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Severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause the hyperproduction of inflammatory cytokines, which have pathological effects in patient including severe or fatal cytokine storms. To characterize the effect of SFTSV and SARS-CoV-2 infection on the production of cytokines in severe fever with thrombocytopenia syndrome (SFTS) and COVID-19 patients, we performed an analysis of cytokines in SFTS and COVID-19 patients and also investigated the role of interleukin-10 (IL-10) in vitro studies: lipopolysaccharide-induced THP-1-derived macrophages, SFTSV infection of THP-1 cells, and SARS-CoV-2 infection of THP-1 cells. In this study, we found that levels of both IL-10 and IL-6 were significantly elevated, the level of transforming growth factor-ß (TGF-ß) was significantly decreased and IL-10 was elevated earlier than IL-6 in severe and critical COVID-19 and fatal SFTS patients, and inhibition of IL-10 signaling decreased the production of IL-6 and elevated that of TGF-ß. Therefore, the hyperproduction of IL-10 and IL-6 and the low production of TGF-ß have been linked to cytokine storm-induced mortality in fatal SFTS and severe and critically ill COVID-19 patients and that IL-10 can play an important role in the host immune response to severe and critical SARS-CoV-2 and fatal SFTSV infection.
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COVID-19 , Febre Grave com Síndrome de Trombocitopenia , Humanos , Síndrome da Liberação de Citocina , Citocinas , Interleucina-10 , Interleucina-6 , SARS-CoV-2 , Fator de Crescimento Transformador betaRESUMO
Shiga toxins (Stxs) produced by Stx-producing Escherichia coli are the primarily virulence factors of hemolytic uremic syndrome and central nervous system (CNS) impairment. Although the precise mechanisms of toxin dissemination remain unclear, Stxs bind to extracellular vesicles (EVs). Exosomes, a subset of EVs, may play a key role in Stx-mediated renal injury. To test this hypothesis, we isolated exosomes from monocyte-derived macrophages in the presence of Stx2a or Stx2 toxoids. Macrophage-like differentiated THP-1 cells treated with Stxs secreted Stx-associated exosomes (Stx-Exo) of 90-130 nm in diameter, which induced cytotoxicity in recipient cells in a toxin receptor globotriaosylceramide (Gb3 )-dependent manner. Stx2-Exo engulfed by Gb3 -positive cells were translocated to the endoplasmic reticulum in the human proximal tubule epithelial cell line HK-2. Stx2-Exo contained pro-inflammatory cytokine mRNAs and proteins and induced more severe inflammation than purified Stx2a accompanied by greater death of target cells such as human renal or retinal pigment epithelial cells. Blockade of exosome biogenesis using the pharmacological inhibitor GW4869 reduced Stx2-Exo-mediated human renal cell death. Stx2-Exo isolated from human primary monocyte-derived macrophages activated caspase 3/7 and resulted in significant cell death in primary human renal cortical epithelial cells. Based on these results, we speculate that Stx-containing exosomes derived from macrophages may exacerbate cytotoxicity and inflammation and trigger cell death in toxin-sensitive cells. Therapeutic interventions targeting Stx-containing exosomes may prevent or ameliorate Stx-mediated acute vascular dysfunction.
Assuntos
Exossomos/metabolismo , Macrófagos/metabolismo , Toxina Shiga II/metabolismo , Toxina Shiga II/toxicidade , Triexosilceramidas/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Exossomos/imunologia , Exossomos/ultraestrutura , Humanos , Inflamação , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Toxina Shiga II/farmacologia , Células THP-1RESUMO
Unfortunately, the concentration unit of plasmids was published incorrectly in the original publication of the article. The concentration unit, 'copies/ml' should be corrected to 'copies/µl'. This changes do not affect to the analytic sensitivity of the method because the detection limits of 50-100 copies/µL and 5-100 copies/µL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively, were as sensitive as other real-time PCR methods [1].
