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Cell Jan 2022HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur...
HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.
Topics: Aged; Base Sequence; Biological Evolution; Chromatin; Clone Cells; DNA, Viral; Epigenesis, Genetic; Female; HIV-1; Humans; Ionomycin; Male; Middle Aged; Phylogeny; Proviruses; RNA, Viral; Tetradecanoylphorbol Acetate; Transcription, Genetic; Virus Integration; Virus Latency
PubMed: 35026153
DOI: 10.1016/j.cell.2021.12.011 -
Cell Nov 2021Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental...
Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.
Topics: Animals; Antibody Specificity; Bacteria; Bacterial Proteins; Biofilms; Cell Line; Chinchilla; DNA, Bacterial; DNA, Cruciform; Deoxyribonucleases; Extracellular Matrix; Extracellular Space; Extracellular Traps; Humans; Tetradecanoylphorbol Acetate
PubMed: 34735796
DOI: 10.1016/j.cell.2021.10.010 -
PloS One 2023THP-1 monocyte, which can be differentiated into macrophages by PMA, is widely used in researches on pathogen infection and host innate immunity, but reports on the...
THP-1 monocyte, which can be differentiated into macrophages by PMA, is widely used in researches on pathogen infection and host innate immunity, but reports on the induction methods of PMA are different and lack a unified standard, and the transcriptome characteristics of macrophage compared with THP-1 cells remains unclear. In this research, we examined the differentiation effect of three factors including induction time, cell seeding density and PMA concentration by detecting the positive rate of CD14 expression. The concentration of 80ng/ml of PMA, the induction time of 24h, and the cell seeding density of 5×105 cells/ml, could respectively facilitates a relatively higher CD14 positive rate in THP-1 cells. Under this optimized conditions, the CD14 positive rate of THP-1 cells can reach 66.52%. Transcriptome sequencing showed that after the above induction, the mRNA expression of 3113 genes which were closely related to cell communication, signal transduction, cell response to stimulus, signaling receptor binding and cytokine activity were up-regulated, and the top 10 genes were RGS1, SPP1, GDF15, IL-1B, HAVCR2, SGK1, EGR2, TRAC, IL-8 and EBI3. While the mRNA expression of 2772 genes which were associated with cell cycle process, DNA binding and replication and cell division, were down-regulated, and the top genes were SERPINB10, TRGC2, SERPINB2, TRGC1, MS4A3, MS4A4E, TRGJP1, MS4A6A, TRGJP2, MS4A4A. This research optimized the induction method on THP-1 cell differentiation from three aspects and delineated the transcriptomic profile of PMA-induced THP-1 cells, laying a foundation for the construction method of cell model and for the functional study of macrophage.
Topics: Humans; Transcriptome; THP-1 Cells; Tetradecanoylphorbol Acetate; Macrophages; Monocytes; Cell Differentiation; RNA, Messenger
PubMed: 37459313
DOI: 10.1371/journal.pone.0286056 -
Cell Aug 2021In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen...
In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.
Topics: Adenosine Diphosphate; Adenosine Monophosphate; Allosteric Regulation; Enzyme Activation; Epithelial Cells; Glycolysis; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Microbial Viability; Models, Molecular; NADPH Oxidases; Neutrophils; Phagocytosis; Phosphate-Binding Proteins; Phosphofructokinase-1, Liver Type; Protein Kinase Inhibitors; Recombinant Proteins; Respiratory Burst; Tetradecanoylphorbol Acetate
PubMed: 34320407
DOI: 10.1016/j.cell.2021.07.004 -
Redox Biology Aug 2023Irisin is a newly discovered myokine which links exercise to inflammation and inflammation-related diseases through macrophage regulation. However, the effect of irisin...
INTRODUCTION
Irisin is a newly discovered myokine which links exercise to inflammation and inflammation-related diseases through macrophage regulation. However, the effect of irisin on the activity of inflammation related immune cells (such as neutrophils) has not been clearly described.
OBJECTIVES
The objective of our study was to explore the effect of irisin on the neutrophil extracellular traps (NETs) formation.
METHODS
Phorbol-12-myristate-13-acetate (PMA) was used to construct a classic neutrophil inflammation model that was used to observe the formation of NETs in vitro. We studied the effect of irisin on NETs formation and its regulation mechanism. Subsequently, acute pancreatitis (AP) was used to verify the protective effect of irisin in vivo, which was an acute aseptic inflammatory response disease model closely related to NETs.
RESULTS
Our study found that addition of irisin significantly reduced the formation of NETs via regulation of the P38/MAPK pathway through integrin αVβ5, which might be the one of key pathways in NETs formation, and which could theoretically offset the immunoregulatory effect of irisin. Systemic treatment with irisin reduced the severity of tissue damage common in the disease and inhibited the formation of NETs in pancreatic necrotic tissue of two classical AP mouse models.
