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Phosphatase and tensin homolog (PTEN) is vital for B cell development, acting as a key negative regulator in the PI3K signaling pathway. We used CD23-cre to generate PTEN-conditional knockout mice (CD23-cKO) to examine the impact of PTEN mutation on peripheral B cells. Unlike mb1-cre-mediated PTEN deletion in early B cells, CD23-cKO mutants exhibited systemic inflammation with increased IL-6 production in mature B cells upon CpG stimulation. Inflammatory B cells in CD23-cKO mice showed elevated phosphatidylinositol 3-phosphate [PI(3)P] levels and increased TLR9 endosomal localization. Pharmacological inhibition of PI(3)P synthesis markedly reduced TLR9-mediated IL-6. Single-cell RNA-sequencing (RNA-seq) revealed altered endocytosis, BANK1, and NF-κB1 expression in PTEN-deficient B cells. Ectopic B cell receptor (BCR) expression on non-inflammatory mb1-cKO B cells restored BANK1 and NF-κB1 expression, enhancing TLR9-mediated IL-6 production. Our study highlights PTEN as a crucial inflammatory checkpoint, regulating TLR9/IL-6 axis by fine-tuning PI(3)P homeostasis. Additionally, BCR downregulation prevents the differentiation of inflammatory B cells in PTEN deficiency.
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Gut microbiota rearrangement induced by cold temperature is crucial for browning in murine white adipose tissue. This study provides evidence that DUSP6, a host factor, plays a critical role in regulating cold-induced gut microbiota rearrangement. When exposed to cold, the downregulation of intestinal DUSP6 increased the capacity of gut microbiota to produce ursodeoxycholic acid (UDCA). The DUSP6-UDCA axis is essential for driving Lachnospiraceae expansion in the cold microbiota. In mice experiencing cold-room temperature (CR) transitions, prolonged DUSP6 inhibition via the DUSP6 inhibitor (E/Z)-BCI maintained increased cecal UDCA levels and cold-like microbiota networks. By analyzing DUSP6-regulated microbiota dynamics in cold-exposed mice, we identified Marvinbryantia as a genus whose abundance increased in response to cold exposure. When inoculated with human-origin Marvinbryantia formatexigens, germ-free recipient mice exhibited significantly enhanced browning phenotypes in white adipose tissue. Moreover, M. formatexigens secreted the methylated amino acid Nε-methyl-L-lysine, an enriched cecal metabolite in Dusp6 knockout mice that reduces adiposity and ameliorates nonalcoholic steatohepatitis in mice. Our work revealed that host-microbiota coadaptation to cold environments is essential for regulating the browning-promoting gut microbiome.
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Microbioma Gastrointestinal , Animais , Humanos , Camundongos , Adiposidade , Temperatura Baixa , Fosfatases de Especificidade Dupla/metabolismo , Microbioma Gastrointestinal/fisiologia , ObesidadeRESUMO
Multidrug resistance (MDR) remains a significant challenge in cancer therapy, with inherent and acquired resistance distinct. While conventional drug selection processes enable the isolation of cancer cells with acquired multidrug resistance, identifying cancer cells with inherent drug resistance remains challenging. Herein, we proposed a molecular beacon (MB)-based strategy to identify and isolate the inherent MDR cancer cells. A lipid/PLGA core-shell nanoparticulate system (DNCP) was designed to deliver MB for intracellular MDR1 mRNA imaging. DNCP-MB - possess a surface potential of -8 mV and a size of 150 nm - demonstrated effective delivery of MB, remarkable selectivity towards the selected intracellular mRNA targets, and low cytotoxicity. Following DNCP transfection, fluorescence-activated cell sorting (FACS) was employed to differentiate MCF-7 cells into two distinct sub-populations: the Top 10 cells with a high level of MDR gene expression and the Bottom 10 cells with a low level of MDR gene expression, which represent inherent drug-resistant and non-drug-resistant cells, respectively. Intriguingly, we observed a positive correlation between elevated MDR1 mRNA expression and increased migration, enhanced proliferation rate, and tighter spheroid formation. Moreover, we conducted RNA sequencing analysis on the Top 10, Bottom 10, and MCF-7/ADR cells. The findings revealed a notable disparity in the gene ontology enrichment analysis of differentially expressed genes between the Top 10 and Bottom 10 cells when compared to the Bottom 10 and MCF-7/ADR cells. This novel approach provides a promising avenue for isolating inherent drug-resistant cells and holds significant potential in unraveling the mechanisms underlying inherent drug resistance.