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Since severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. Therefore, the aim of this study was to develop a duplex real-time reverse transcription (RT)-PCR method for the simultaneous detection of these viruses. Primers and probes that target the conserved spike S2 region of human SARS-CoV, MERS-CoV, and their related bat CoVs were designed. The results of real-time RT-PCR showed specific reactions for each virus with adequate detection limits of 50-100 copies/mL and 5-100 copies/mL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively. In addition, this real-time RT-PCR system was able to detect the target viruses SARS-like bat CoV and MERS-CoV in bat fecal samples and sputum of MERS patients, respectively. Therefore, this newly developed real-time RT-PCR method is expected to detect not only SARS-CoV and MERS-CoV in humans but also several bat CoVs that are closely related to these viruses in bats.
Assuntos
Coronaviridae/isolamento & purificação , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/isolamento & purificação , Animais , Quirópteros/virologia , Coronaviridae/genética , Infecções por Coronavirus/virologia , Diagnóstico Diferencial , Fezes/virologia , Humanos , Limite de Detecção , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , RNA Viral/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Escarro/virologiaRESUMO
Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1ß (IL-1ß), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1ß secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1ß. Processing and release of both caspase-1 and IL-1ß were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1ß as well as to promote apoptotic cell death.
Assuntos
Proteínas de Transporte/imunologia , Caspase 1/imunologia , Interleucina-1beta/biossíntese , Piroptose/imunologia , Toxinas Shiga/imunologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Transporte/genética , Caspase 1/genética , Caspase 3/imunologia , Caspase 8/imunologia , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Ativação Enzimática/imunologia , Glicoesfingolipídeos/biossíntese , Humanos , Inflamação/imunologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Interferência de RNA , RNA Interferente Pequeno , Escherichia coli Shiga Toxigênica/metabolismo , Transdução de Sinais/imunologia , Triexosilceramidas/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.
Assuntos
Quimiotaxia , Escherichia coli Êntero-Hemorrágica/fisiologia , Síndrome Hemolítico-Urêmica/microbiologia , Síndrome Hemolítico-Urêmica/patologia , Rim/patologia , Papio/microbiologia , Toxinas Shiga/metabolismo , Animais , Quimiocinas/genética , Quimiocinas/urina , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Eosinófilos/patologia , Regulação da Expressão Gênica , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/urina , Humanos , Inflamação/patologia , Rim/metabolismo , Rim/microbiologia , Rim/ultraestrutura , Células Mesangiais/metabolismo , Células Mesangiais/microbiologia , Células Mesangiais/patologia , Células Mesangiais/ultraestrutura , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismoRESUMO
In contrast to normal cells, cancer cells predominantly utilise glycolysis for ATP generation under aerobic conditions, facilitating proliferation and metastasis. Targeting glycolysis is effective for cancer treatment. Prodigiosin (PDG) is a natural compound with various bioactivities, including anticancer effects. However, the precise action mechanisms and molecular targets of PDG, which has demonstrated efficacy in regulating glucose metabolism in cancer cells, remain elusive. Here, we aimed to investigate the anti-cancer activity of PDG and mechanism in cancer metabolism. PDG regulated cancer metabolism by suppressing intracellular ATP production rate and levels. It inhibited glycolysis and mitochondrial oxidative phosphorylation, impeding ATP production dependent on both glycolysis and mitochondrial respiration. Moreover, it inhibited cellular glucose uptake by directly interacting with glucose transporter 1 without affecting its mRNA or protein levels in HCT116 cells. We provide insights into the anti-cancer effects of PDG mediated via cancer metabolism regulation, suggesting its therapeutic potential for cancer.
