Humanos , Inibidores da Topoisomerase II/farmacologiaRESUMO
Uric acid is an effective antioxidant. Oxidized low-density lipoprotein (ox-LDL) is derived from circulating LDL and promotes atherosclerosis. The Keap1-Nrf2-ARE pathway is a key body pathway involved in protection against internal and external oxidative damages. The role of uric acid on vascular endothelial function damaged by ox-LDL, and its effect on the Keap1-Nrf2-ARE pathway has not been fully explored. HUVECs were treated with different concentrations of uric acid and ox-LDL to explore the effect of uric acid in vitro. Cell phenotype was determined by cytometry and Western blot. Nuclear translocation of Nrf2 was determined by immunofluorescence. Coimmunoprecipitation was used to determine the level of Nrf2 ubiquitination. A microfluidic device was used to mimic the vascular environment in the body, and the level of mRNA levels of inflammatory factors was determined by RT-PCR. The findings of this study show that suitable uric acid can significantly reduce endothelial damage caused by ox-LDL, such as oxidative stress, inflammation, and increased adhesion. In addition, uric acid reduced Nrf2 ubiquitination and increased nuclear translocation of Nrf2 protein, thus activating the Keap1-Nrf2-ARE pathway and playing a protective role. Interestingly, the effects of UA were significantly inhibited by administration of Brusatol, an inhibitor of Nrf2. In summary, suitable concentrations of uric acid can alleviate the oxidative stress level of endothelial cells through Nrf2 nuclear translocation and further protect cells from damage.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lipoproteínas LDL/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Substâncias Protetoras/farmacologia , Ácido Úrico/farmacologia , Antioxidantes/metabolismo , Aterosclerose/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The use of the plasticizer bis(2-ethylhexyl)phthalate (DEHP) and other plasticizers in the manufacture of plastic products has been restricted due to adverse health outcomes such as obesity, metabolic syndrome, and asthma, for which inflammation has been described to be a driving factor. The emerging alternative plasticizer 1,2-cyclohexanedioic acid diisononyl ester (DINCH) still lacks information regarding its potential effects on the immune system. Here, we investigated the effects of DINCH and its naturally occurring metabolite monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) on the innate immune response. Human THP-1 macrophages were exposed to 10 nM-10 µM DINCH or MINCH for 4 h, 16 h, and 24 h. To decipher the underlying mechanism of action, we applied an untargeted proteomic approach that revealed xenobiotic-induced activation of immune-related pathways such as the nuclear factor κB (NF-κB) signaling pathway. Key drivers were associated with oxidative stress, mitochondrial dysfunction, DNA damage repair, apoptosis, and autophagy. We verified increased reactive oxygen species (ROS) leading to cellular damage, NF-κB activation, and subsequent TNF and IL-1ß release, even at low nM concentrations. Taken together, DINCH and MINCH induced cellular stress and pro-inflammatory effects in macrophages, which may lead to adverse health effects.
Assuntos
Ácidos Dicarboxílicos/metabolismo , Ésteres/metabolismo , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Plastificantes/farmacologia , Células THP-1/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Macrófagos/metabolismo , NF-kappa B/metabolismo , Ácidos Ftálicos/metabolismo , Proteômica , Transdução de Sinais/efeitos dos fármacos , Células THP-1/metabolismoRESUMO
The soybean allergen Gly m 4 is known to cause severe allergic reactions including anaphylaxis, unlike other Bet v 1 homologues, which induce mainly local allergic reactions. In the present study, we aimed to investigate whether the food Bet v 1 homologue Gly m 4 can be a sensitizer of the immune system. Susceptibility to gastrointestinal digestion was assessed in vitro. Transport through intestinal epithelium was estimated using the Caco-2 monolayer. Cytokine response of different immunocompetent cells was evaluated by using Caco-2/Immune cells co-culture model. Absolute levels of 48 cytokines were measured by multiplex xMAP technology. It was shown that Gly m 4 can cross the epithelial barrier with a moderate rate and then induce production of IL-4 by mature dendritic cells in vitro. Although Gly m 4 was shown to be susceptible to gastrointestinal enzymes, some of its proteolytic fragments can selectively cross the epithelial barrier and induce production of Th2-polarizing IL-5, IL-10, and IL-13, which may point at the presence of the T-cell epitope among the crossed fragments. Our current data indicate that Gly m 4 can potentially be a sensitizer of the immune system, and intercommunication between immunocompetent and epithelial cells may play a key role in the sensitization process.