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Técnicas Biossensoriais , Neoplasias , Humanos , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos/genética , Resistência a Múltiplos Medicamentos/genética , Células MCF-7 , RNA Mensageiro , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Gut dysbiosis can induce chronic inflammation and contribute to atherosclerosis and vascular calcification. The aortic arch calcification (AoAC) score is a simple, noninvasive, and semiquantitative assessment tool to evaluate vascular calcification on chest radiographs. Few studies have discussed the relationship between gut microbiota and AoAC. Therefore, this study aimed to compare the microbiota composition between patients with chronic diseases and high or low AoAC scores. A total of 186 patients (118 males and 68 females) with chronic diseases, including diabetes mellitus (80.6%), hypertension (75.3%), and chronic kidney disease (48.9%), were enrolled. Gut microbiota in fecal samples were analyzed by sequencing of the 16S rRNA gene, and differences in microbial function were examined. The patients were divided into three groups according to AoAC score, including 103 patients in the low AoAC group (AoAC ≤ 3), 40 patients in the medium AoAC group (3 < AoAC ≤ 6), and 43 patients in the high AoAC group (AoAC > 6). Compared to the low AoAC group, the high AoAC group had a significantly lower microbial species diversity (Chao1 index and Shannon index) and increased microbial dysbiosis index. Beta diversity showed that the microbial community composition was significantly different among the three groups (p = 0.041, weighted UniFrac PCoA). A distinct microbial community structure was found in the patients with a low AoAC, with an increased abundance at the genus level of Agathobacter, Eubacterium coprostanoligenes group, Ruminococcaceae UCG-002, Barnesiella, Butyricimonas, Oscillibacter, Ruminococcaceae DTU089, and Oxalobacter. In addition, there was an increased relative abundance of class Bacilli in the high AoAC group. Our findings support the association between gut dysbiosis and the severity of AoAC in patients with chronic diseases.
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Microbioma Gastrointestinal , Insuficiência Renal Crônica , Calcificação Vascular , Masculino , Feminino , Humanos , Microbioma Gastrointestinal/genética , Aorta Torácica , Disbiose/microbiologia , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND: Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB). Evidence has linked the DM-related dysbiosis of gut microbiota to modifiable host immunity to Mycobacterium tuberculosis infection. However, the crosslinks between gut microbiota composition and immunological effects on the development of latent TB infection (LTBI) in DM patients remain uncertain. METHODS: We prospectively obtained stool, blood samples, and medical records from 130 patients with poorly-controlled DM (pDM), defined as ever having an HbA1c > 9.0% within previous 1 year. Among them, 43 had LTBI, as determined by QuantiFERON-TB Gold in-Tube assay. The differences in the taxonomic diversity of gut microbiota between LTBI and non-LTBI groups were investigated using 16S ribosomal RNA sequencing, and a predictive algorithm was established using a random forest model. Serum cytokine levels were measured to determine their correlations with gut microbiota. RESULTS: Compared with non-LTBI group, the microbiota in LTBI group displayed a similar alpha-diversity but different beta-diversity, featuring decrease of Prevotella_9, Streptococcus, and Actinomyces and increase of Bacteroides, Alistipes, and Blautia at the genus level. The accuracy was 0.872 for the LTBI prediction model using the aforementioned 6 microbiome-based biomarkers. Compared with the non-LTBI group, the LTBI group had a significantly lower serum levels of IL-17F (p = 0.025) and TNF-α (p = 0.038), which were correlated with the abundance of the aforementioned 6 taxa. CONCLUSIONS: The study results suggest that gut microbiome composition maybe associated with host immunity relevant to TB status, and gut microbial signature might be helpful for the diagnosis of LTBI.