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Shiga toxin (Stxs)-producing enterohaemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are major causative agents of severe bloody diarrhea (known as hemorrhagic colitis) and hemolytic uremic syndrome (HUS) associated with extraintestinal complications such as acute renal failure and neurologic impairment in infected patients under 9 years of age. Extreme nephrotoxicity of Stxs in HUS patients is associated with severe outcomes, highlighting the need to develop technologies to detect low levels of the toxin in environmental or food samples. Currently, the conventional polymerase chain reaction (PCR) or immunoassay is the most broadly used assay to detect the toxin. However, these assays are laborious, time-consuming, and costly. More recently, numerous studies have described novel, highly sensitive, and portable methods for detecting Stxs from EHEC. To contextualize newly emerging Stxs detection methods, we briefly explain the basic principles of these methods, including lateral flow assays, optical detection, and electrical detection. We subsequently describe existing and newly emerging rapid detection technologies to identify and measure Stxs.
Assuntos
Escherichia coli Êntero-Hemorrágica , Síndrome Hemolítico-Urêmica , Humanos , Toxinas Shiga/genética , Toxinas Shiga/toxicidade , Toxina Shiga/genética , Síndrome Hemolítico-Urêmica/diagnóstico , Escherichia coli Êntero-Hemorrágica/genética , Shigella dysenteriaeRESUMO
Endemic human coronaviruses (HCoVs) have been evidenced to be cross-reactive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although a correlation exists between the immunological memory to HCoVs and coronavirus disease 2019 (COVID-19) severity, there is little experimental evidence for the effects of HCoV memory on the efficacy of COVID-19 vaccines. Here, we investigated the Ag-specific immune response to COVID-19 vaccines in the presence or absence of immunological memory against HCoV spike Ags in a mouse model. Pre-existing immunity against HCoV did not affect the COVID-19 vaccine-mediated humoral response with regard to Ag-specific total IgG and neutralizing Ab levels. The specific T cell response to the COVID-19 vaccine Ag was also unaltered, regardless of pre-exposure to HCoV spike Ags. Taken together, our data suggest that COVID-19 vaccines elicit comparable immunity regardless of immunological memory to spike of endemic HCoVs in a mouse model.
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Prodigiosin, a red pigment produced by Hahella chejuensis, a marine-derived microorganism, has several biological functions, including antimicrobial activity and inflammatory relief. In this study, the antibacterial activity of prodigiosin against skin microorganisms was explored. Paper disc assay on skin bacterial cells revealed that Cutibacterium acnes related to acne vulgaris highly susceptible to prodigiosin. MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) were determined on Cutibacterium species. The RNA-seq analysis of prodigiosin-treated C. acnes cells was performed to understand the antibacterial mechanism of prodigiosin. Among changes in the expression of hundreds of genes, the expression of a stress-responsive sigma factor encoded by sigB increased. Conversely, the gene expression of cell wall biosynthesis and energy metabolism was inhibited by prodigiosin. Specifically, the expression of genes related to the metabolism of porphyrin, a pro-inflammatory metabolite, was significantly reduced. Therefore, prodigiosin could be used to control C. acnes. Our study provided new insights into the antimicrobial mechanism of prodigiosin against C. acnes strains.
Assuntos
Acne Vulgar , Prodigiosina , Humanos , Prodigiosina/farmacologia , Transcriptoma , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Acne Vulgar/microbiologia , Testes de Sensibilidade Microbiana , Propionibacterium acnes/genéticaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) is a specific subset of Shiga toxin-producing Escherichia coli (STEC) strains that are characterized by their ability to cause bloody diarrhea (hemorrhagic colitis) and potentially life-threatening, extraintestinal complications such as hemolytic uremic syndrome (HUS), which is associated with acute renal failure., contributing to severe clinical outcomes. The Shiga toxins (Stxs), produced by EHEC, are primary virulence factors. These potent cytotoxins are composed of one enzymatically active A subunit (StxA) and five receptor-binding B subunits (StxB). Although the toxins are primarily associated with cytotoxic effects, they also elicit other pathogenic consequences due to their induction of a number of biological processes, including apoptosis through ER-stress, pro-inflammatory responses, autophagy, and post-translational modification (PTM). Moreover, several studies have reported the association between Stxs and extracellular vesicles (EVs), including microvesicles and exosomes, demonstrating that Stx-containing EVs secreted by intoxicated macrophages are taken up by recipient cells, such as toxin-sensitive renal proximal tubular epithelial cells. This mechanism likely contributes to the spreading of Stxs within the host, and may exacerbate gastrointestinal illnesses and kidney dysfunction. In this review, we summarize recent findings relating to the host responses, in different types of cells in vitro and in animal models, mediated by Stxs-containing exosomes. Due to their unique properties, EVs have been explored as therapeutic agents, drug delivery systems, and diagnostic tools. Thus, potential therapeutic applications of EVs in EHEC Stxs-mediated pathogenesis are also briefly reviewed.