Assuntos
Antígenos de Plantas/farmacologia , Hipersensibilidade Alimentar/terapia , Imunização/métodos , Antígenos de Plantas/imunologia , Células CACO-2/efeitos dos fármacos , Células CACO-2/imunologia , Quimiocinas/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , Escherichia coli/metabolismo , Hipersensibilidade Alimentar/imunologia , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Biológicos , Organismos Geneticamente Modificados , Células THP-1/efeitos dos fármacos , Células THP-1/imunologiaRESUMO
Platelets can modulate cancer through budding of platelet microparticles (PMPs) that can transfer a plethora of bioactive molecules to cancer cells upon internalization. In acute myelogenous leukemia (AML) this can induce chemoresistance, partially through a decrease in cell activity. Here we investigated if the internalization of PMPs protected the monocytic AML cell line, THP-1, from apoptosis by decreasing the initial cellular damage inflicted by treatment with daunorubicin, or via direct modulation of the apoptotic response. We examined whether PMPs could protect against apoptosis after treatment with a selection of inducers, primarily associated with either the intrinsic or the extrinsic apoptotic pathway, and protection was restricted to the agents targeting intrinsic apoptosis. Furthermore, levels of daunorubicin-induced DNA damage, assessed by measuring gH2AX, were reduced in both 2N and 4N cells after PMP co-incubation. Measuring different BCL2-family proteins before and after treatment with daunorubicin revealed that PMPs downregulated the pro-apoptotic PUMA protein. Thus, our findings indicated that PMPs may protect AML cells against apoptosis by reducing DNA damage both dependent and independent of cell cycle phase, and via direct modulation of the intrinsic apoptotic pathway by downregulating PUMA. These findings further support the clinical relevance of platelets and PMPs in AML.
Assuntos
Apoptose/fisiologia , Micropartículas Derivadas de Células/fisiologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Daunorrubicina/farmacologia , Células THP-1/fisiologia , Apoptose/efeitos dos fármacos , Plaquetas , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismoRESUMO
Envenomation by the Loxosceles genus spiders is a recurring health issue worldwide and specially in the Americas. The physiopathology of the envenomation is tightly associated to the venom's rich toxin composition, able to produce a local dermonecrotic lesion that can evolve systemically and if worsened, might result in multiple organ failure and lethality. The cellular and molecular mechanisms involved with the physiopathology of Loxoscelism are not completely understood, however, the venom's Phospholipases D (PLDs) are known to trigger membrane injury in various cell types. Here, we report for the first time the Loxosceles venom's ability to stimulate the production of extracellular vesicles (EVs) in various human cell lineages. Components of the Loxosceles venom were also detectable in the cargo of these vesicles, suggesting that they may be implicated in the process of extracellular venom release. EVs from venom treated cells exhibited phospholipase D activity and were able to induce in vitro hemolysis in human red blood cells and alter the HEK cell membranes' permeability. Nonetheless, the PLD activity was inhibited when an anti-venom PLDs monoclonal antibody was co-administered with the whole venom. In summary, our findings shed new light on the mechanisms underlying cellular events in the context of loxoscelism and suggest a crucial role of EVs in the process of envenomation.
Assuntos
Células Cultivadas/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Células HEK293/efeitos dos fármacos , Picada de Aranha/fisiopatologia , Venenos de Aranha/metabolismo , Venenos de Aranha/toxicidade , Células THP-1/efeitos dos fármacos , Animais , HumanosRESUMO
Naringenin (Nar) is a flavanone that has been suggested to provide human health benefits such as anti-inflammatory, anti-oxidant and anti-cancer properties. However, the mechanisms underlying these benefits are complex and still not fully understood. In this study, we investigated the effect of Nar on the inflammatory response of macrophages and its underlying mechanism. In lipopolysaccharide (LPS)-stimulated human macrophages, Nar inhibited the activation of NF-κB pathway and suppressed the downstream expression of pro-inflammatory factors. In addition, Nar was also able to induce metallothionein 1 G (MT1G) expression, and the inhibitory effects of Nar on the production of pro-inflammatory cytokines was dependent on MT1G. Mechanistically, we found that MT1G-mediated inhibition of pro-inflammatory cytokines responses might be through repressing NF-κB activation via zinc chelation. Overall, this study reveals a novel mechanism of Nar on inflammatory responses, the suppression of NF-κB activation through upregulation of MT1G.
Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/metabolismo , Flavanonas/farmacologia , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Antioxidantes/farmacologia , Células Cultivadas , Células HEK293 , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Metalotioneína/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing (NLRP) 3 inflammasome is a multiprotein complex that triggers Caspase-1-mediated IL-1ß production and pyroptosis, and its dysregulation is associated with the pathogenesis of inflammatory diseases. 1'-Acetoxychavicol acetate (ACA) is a natural compound in the rhizome of tropical ginger Alpinia species with anti-microbial, anti-allergic and anti-cancer properties. In this study, we found that ACA suppressed NLRP3 inflammasome activation in mouse bone marrow-derived macrophages and human THP-1 monocytes. ACA inhibited Caspase-1 activation and IL-1ß production by NLRP3 agonists such as nigericin, monosodium urate (MSU) crystals, and ATP. Moreover, it suppressed oligomerization of the adapter molecule, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1-mediated cleavage of pyroptosis executor Gasdermin D. Mechanistically, ACA inhibited generation of mitochondrial reactive oxygen species (ROS) and prevented release of oxidized mitochondrial DNA, which trigger NLRP3 inflammasome activation. ACA also prevented NLRP3 inflammasome activation in vivo, as evidenced in the MSU crystal-induced peritonitis and dextran sodium sulfate-induced colitis mouse models accompanied by decreased Caspase-1 activation. Thus, ACA is a potent inhibitor of the NLRP3 inflammasome for prevention of NLRP3-associated inflammatory diseases.
Assuntos
Álcoois Benzílicos/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Caspase 1/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Peritonite/tratamento farmacológico , Peritonite/metabolismo , Fagocitose/efeitos dos fármacos , Piroptose/efeitos dos fármacos , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismoRESUMO
BACKGROUND: Monosodium urate (MSU)-mediated inflammatory response is a crucial inducing factor in gouty arthritis. Here, we explored the underlying mechanism of total glucosides of paeony (TGP) in MSU-induced inflammation of THP-1 macrophages in gouty arthritis. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability. Enzyme-linked immunosorbent assay (ELISA) was utilized to measure the production of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α). Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were conducted to determine RNA and protein expression. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull down assay were used to confirm the interaction between miR-876-5p and MALAT1 or NLR family pyrin domain containing 3 (NLRP3). RESULTS: MSU-induced damage and inflammatory response in THP-1 macrophages were alleviated by the treatment of TGP in a dose-dependent manner. Overexpression of NLRP3 or MALAT1 reversed the protective effects of TGP in MSU-induced THP-1 macrophages. The binding relation between miR-876-5p and MALAT1 or NLRP3 was identified in THP-1 macrophages. MALAT1 up-regulated the expression of NLRP3 by sponging miR-876-5p in THP-1 macrophages. TGP suppressed MSU-induced inflammation in THP-1 macrophages through regulating MALAT1/miR-876-5p/NLRP3 axis. TGP suppressed MSU-induced activation of TLR4/MyD88/NF-κB pathway through regulating MALAT1/miR-876-5p/NLRP3 axis. CONCLUSION: In conclusion, TGP suppressed MSU-induced inflammation in THP-1 macrophages through regulating MALAT1/miR-876-5p/NLRP3 axis and TLR4/MyD88/NF-κB pathway, suggesting that TGP was a promising active ingredient for gouty arthritis treatment.