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Diabetes Mellitus Tipo 2 , Microbioma Gastrointestinal , Tuberculose Latente , Humanos , Microbioma Gastrointestinal/imunologia , Imunidade , Tuberculose Latente/imunologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/imunologiaRESUMO
Single-cell RNA sequencing (scRNA-seq) approach can broadly and specifically evaluate the individual cells with minimum detection bias. To explore the individual compositional and transcriptional alteration of intestinal leukocytes in the Dual Specificity Phosphatase six knockout (D6KO) mice, we performed a scRNA-seq followed by the cell type annotation based on ImmGen database. Composition assessments found that D6KO-derived intestinal leukocytes tend to stay inactivate or immature status. The enrichment analysis showed that D6KO-derived intestinal leukocytes are less sensitive to microbes. The mod PhEA phenotypic analysis showed that the D6KO leukocytes may link to not only immune-associated but also diverse previously non-immune-related diseases. Integrating our dataset with the published dataset GSE124880 generated a comprehensive dataset for exploring intestinal immunity. Down-regulation of Ccl17 gene was found in the D6KO-derived dendritic cells. Our results demonstrated the advantage of applying scRNA-seq for dissecting the individual alteration of intestinal leukocytes, particularly in the D6KO mice at a naive state.
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Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases.
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Colite/microbiologia , Fosfatase 6 de Especificidade Dupla/metabolismo , Microbioma Gastrointestinal/fisiologia , Animais , Células CACO-2 , Colite/prevenção & controle , Colo/metabolismo , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Fosfatase 6 de Especificidade Dupla/deficiência , Fosfatase 6 de Especificidade Dupla/genética , Disbiose/metabolismo , Células Epiteliais/metabolismo , Fezes , Feminino , Humanos , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Ribossômico 16S/metabolismoRESUMO
Antrodia cinnamomea (AC) is a nutraceutical fungus and studies have suggested that AC has the potential to prevent or alleviate diseases. However, little is known about the AC-induced phenotypes on the intestine-liver axis and gut microbial alterations. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-Biotyper to elaborate the AC-induced phenotypes on the intestine-liver axis and gut microbial distribution of C57BL/6 mice. The experimental outcomes showed that the hepatic density may increase by elevating hepatic redox regulation, lipid degradation and glycolysis-related proteins and alleviating cholesterol biosynthesis and transport-related proteins in C57BL/6 mice with AC treatment. Moreover, AC facilitates intestinal glycolysis, TCA cycle, redox and cytoskeleton regulation-related proteins, but also reduces intestinal vesicle transport-related proteins in C57BL/6 mice. However, the body weight, GTT, daily food/water intake, and fecal/urine weight were unaffected by AC supplementation in C57BL/6 mice. Notably, the C57BL/6-AC mice had a higher gut microbial abundance of Alistipes shahii (AS) than C57BL/6-Ctrl mice. In summary, the AC treatment affects intestinal permeability by regulating redox and cytoskeleton-related proteins and elevates the gut microbial abundance of AS in C57BL/6 mice that might be associated with increasing hepatic density and metabolism-related proteins of the liver in C57BL/6 mice. Our study provides an insight into the mechanisms of AC-induced phenotypes and a comprehensive assessment of AC's nutraceutical effect in C57BL/6 mice.