Assuntos
Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Vesículas Extracelulares , Síndrome Hemolítico-Urêmica , Escherichia coli Shiga Toxigênica , Animais , Toxina Shiga , Toxinas Shiga/toxicidade , Infecções por Escherichia coli/patologiaRESUMO
Atmospheric particulate matter (PM) contains a mixture of chemical and biological elements that pose threat to human health by increasing susceptibility to respiratory diseases. Although the identification of the microorganisms composing the PM has been assessed, their immunological impacts are still questionable. Here, we examined the mechanisms responsible for the pathogenicity of Pseudomonas stutzeri PM101005 (PMPS), a bacterium isolated from fine dust, in lung epithelial cells, alveolar cells, and macrophages. Relative to its comparative strain Pseudomonas stutzeri (PS), infections with PMPS induced higher production of inflammatory cytokines and chemokines, mediated by the activation of NF-κB and MAPK signaling pathways. Additionally, with three-dimensional (3D) airway spheroids which mimic the human bronchial epithelium, we confirmed that PMPS infections lead to relatively higher induction of pro-inflammatory cytokines than PM infections. Consistent results were observed in murine models as the infections with PMPS provoked greater inflammatory responses than the infections with PS. These PMPS-induced responses were mediated by the signaling pathways of the Toll-like receptors (TLRs), which regulated PMPS infection and played an important role in the expression of the antibiotic peptide ß-defensin 3 (BD3) that suppressed PMPS proliferation. Moreover, PM pretreatment enhanced inflammatory responses and tissue damage of PMPS, while reducing BD3 expression. Overall, these results indicate that PM-isolated PMPS induce TLR-mediated inflammatory responses in lung tissues, and contributes to the understanding of the etiology of PM-induced respiratory damage.
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Material Particulado , Pseudomonas stutzeri , Camundongos , Humanos , Animais , Material Particulado/toxicidade , Material Particulado/metabolismo , Pseudomonas stutzeri/metabolismo , Pulmão/metabolismo , Citocinas/metabolismo , Transdução de SinaisRESUMO
Background: A growing body of evidence suggests that particulate matter (PM10) enters the gastrointestinal (GI) tract directly, causing the GI epithelial cells to function less efficiently, leading to inflammation and an imbalance in the gut microbiome. PM10 may, however, act as an exacerbation factor in patients with inflamed intestinal epithelium, which is associated with inflammatory bowel disease. Objective: The purpose of this study was to dissect the pathology mechanism of PM10 exposure in inflamed intestines. Methods: In this study, we established chronically inflamed intestinal epithelium models utilizing two-dimensional (2D) human intestinal epithelial cells (hIECs) and 3D human intestinal organoids (hIOs), which mimic in vivo cellular diversity and function, in order to examine the deleterious effects of PM10 in human intestine-like in vitro models. Results: Inflamed 2D hIECs and 3D hIOs exhibited pathological features, such as inflammation, decreased intestinal markers, and defective epithelial barrier function. In addition, we found that PM10 exposure induced a more severe disturbance of peptide uptake in inflamed 2D hIECs and 3D hIOs than in control cells. This was due to the fact that it interferes with calcium signaling, protein digestion, and absorption pathways. The findings demonstrate that PM10-induced epithelial alterations contribute to the exacerbation of inflammatory disorders caused by the intestine. Conclusions: According to our findings, 2D hIEC and 3D hIO models could be powerful in vitro platforms for the evaluation of the causal relationship between PM exposure and abnormal human intestinal functions.