Assuntos
Artrite Gotosa/metabolismo , Glucosídeos/uso terapêutico , MicroRNAs/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Paeonia , RNA Longo não Codificante/metabolismo , Ácido Úrico/toxicidade , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/prevenção & controle , Glucosídeos/isolamento & purificação , Glucosídeos/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismoRESUMO
Thrombocytopenia is common among patients with viral hepatitis, limiting the use of antiviral therapy. Eltrombopag (EP) is a thrombopoietin receptor (TPO-R) agonist that has been approved for treatment of immune thrombocytopenia patients with hepatitis virus infection. Interferon-α (IFN-α) plays a crucial role in the antiviral response, and is recommended as the first-line agent for chronic hepatitis B patients. Here, we investigated whether EP inhibits the production of IFN-stimulated genes (ISGs) induced by IFN-α through the TPO-R-independent pathway by mediating reactive oxygen species production by iron chelation. Our results assessed the inhibitory effect of EP on IFN-α signaling, which contributes to the downregulation of ISGs produced by monocytes and sheds light on the underlying mechanisms using iron chelation to treat patients with hepatitis-related immunological thrombocytopenia.
Assuntos
Antivirais/metabolismo , Benzoatos/farmacologia , Hidrazinas/farmacologia , Interferon-alfa/metabolismo , Ferro/metabolismo , Leucócitos Mononucleares/metabolismo , Pirazóis/farmacologia , Adulto , Animais , Antivirais/antagonistas & inibidores , Benzoatos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/metabolismo , Humanos , Hidrazinas/uso terapêutico , Interferon-alfa/antagonistas & inibidores , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Pirazóis/uso terapêutico , Receptores de Trombopoetina/agonistas , Receptores de Trombopoetina/metabolismo , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismo , Trombocitopenia/tratamento farmacológico , Trombocitopenia/metabolismoRESUMO
Macrophages are essential immune cells of the innate immune system. They participate in the development and regulation of inflammation. Macrophages play a fundamental role in fighting against bacterial infections by phagocytosis of bacteria, and they also have a specific role in immunomodulation by secreting pro-inflammatory cytokines. In bacterial infection, macrophages decrease the serum iron concentration by removing iron from the blood, acting as one of the most important regulatory cells of iron homeostasis. We examined whether the Gram-positive and Gram-negative cell wall components from various bacterial strains affect the cytokine production and iron transport, storage and utilization of THP-1 monocytes in different ways. We found that S. aureus lipoteichoic acid (LTA) was less effective in activating pro-inflammatory cytokine expression that may related to its effect on fractalkine production. LTA-treated cells increased iron uptake through divalent metal transporter-1, but did not elevate the expression of cytosolic and mitochondrial iron storage proteins, suggesting that the cells maintained iron efflux via the ferroportin iron exporter. E. coli and P. aeruginosa lipopolysaccharides (LPSs) acted similarly on THP-1 cells, but the rates of the alterations of the examined proteins were different. E. coli LPS was more effective in increasing the pro-inflammatory cytokine production, meanwhile it caused less dramatic alterations in iron metabolism. P. aeruginosa LPS-treated cells produced a smaller amount of pro-inflammatory cytokines, but caused remarkable elevation of both cytosolic and mitochondrial iron storage proteins and intracellular iron content compared to E. coli LPS. These results prove that LPS molecules from different bacterial sources alter diverse molecular mechanisms in macrophages that prepossess the outcome of the bacterial infection.
Assuntos
Parede Celular/química , Citocinas/metabolismo , Escherichia coli/química , Ferro/metabolismo , Lipopolissacarídeos/farmacologia , Pseudomonas aeruginosa/química , Staphylococcus aureus/química , Células THP-1/metabolismo , Ácidos Teicoicos/farmacologia , Transporte Biológico , Receptor 1 de Quimiocina CX3C/biossíntese , Receptor 1 de Quimiocina CX3C/genética , Quimiocina CX3CL1/metabolismo , Citocinas/biossíntese , Citosol/metabolismo , Ferritinas/biossíntese , Ferritinas/genética , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Hepcidinas/biossíntese , Hepcidinas/genética , Humanos , Mitocôndrias/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Oxirredutases/biossíntese , Oxirredutases/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/genética , Células THP-1/efeitos dos fármacosRESUMO