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Suplementos Nutricionais , Microbioma Gastrointestinal/efeitos dos fármacos , Polyporales , Proteoma/metabolismo , Animais , Hepatócitos/metabolismo , Intestinos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BLRESUMO
Predictive metabolic biomarkers for the recurrent luminal breast cancer (BC) with hormone receptor (HR)-positive and human epidermal growth factor receptor type 2 (HER2)-negative are lacking. High levels of O-GlcNAcylation (O-GlcNAc) and pyruvate kinase isoenzyme M2 (PKM2) are associated with malignancy in BC; however, the association with the recurrence risk remains unclear. We first conduct survival analysis by using the METABRIC dataset to assess the correlation of PKM2 expression with BC clinical outcomes. Next, patients with HR+/HER2- luminal BC were recruited for PKM2/O-GlcNAc testing. Logistic regression and receiver operating characteristic curve analysis were performed to evaluate the 10-year DFS predicted outcome. Survival analysis of the METABRIC dataset revealed that high expression of PKM2 was significantly associated with worse overall survival in luminal BC. The high expression of O-GlcNAc or PKM2 was a significant independent marker for poor 10-year DFS using immunohistochemical analysis. The PKM2 or O-GlcNAc status was a significant predictor of DFS, with the combination of PKM2-O-GlcNAc status and T stage greatly enhancing the predictive outcome potential. In summary, O-GlcNAc, PKM2, and T stage serve as good prognostic discriminators in HR+/HER2- luminal BC.
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Isolation and enumeration of bacteria at ultralow concentrations and antibiotic resistance profiling are of great importance for early diagnosis and treatment of bacteremia. In this work, we describe a simple, rapid, and versatile magnetic-assisted microfluidic method for rapid bacterial detection. The developed method enables magnetophoretic loading of bead-captured bacteria into the microfluidic chamber under external static and dynamic magnetic fields in 4 min. A shallow microfluidic chamber design that enables the monolayer orientation and transportation of the beads and a glass substrate with a thickness of 0.17 mm was utilized to allow high-resolution fluorescence imaging for quantitative detection. Escherichia coli (E. coli) with green fluorescent protein (GFP)-expressing gene and streptavidin-modified superparamagnetic microbeads were used as model bacteria and capturing beads, respectively. The specificity of the method was validated using Lactobacillus gasseri as a negative control group. The limit of detection and limit of quantification values were determined as 2 CFU/ml and 10 CFU/ml of E. coli, respectively. The magnetic-assisted microfluidic method is a versatile tool for the detection of ultralow concentrations of viable bacteria with the linear range of 5-5000 CFU/ml E. coli in 1 h, and providing growth curves and phenotypic characterization bead-captured E. coli in the following 5 h of incubation. Our results are promising for future rapid and sensitive antibiotic susceptibility testing of ultralow numbers of viable cells.
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Escherichia coli , Microfluídica , Bactérias , Fenômenos Magnéticos , EstreptavidinaRESUMO
Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood-brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the ß3 and ß4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.
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Anticorpos Monoclonais/administração & dosagem , Lipocalina-2/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Cérebro/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/química , Masculino , Camundongos , Neutrófilos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/metabolismoRESUMO
Obesity is associated with metabolic disorders. Thus, obesity prevention and treatment are essential for health. Antrodia cinnamomea (AC) is a multifunctional medicinal fungus used for the treatment of various diseases and for preventing diet-induced obesity. Leptin deficiency causes over-eating and spontaneous obesity. The concomitant metabolic symptoms are more severe than diet-induced obesity. Here, we used leptin-deficient (ob/ob) mice as an animal model for over-feeding to study the effect of AC on obesity. We fed C57BL/6 mice (WT, ob+/+) and ob/ob mice with AC for four weeks before performing qRT-PCR and immunoblot analysis to elaborate AC-modulated mechanisms. Further, we used Caco-2 cells as a human intestinal epithelial barrier model to examine the effect of AC on intestinal permeability. Our results suggested that AC reduces lipid deposits of the liver and epididymal white adipose tissue (EWAT) by promoting lipid metabolism and inhibiting lipogenesis-associated genes and proteins in ob/ob mice. Moreover, AC effectively repaired intestinal-barrier injury caused by leptin deficiency and enhanced intestinal barrier integrity in Caco-2 cells. Interestingly, AC significantly reduced body weight and EWAT with no compromise on food intake in ob/ob mice. Thus, AC effectively reduced obesity caused by leptin-deficiency and can potentially be used as a nutraceutical for treating obesity.