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Células Epiteliais , Intestinos , Humanos , Organoides , Sinalização do Cálcio , Inflamação , Material Particulado/efeitos adversosRESUMO
Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.
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Histonas , Neoplasias Pulmonares , Humanos , Histonas/metabolismo , Histona Metiltransferases/metabolismo , Epigênese Genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proliferação de Células , Neoplasias Pulmonares/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
Shiga toxins (Stxs) are cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli (STEC). Stxs bind to a membrane glycolipid receptor, enter cells, and undergo retrograde transport to ultimately reach the cytosol, where the toxins exert their protein synthesis-inhibitory activity by depurination of a single adenine residue from the 28S rRNA component of eukaryotic ribosomes. The depurination reaction activates the ribotoxic stress response, leading to signaling via the mitogen-activated protein kinase (MAPK) pathways (Jun N-terminal protein kinase [JNK], p38, and extracellular signal-regulated kinase [ERK]) in human epithelial, endothelial, and myeloid cells. We previously showed that treatment of human macrophage-like THP-1 cells with Stxs resulted in increased cytokine and chemokine expression. In the present study, we show that individual inactivation of ERK, JNK, and p38 MAPKs using pharmacological inhibitors in the presence of Stx1 resulted in differential regulation of the cytokines tumor necrosis factor alpha and interleukin-1ß (IL-1ß) and chemokines IL-8, growth-regulated protein-ß, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß. THP-1 cells exposed to Stx1 upregulate the expression of select dual-specificity phosphatases (DUSPs), enzymes that dephosphorylate and inactivate MAPKs in mammalian cells. In this study, we confirmed DUSP1 protein production by THP-1 cells treated with Stx1. DUSP1 inhibition by triptolide showed that ERK and p38 phosphorylation is regulated by DUSP1, while JNK phosphorylation is not. Inhibition of p38 MAPK signaling blocked the ability of Stx1 to induce DUSP1 mRNA expression, suggesting that an autoregulatory signaling loop may be activated by Stxs. Thus, Stxs appear to be capable of eliciting signals which both activate and deactivate signaling for increased cytokine/chemokine production in human macrophage-like cells.
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Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Toxina Shiga I/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Antracenos , Linhagem Celular Tumoral , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Flavonoides , Humanos , Imidazóis , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Piridinas , Fatores de TempoRESUMO
The bacterial virulence factors Shiga toxins (Stxs) are expressed by Shigella dysenteriae serotype 1 and certain Escherichia coli strains. Stxs are protein synthesis inhibitors and induce apoptosis in many cell types. Stxs induce apoptosis via prolonged endoplasmic reticulum stress signalling to activate both extrinsic and intrinsic pathways in human myeloid cells. Studies have shown that autophagy, a lysosome-dependent catabolic process, may be associated with activation of pro-survival or death processes. It is currently unknown if autophagy contributes to apoptosis or protects cells from Stxs. To study cellular responses to Stxs, we intoxicated toxin-sensitive cells (THP-1 and HK-2 cells), and toxin-resistant cells (primary human monocyte-derived macrophages) and examined toxin intracellular trafficking and autophagosome formation. Stxs translocated to different cell compartments in toxin-resistant versus toxin-sensitive cells. Confocal microscopy revealed autophagosome formation in both toxin-resistant and toxin-sensitive cells. Proteolytic cleavage of Atg5 and Beclin-1 plays pivotal roles in switching non-cytotoxic autophagy to cell death signalling. We detected cleaved forms of Atg5 and Beclin-1 in Stx-treated toxin-sensitive cells, while cleaved caspases, calpains, Atg5 and Beclin-1 were not detected in toxin-resistant primary human monocytes and macrophages. These findings suggest that toxin sensitivity correlates with caspase and calpain activation, leading to Atg5 and Beclin-1 cleavage.
