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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 -
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 -
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 -
Biomedicine & Pharmacotherapy =... Jul 2023Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung... (Review)
Review
Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung disorders. TRPV4 expresses in lung tissue and plays an important role in the maintenance of respiratory homeostatic function. TRPV4 is upregulated in life-threatening respiratory diseases like pulmonary hypertension, asthma, cystic fibrosis, and chronic obstructive pulmonary diseases. TRPV4 is linked to several proteins that have physiological functions and are sensitive to a wide variety of stimuli, such as mechanical stimulation, changes in temperature, and hypotonicity, and responds to a variety of proteins and lipid mediators, including anandamide (AA), the arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid (5,6-EET), a plant dimeric diterpenoid called bisandrographolide A (BAA), and the phorbol ester 4-alpha-phorbol-12,13-didecanoate (4α-PDD). This study focused on relevant research evidence of TRPV4 in lung disorders and its agonist and antagonist effects. TRPV4 can be a possible target of discovered molecules that exerts high therapeutic potential in the treatment of respiratory diseases by inhibiting TRPV4.
Topics: Humans; Transient Receptor Potential Channels; TRPV Cation Channels; Phorbol Esters; Hypertension, Pulmonary
PubMed: 37178575
DOI: 10.1016/j.biopha.2023.114861 -
Cells Sep 2023Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the...
Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.
Topics: Neutrophils; Calcimycin; Microscopy, Atomic Force; Extracellular Traps; Cell Nucleus; Tetradecanoylphorbol Acetate
PubMed: 37681931
DOI: 10.3390/cells12172199 -
Scientific Reports Jul 2023To further elucidate the expression, regulation and function of Signaling Lymphocytic Activation Molecule Family (SLAMF) protein members in human monocytes and...
To further elucidate the expression, regulation and function of Signaling Lymphocytic Activation Molecule Family (SLAMF) protein members in human monocytes and macrophages. Un-differentiated monocytic THP-1 cell (u-THP-1) and differentiated THP-1 macrophage (d-THP-1) were used as culture models in the study. Responses of cells to the differentiation agents phorbol ester (25 ng/ml) and TLR (Toll-like receptor) ligands were assessed. RT-PCR and Western blot analysis were used to determine mRNA and protein level. Pro-inflammatory cytokine mRNA expression levels and phagocytosis were used as functional markers. Data analyzed using t-test, one-way or two-way ANOVA followed by post hoc test. SLAMFs were differentially expressed in THP-1 cells. Differentiation of u-THP-1 to d-THP-1 led to significantly higher SLAMF7 mRNA and protein levels than other SLAMF. In addition, TLR stimuli increased SLAMF7 mRNA expression but not protein expression. Importantly, SLAMF7 agonist antibody and TLR ligands synergistically increased the mRNA expression levels of IL-1β, IL-6 and TNF-α, but had no effect on phagocytosis. SLAMF7 knocked-down in d-THP-1 significantly lowered TLR-induced mRNA expressions of pro-inflammatory markers. SLAM family proteins are differentially regulated by differentiation and TLRs. SLAMF7 enhanced TLR-mediated induction of pro-inflammatory cytokines in monocytes and macrophages but not phagocytosis.
Topics: Humans; Cytokines; Family; Ligands; Lipopolysaccharides; Macrophages; Monocytes; RNA, Messenger; Signaling Lymphocytic Activation Molecule Family; Toll-Like Receptors
PubMed: 37420084
DOI: 10.1038/s41598-023-37040-0 -
BioRxiv : the Preprint Server For... Aug 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.
PubMed: 37333286
DOI: 10.1101/2023.06.02.543509 -
Cell Reports Jun 2023Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic...
Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, our analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A's calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity.
Topics: Animals; Drosophila Proteins; Calmodulin; Presynaptic Terminals; Drosophila; Synaptic Transmission; Synapses; Neuronal Plasticity
PubMed: 37243591
DOI: 10.1016/j.celrep.2023.112541 -
Arteriosclerosis, Thrombosis, and... Sep 2023Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic...
BACKGROUND
Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches.
METHODS
Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbβ3.
RESULTS
We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca] rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide.
CONCLUSIONS
Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.
Topics: Blood Platelets; Platelet Glycoprotein GPIIb-IIIa Complex; Chemokines; Thrombosis; Neutrophil Activation; CD40 Ligand; Neutrophils; Cell Adhesion; Humans; Arteries
PubMed: 37409530
DOI: 10.1161/ATVBAHA.122.318767 -
Pflugers Archiv : European Journal of... Jan 2024Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal...
Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal phosphate and vitamin D homeostasis. Soluble Klotho (sKL) is released from the transmembrane form and controls various cellular functions as a paracrine and endocrine factor. αKlotho deficiency accelerates aging, whereas its overexpression favors longevity. Higher αKlotho abundance confers a better prognosis in cardiovascular and renal disease owing to anti-inflammatory, antifibrotic, or antioxidant effects and tumor suppression. Serine/threonine protein kinase C (PKC) is ubiquitously expressed, affects several cellular responses, and is also implicated in heart or kidney disease as well as cancer. We explored whether PKC is a regulator of αKlotho. Experiments were performed in renal MDCK or NRK-52E cells and PKC isoform and αKlotho expression determined by qRT-PCR and Western Blotting. In both cell lines, PKC activation with phorbol ester phorbol-12-myristate-13-acetate (PMA) downregulated, while PKC inhibitor staurosporine enhanced αKlotho mRNA abundance. Further experiments with PKC inhibitor Gö6976 and RNA interference suggested that PKCγ is the major isoform for the regulation of αKlotho gene expression in the two cell lines. In conclusion, PKC is a negative regulator of αKlotho gene expression, an effect which may be relevant for the unfavorable effect of PKC on heart or kidney disease and tumorigenesis.
Topics: Humans; Protein Kinase C; Glucuronidase; Fibroblast Growth Factors; Protein Isoforms; Gene Expression; Kidney Diseases
PubMed: 37773536
DOI: 10.1007/s00424-023-02863-3