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Tecido Adiposo Branco , Antrodia/química , Mucosa Intestinal , Leptina/deficiência , Obesidade , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Células CACO-2 , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Leptina/metabolismo , Camundongos , Camundongos Knockout , Camundongos Obesos , Obesidade/tratamento farmacológico , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologiaRESUMO
There is a positive feedback loop driving tumorigenesis and tumor growth through coordinated regulation of epigenetics, inflammation, and stemness. Nevertheless, the molecular mechanism linking these processes is not well understood. In this study, we analyzed the correlation of de-ubiquitinases (DUBs) expression with survival data from the OncoLnc database. Among the DUBs analyzed, ubiquitin specific protease 4 (USP4) had the lowest negative Cox coefficient. Low expression of USP4 was associated with poor survival among lung cancer patients and was inversely correlated with expression of stemness and inflammation markers. Expression of USP4 were reduced at more advanced stages of lung cancer. Mechanistically, expression of USP4 was downregulated in snail1-overexpressing and stemness-enriched lung cancer cells. Snail1 was induced in lung cancer cells by interaction with macrophages, and epigenetically suppressed USP4 expression by promoter methylation. Stable knockdown of USP4 in lung cancer cells enhanced inflammatory responses, stemness properties, chemotherapy resistance, and the expression of molecules allowing escape from immunosurveillance. Further, mice injected with USP4 knockdown lung cancer cells demonstrated enhanced tumorigenesis and tumor growth. These results reveal that the Snail1-mediated suppression of USP4 is a potential mechanism to orchestrate epigenetic regulation, inflammation and stemness for macrophage-promoted tumor progression.
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Increasing evidences have shown strong associations between gut microbiota and many human diseases, and understanding the dynamic crosstalks of host-microbe interaction in the gut has become necessary for the detection, prevention, or therapy of diseases. Many reports have showed that diet, nutrient, pharmacologic factors and many other stimuli play dominant roles in the modulation of gut microbial compositions. However, it is inappropriate to neglect the impact of host factors on shaping the gut microbiota. In this review, we highlighted the current findings of the host factors that could modulate the gut microbiota. Particularly the epithelium-associated factors, including the innate immune sensors, anti-microbial peptides, mucus barrier, secretory IgAs, epithelial microvilli, epithelial tight junctions, epithelium metabolism, oxygen barrier, and even the microRNAs are discussed in the context of the microbiota shaping. With these shaping factors, the gut epithelial cells could select the residing microbes and affect the microbial composition. This knowledge not only could provide the opportunities to better control many diseases, but may also be used for predicting the success of fecal microbiota transplantation clinically.
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Peptídeos Catiônicos Antimicrobianos/fisiologia , Epitélio/fisiologia , Microbioma Gastrointestinal/fisiologia , Imunidade Inata , Imunoglobulina A Secretora/fisiologia , Muco/fisiologia , Humanos , MicroRNAs/fisiologia , Microvilosidades/fisiologia , Junções Íntimas/fisiologiaRESUMO
With the significant global obesity epidemic and emerging strong scientific evidence that connected gut microbiota to obesity, intervening obesity by targeting gut microbiota has become a trendy strategy. Particularly the application of probiotics has become remarkably popular because of their expected association with gut microbiota modulation. Although there are many literatures on the effects of probiotics in obese animal models, most of them reported the effects of probiotic bacteria on metabolic indications with limited information on anti-obesity itself. Besides, some probiotics have been shown to reduce certain metabolic symptoms but they failed to achieve weight loss. This report reviewed the current literatures on the anti-obesity effects of next-generation probiotics in various animal obesity models and discussed the beneficial potential of fecal microbiota transplantation in treating obesity in humans. The purpose of this article is to help guide further research improve the probiotic bacteria experiments in more precise animal obesity models by standardizing the anti-obesogenesis, obesity control, and treatment assays and hopefully the evidence-based investigations on harnessing gut microbiota through next-generation probiotics or fecal microbiota transplantation will develop new interventions to promote and achieve anti-obesity.