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Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Escherichia coli/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Toxinas Shiga/toxicidade , Shigella dysenteriae/patogenicidade , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Calpaína/metabolismo , Caspases/metabolismo , Células Cultivadas , Humanos , Toxina Shiga , Transdução de SinaisRESUMO
Host immune responses, such as those initiated by pattern recognition receptor (PRR) activation, are important for viral clearance and pathogenesis. However, little is known about the interactions of viral proteins with surface PRRs or, more importantly, the association of innate immune activation with viral pathogenesis. In this study, we showed that internal influenza virus proteins were released from infected cells. Among these proteins, nucleoprotein (NP) played a critical role in viral pathogenesis by stimulating neighboring cells through toll-like receptor (TLR)2, TLR4, and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Through the activation of these PRRs, NP induced the production of interleukin (IL)-1ß and IL-6, which subsequently led to the induction of trypsin. Trypsin induced by NP increased the infectivity of influenza virus, leading to increases in viral replication and pathology upon subsequent viral infection. These results reveal the role of released NP in influenza pathogenesis and highlight the importance of the interactions of internal viral proteins with PRRs in the extracellular compartment during viral pathogenesis.
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Influenza Humana , Orthomyxoviridae , Receptor 4 Toll-Like , Humanos , Inflamassomos/metabolismo , Influenza Humana/metabolismo , Influenza Humana/virologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleoproteínas , Orthomyxoviridae/metabolismo , Receptor 4 Toll-Like/metabolismo , Tripsina/metabolismoRESUMO
Particulate matter (PM) can be categorized by particle size (PM10, PM2.5 and PM1.0), which is an important factor affecting the biological response. Exposure to PM in the air (dust, smoke, dirt and biological contaminants) is clearly associated with lung disease (lung cancer, pneumonia and asthma). Although PM primarily affects lung epithelial cells, the specific response of related cell types to PM remains to be elucidated. The present study performed Gene Ontology (GO) analysis programs (Clustering GO and Database for Annotation, Visualization and Integrated Discovery) on differentially expressed genes in lung epithelial cells (WI38 VA13) and fibroblasts (WI38) following treatment with PM10 and evaluated the cellspecific biological responses related to cell proliferation, apoptosis, adhesion and extracellular matrix production. The results suggested that short or longterm exposure to PM may affect cell condition and may consequently be related to several human diseases, including lung cancer and cardiopulmonary disease.
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Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Material Particulado/efeitos adversos , Transcriptoma , Poluentes Atmosféricos , Poluição do Ar , Adesão Celular , Linhagem Celular , Matriz Extracelular/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pulmão , RNA-SeqRESUMO
Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC) are the major virulence factors responsible for hemorrhagic colitis, which can lead to life-threatening systemic complications including acute renal failure (hemolytic uremic syndrome) and neuropathy. Here, we report that O-GlcNAcylation, a type of post-translational modification, was acutely increased upon induction of endoplasmic reticulum (ER) stress in host cells by Stxs. Suppression of the abnormal Stx-mediated increase in O-GlcNAcylation effectively inhibited apoptotic and inflammatory responses in Stx-susceptible cells. The protective effect of O-GlcNAc inhibition for Stx-mediated pathogenic responses was also verified using three-dimensional (3D)-cultured spheroids or organoids mimicking the human kidney. Treatment with an O-GlcNAcylation inhibitor remarkably improved the major disease symptoms and survival rate for mice intraperitoneally injected with a lethal dose of Stx. In conclusion, this study elucidates O-GlcNAcylation-dependent pathogenic mechanisms of Stxs and demonstrates that inhibition of aberrant O-GlcNAcylation is a potential approach to treat Stx-mediated diseases.
Assuntos
Infecções por Escherichia coli , Síndrome Hemolítico-Urêmica , Animais , Estresse do Retículo Endoplasmático , Síndrome Hemolítico-Urêmica/patologia , Rim/patologia , Camundongos , Toxina Shiga/metabolismo , Toxinas ShigaRESUMO
Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.