CONCLUSION
The findings confirmed for the first time that irisin could inhibit NETs formation and protect mice from pancreatic injury, which further elucidated the protective effect of exercise on acute inflammatory injury.
Topics: Mice; Animals; Extracellular Traps; Pancreatitis; Fibronectins; Acute Disease; Neutrophils; Inflammation; Tetradecanoylphorbol Acetate
PubMed: 37392517
DOI: 10.1016/j.redox.2023.102787 -
Immunology and Cell Biology Nov 2022For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate,...
For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ), after which it differentiates and polarizes into proinflammatory M1-like macrophages. For the quantification of this and the effect of different factors affecting these processes, the expression levels of various maturation markers are determined using reverse transcription-quantitative PCR. For this purpose, stably expressed reference genes are crucial. However, no studies evaluating the stability of reference genes in THP-1 cells stimulated with LPS and IFN-γ have been performed. Therefore, this paper describes the selection of the most used reference genes [RPL37A (ribosomal protein L37a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), UBC (ubiquitin C), B2M (0β2-microbulin), ACTB (β-actin) and PPIA (cyclophilin A)], the in silico primer design, the analysis and the validation of these in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and more recent recommendations for the validation of the stability of reference genes. Using the RefFinder platform, including the four most popular algorithms for reference gene validation, the Delta CT, BestKeeper, NormFinder and geNorm, we find the reference genes GAPDH and UBC to be the most stable. Furthermore, we demonstrate that the normalization of gene expression data using the least stable reference genes, ACTB and B2M, dramatically affects the interpretation of experimental data. Taken together, it is vital to validate the stability of reference genes under the specific experimental conditions used when utilizing the THP-1 monocyte model system.
Topics: Humans; Real-Time Polymerase Chain Reaction; THP-1 Cells; Lipopolysaccharides; Macrophages; Monocytes; Tetradecanoylphorbol Acetate; Gene Expression Profiling
PubMed: 36184577
DOI: 10.1111/imcb.12590 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... Sep 2023Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According... (Review)
Review
Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According to the different functional groups, they can be classified into types of phorbol esters, C-4 deoxyphorbol esters, C-12 deoxyphorbol esters, C-16 or C-17 substituted phorbol esters and others. Most of them present promising antiviral activities and cytotoxic activities and are expected to be developed as candidates for anti-AIDS, anti-tuberculosis, and anti-tumor clinical trials, demonstrating great potential for the application in healthcare. This paper reviews 115 novel tigliane-type diterpenoids discovered since 2013 and summarize their chemical structures and bioactivities, aiming to lay a foundation for further development and utilization of these compounds and provide new ideas for the development of clinical drugs.
Topics: Phorbols; Molecular Structure; Diterpenes; Antiviral Agents; Phorbol Esters
PubMed: 37802801
DOI: 10.19540/j.cnki.cjcmm.20230427.201 -
ELife Oct 2023Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt...
Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.
Topics: Female; Pregnancy; Humans; Animals; Mice; Carcinogens; Wnt Signaling Pathway; Glycogen Synthase Kinase 3; Phorbol Esters; Esters; Neoplasms
PubMed: 37902809
DOI: 10.7554/eLife.89141 -
Frontiers in Immunology 2022Neutrophil extracellular traps (NETs)-as double-edged swords of innate immunity-are involved in numerous processes such as infection, inflammation and tissue repair....
BACKGROUND
Neutrophil extracellular traps (NETs)-as double-edged swords of innate immunity-are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs.
METHODS
We analysed the prolongation of neutrophil survival under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO).
RESULTS
Untreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α.
CONCLUSIONS
G-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions.
Topics: Cytokines; Granulocyte Colony-Stimulating Factor; Lipopolysaccharides; Neutrophils; Peroxidase; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha
PubMed: 35242132
DOI: 10.3389/fimmu.2022.815412 -
Bioorganic & Medicinal Chemistry Letters Apr 2020TRPV4 is a ubiquitously expressed, non-selective cation channel activated by a range of stimuli including hypotonicity, temperature, pH, stretch and endogenous ligands.... (Review)
Review
TRPV4 is a ubiquitously expressed, non-selective cation channel activated by a range of stimuli including hypotonicity, temperature, pH, stretch and endogenous ligands. Agents that modulate TRPV4 are sought as potential therapeutics for the treatment of many diseases including osteoarthritis, respiratory illnesses, gastrointestinal disorders, pain and congestive heart failure. In recent years, significant advances in TRPV4 drug discovery have been realized as at least seven novel TRPV4 agonist or antagonist templates were reported and the first selective TRPV4 antagonist was evaluated in early clinical trials.
Topics: Biological Products; Drug Discovery; Humans; Models, Molecular; Molecular Structure; Phorbols; TRPV Cation Channels
PubMed: 32063431
DOI: 10.1016/j.bmcl.2020.127022