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Microbioma Gastrointestinal , Obesidade/microbiologia , Animais , Fármacos Antiobesidade/uso terapêutico , Transplante de Microbiota Fecal , Humanos , Obesidade/genética , Obesidade/terapia , Probióticos/uso terapêuticoRESUMO
Activated T cells undergo metabolic reprogramming and effector-cell differentiation but the factors involved are unclear. Utilizing mice lacking DUSP6 (DUSP6-/-), we show that this phosphatase regulates T cell receptor (TCR) signaling to influence follicular helper T (TFH) cell differentiation and T cell metabolism. In vitro, DUSP6-/- CD4+ TFH cells produced elevated IL-21. In vivo, TFH cells were increased in DUSP6-/- mice and in transgenic OTII-DUSP6-/- mice at steady state. After immunization, DUSP6-/- and OTII-DUSP6-/- mice generated more TFH cells and produced more antigen-specific IgG2 than controls. Activated DUSP6-/- T cells showed enhanced JNK and p38 phosphorylation but impaired glycolysis. JNK or p38 inhibitors significantly reduced IL-21 production but did not restore glycolysis. TCR-stimulated DUSP6-/- T cells could not induce phosphofructokinase activity and relied on glucose-independent fueling of mitochondrial respiration. Upon CD28 costimulation, activated DUSP6-/- T cells did not undergo the metabolic commitment to glycolysis pathway to maintain viability. Unexpectedly, inhibition of fatty acid oxidation drastically lowered IL-21 production in DUSP6-/- TFH cells. Our findings suggest that DUSP6 connects TCR signaling to activation-induced metabolic commitment toward glycolysis and restrains TFH cell differentiation via inhibiting IL-21 production.
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Diferenciação Celular/fisiologia , Fosfatase 6 de Especificidade Dupla , Glicólise/fisiologia , Receptores de Antígenos de Linfócitos T , Transdução de Sinais/fisiologia , Linfócitos T Auxiliares-Indutores , Animais , Formação de Anticorpos/fisiologia , Antígenos CD28/genética , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/imunologia , Fosfatase 6 de Especificidade Dupla/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Interleucinas/genética , Interleucinas/imunologia , Interleucinas/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Autophagy is an evolutionarily conserved intracellular system that maintains cellular homeostasis by degrading and recycling damaged cellular components. The transcription factor HLH-30/TFEB-mediated autophagy has been reported to regulate tolerance to bacterial infection, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we reveal that bacterial membrane pore-forming toxin (PFT) induces autophagy in an HLH-30-dependent manner in Caenorhabditis elegans. Moreover, autophagy controls the susceptibility of animals to PFT toxicity through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously in the PFT-targeted intestinal cells in C. elegans. These results demonstrate that autophagic pathways and autophagy are induced partly at the transcriptional level through HLH-30 activation and are required to protect metazoan upon PFT intoxication. Together, our data show a new and powerful connection between HLH-30-mediated autophagy and epithelium intrinsic cellular defense against the single most common mode of bacterial attack in vivo.