The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay is one of the most commonly used tests of cell proliferation. Hydralazine has been reported to interfere with the performance of the MTS assay when used on adherent cells. This study aimed to investigate whether hydralazine interferes with the performance of the MTS assay on suspended cells. THP-1 (a monocytic leukemia cell line) cells were cultured in the presence or absence of hydralazine (0, 10, 50, 100, and 500 µM) for 2 or 24 h. Cell numbers were analyzed using the MTS, trypan blue exclusion, or microscopic assays. A modified version of the standard MTS assay was established by centrifuging the cells and replacing the test medium with fresh culture medium immediately before the addition of the MTS reagent. Culture of THP-1 cells with hydralazine at concentrations of 50, 100, and 500 µM for 2 h increased absorbance (p < 0.001) in the standard MTS assay, whereas both the trypan blue exclusion assay and microscopy suggested no change in cell numbers. Culture of THP-1 cells with 100 and 500 µm hydralazine for 24 h increased absorbance (p < 0.05) in the standard MTS assay; however, trypan blue exclusion and microscopy suggested a decrease in cell numbers. In a cell-free system, hydralazine (100 and 500 µM) increased absorbance in a time- and concentration-dependent manner. The modified MTS assay produced results consistent with trypan blue exclusion and microscopy using THP-1 cells. In addition, the modified MTS assay produced reliable results when K562 and Jurkat cells were incubated with hydralazine or ß-mercaptoethanol (ßME). In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and ßME when assessing suspended cells.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hidralazina/farmacologia , Mercaptoetanol/farmacologia , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Sistema Livre de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células Jurkat/efeitos dos fármacos , Microscopia , Células THP-1/efeitos dos fármacosRESUMO
BACKGROUND: Smoking induces excessive inflammation which is associated with all the stages of atherosclerosis. Earlier, we reported Nod-like receptor protein 3 (NLRP3) inflammasome activation as a pro-atherosclerotic property of cigarette smoking. In the present study, we aimed to explore the underlying detailed upstream mechanism and the cellular status of putative downstream molecules of cigarette smoke condensate (CSC)-activated NLRP3 inflammasome in atherosclerotic disease. METHODS AND RESULTS: THP-1 monocytes, macrophages and foam cells represent crucial stages of atherogenesis as initiation, progression and development. To determine the upstream molecular regulators of smoking-induced NLRP3 inflammasome in atherogenesis, Myeloid differentiation primary response 88/Nuclear Factor kappa-light-chain-enhancer of activated B cells (MyD88/NF-κB) and Suppressor of cytokine signaling 3/Signal transduction and activator of transcription 3 (SOCS3/STAT3) pathways were elucidated. Stage-specific THP-1 cells were treated with MyD88 and SOCS3/STAT3 inhibitors. The results showed that MyD88 inhibition markedly attenuated the expression of NLRP3 markers (NLRP3, caspase-1, Interleukin (IL)-1ß and IL-18), IL-6, SOCS3 and NF-κB. Moreover, the secretory levels of pro-cytokines were also significantly reduced in culture media. In contrast, no changes were observed with SOCS3/STAT3 inhibitor. Further, ac-vyad-cmk, an inflammasome inhibitor was used to explore the downstream targets of CSC-activated NLRP3 inflammasome in atherosclerotic process. The transcriptional profiling of 25 atherosclerotic markers was carried out using ExProfile™ Custom Gene qPCR Arrays. CSC exposure upregulated the expression of 17 genes and downregulated 4 genes in a stage-specific manner. Inhibitory experiments showed aberrant changes in CSC-regulated genes. Altogether, 15 molecules were common in all three stages. CONCLUSION: The findings may suggest that MyD88/NF-κB pathway is an upstream regulator of NLRP3 inflammasome underlying smoking-induced atherosclerosis. Notably, 15 atherosclerotic molecules associated with endothelial dysfunction, scavenger receptors, cholesterol esterification and matrix-metalloproteins were found downstream to CSC-activated NLRP3 inflammasome.
Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nicotiana/química , Fumaça/análise , Células THP-1/efeitos dos fármacos , Aterosclerose , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/antagonistas & inibidores , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Células THP-1/metabolismoRESUMO
Osteopontin (OPN) mediates bone remodeling and tissue debridement. The OPN protein is cleaved, but it is unclear how full-length (FL)-OPN or its cleaved form perform their biological activities in target cells. We, therefore, performed the molecular characterization of OPN in exosomes (Exo). The Exo were isolated from lipopolysaccharide (LPS)-stimulated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages. The Exo were also isolated from PMA-differentiated THP-1 macrophages. The Exo were identified using the qNano multiple analyzer (diameter 59-315 nm) and western blotting with a CD9 antibody. LPS-stimulated cells produced more particles than non-stimulated cells. The presence of the FL or the cleaved form of OPN was confirmed using western blot analysis. A mixture of FL and cleaved OPN was also measured using an ELISA system (Ud-OPN) and their presence in the Exo was confirmed. Ud/FL ratios became low after LPS stimulation, indicating the enhanced encapsulation of FL-OPN in the Exo by LPS. These findings suggest that LPS stimulation of human macrophages facilitates the synthesis of FL-OPN, which is cleaved in cells or the Exo after release. These findings indicate that Exo is a suitable vehicle to transfer OPN to the target cells.