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Autofagia , Proteínas de Bactérias/toxicidade , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/microbiologia , Endotoxinas/toxicidade , Epitélio/metabolismo , Proteínas Hemolisinas/toxicidade , Animais , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Toxinas de Bacillus thuringiensis , Sequência de Bases , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Intestinos/microbiologia , Intestinos/patologia , Modelos Biológicos , Transcrição Gênica/efeitos dos fármacosRESUMO
The gut microbiota plays profound roles in host metabolism and the inflammatory response associated with the development of obesity. Dusp6-deficient mice have been shown to be resistant to diet-induced obesity, but the mechanism behind this remains unclear. 16S ribosomal RNA gene analysis demonstrated that dusp6-deficient mice harbour unique gut microbiota with resistance to diet-induced-obesity-mediated alteration of the gut microbiome. Using a germ-free mouse model, we found that faecal/gut microbiota derived from dusp6-deficient mice significantly increased energy expenditure and reduced weight gain in recipient wild-type mice fed on a high-fat diet. On analysis of the intestinal transcriptome of dusp6-deficient mice, we found that dusp6 deficiency mainly induced biological processes involved in metabolism and the extracellular matrix, particularly the peroxisome proliferator-activated receptor gamma (Pparγ) pathway and tight-junction genes. Furthermore, dusp6-deficient mice have a high-fat-diet-specific transcriptomic response to reverse the expression of genes associated with intestinal barrier functions and mucosal immunity involved in microbiome homeostasis. This study demonstrates that dusp6 deficiency is a strong genetic factor shaping gut microbiota, and that it confers obesity protection by ameliorating the gut microbiota response to diet-mediated stress.
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Dieta , Fosfatase 6 de Especificidade Dupla/deficiência , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Perfilação da Expressão Gênica , Obesidade , Animais , Análise por Conglomerados , DNA Ribossômico/química , DNA Ribossômico/genética , Metabolismo Energético , Camundongos , Camundongos Knockout , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Activation of TLR7-9 has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. Thus, therapeutic applications of antagonists of these TLRs for such disorders are being investigated. Bortezomib (Velcade) is a proteasome inhibitor known to suppress activation of these TLRs. To identify novel TLR7-9 inhibitors, we searched the Gene Expression Omnibus database for gene expression profiles of bortezomib-treated cells. These profiles were then used to screen the Connectivity Map database for chemical compounds with similar functions as bortezomib. A natural antibiotic, thiostrepton, was identified for study. Similar to bortezomib, thiostrepton effectively inhibits TLR7-9 activation in cell-based assays and in dendritic cells. In contrast to bortezomib, thiostrepton does not inhibit NF-κB activation induced by TNF-α, IL-1, and other TLRs, and it is less cytotoxic to dendritic cells. Thiostrepton inhibits TLR9 localization in endosomes for activation via two mechanisms, which distinguish it from currently used TLR7-9 inhibitors. One mechanism is similar to the proteasome inhibitory function of bortezomib, whereas the other is through inhibition of endosomal acidification. Accordingly, in different animal models, thiostrepton attenuated LL37- and imiquimod-induced psoriasis-like inflammation. These results indicated that thiostrepton is a novel TLR7-9 inhibitor, and compared with bortezomib, its inhibitory effect is more specific to these TLRs, suggesting the potential therapeutic applications of thiostrepton on immunologic disorders elicited by inappropriate activation of TLR7-9.
Assuntos
Glicoproteínas de Membrana/antagonistas & inibidores , Psoríase/tratamento farmacológico , Tioestreptona/farmacologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor Toll-Like 9/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Inflamação/imunologia , Inflamação/patologia , Interleucina-1/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Psoríase/imunologia , Psoríase/patologia , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Pneumonia remains one of the leading causes of death in both adults and children worldwide. Despite the adoption of a wide variety of therapeutics, the mortality from community-acquired pneumonia has remained relatively constant. Although viral and fungal acute airway infections can result in pneumonia, bacteria are the most common cause of community-acquired pneumonia, with Streptococcus pneumoniae isolated in nearly 50% of cases. Pneumolysin is a cholesterol-dependent cytolysin or pore-forming toxin produced by Streptococcus pneumonia and has been shown to play a critical role in bacterial pathogenesis. Airway epithelium is the initial site of many bacterial contacts and its barrier and mucosal immunity functions are central to infectious lung diseases. In our studies, we have shown that the prior exposure to statins confers significant resistance of airway epithelial cells to the cytotoxicity of pneumolysin. We decided to take this study one step further, assessing changes in both the transcriptome and lipidome of human airway epithelial cells exposed to toxin, statin or both. Our current work provides the first global view in human airway epithelial cells of both the transcriptome and the lipid interactions that result in cellular protection from pneumolysin.