Assuntos
Exossomos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Osteopontina/metabolismo , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Ácidos Polimetacrílicos/químicaRESUMO
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 fármacosRESUMO
There is little literature showing the effect of urocortin (UCN) on macrophage apoptosis. The underlying mechanism is also unclear. This work was to investigate the involvement of UCN in the regulation of LPS-induced macrophage apoptosis and hence in the prevention from the atherosclerotic lesion development through targeting PLA2. Flow cytometry analysis showed that cell apoptosis was increased by more than 50% after LPS treatment in human THP-1 macrophage. Lp-PLA2 and cPLA2 were found to mediate LPS-induced macrophage apoptosis and NF-κB differentially influenced the expression of Lp-PLA2 and cPLA2. However, the reverse regulation of the expression of Lp-PLA2 and cPLA2 by NF-κB suggested that NF-κB may not be a key target for regulating macrophage apoptosis. Interestingly, we found that the approximate three folds upregulation of cPLA2 was in line with the induction of S1P formation and cell apoptosis by LPS. Inversely, LPS obviously decreased UCN expression by about 50% and secretion by about 25%. Both the enzyme inhibitor and knockdown expression of cPLA2 could completely abolish LPS-induced cell apoptosis. In addition, suppression of S1P synthesis by Sphk1 inhibitor PF-543 reduced the expression of cPLA2 and cell apoptosis but at the same time restored the normal level of UCN in cell culture supernatant. Furthermore, addition of exogenous UCN also reversed LPS-induced expression of cPLA2 and apoptosis. Taken together, UCN may be the reverse regulator of LPS-S1P-cPLA2-apoptosis pathway, thereby contributing to the prevention from the formation of unstable plaques.
Assuntos
Apoptose/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fosfolipases A2 Citosólicas/efeitos dos fármacos , Pró-Proteína Convertases/efeitos dos fármacos , Serina Endopeptidases/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células THP-1/efeitos dos fármacos , Urocortinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metanol/farmacologia , Proteínas Quinases Ativadas por Mitógeno/farmacologia , NF-kappa B/efeitos dos fármacos , Fosfolipases A2 Citosólicas/biossíntese , Pirrolidinas/farmacologia , Sulfonas/farmacologia , Urocortinas/farmacologiaRESUMO
The anti-inflammatory effects of curcumin are well documented. However, the bioavailability of curcumin is a major barrier to its biological efficacy. Low-dose combination of complimentary bioactives appears to be an attractive strategy for limiting barriers to efficacy of bioactive compounds. In this study, the anti-inflammatory potential of curcumin in combination with chlorogenic acid (CGA), was investigated using human THP-1 macrophages stimulated with lipopolysaccharide (LPS). Curcumin alone suppressed TNF-α production in a dose-dependent manner with a decrease in cell viability at higher doses. Although treatment with CGA alone had no effect on TNF-α production, it however enhanced cell viability and co-administration with curcumin at a 1:1 ratio caused a synergistic reduction in TNF-α production with no impact on cell viability. Furthermore, an qRT-PCR analysis of NF-κB pathway components and inflammatory biomarkers indicated that CGA alone was not effective in reducing the mRNA expression of any of the tested inflammatory marker genes, except TLR-4. However, co-administration of CGA with curcumin, potentiated the anti-inflammatory effects of curcumin. Curcumin and CGA together reduced the mRNA expression of pro-inflammatory cytokines [TNF-α (~88%) and IL-6 (~99%)], and COX-2 (~92%), possibly by suppression of NF-κB (~78%), IκB-ß-kinase (~60%) and TLR-4 receptor (~72%) at the mRNA level. Overall, co-administration with CGA improved the inflammation-lowering effects of curcumin in THP-1 cells.