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Frontiers in Pharmacology 2021Qingfei Paidu decoction (QPD) and Xuanfei Baidu decoction (XBD) are two typical traditional Chinese medicines with proven efficacy for the treatment of SARS-CoV-2,...
Traditional Chinese Medicine, Qingfei Paidu Decoction and Xuanfei Baidu Decoction, Inhibited Cytokine Production NF-κB Signaling Pathway in Macrophages: Implications for Coronavirus Disease 2019 (COVID-19) Therapy.
Qingfei Paidu decoction (QPD) and Xuanfei Baidu decoction (XBD) are two typical traditional Chinese medicines with proven efficacy for the treatment of SARS-CoV-2, although the underlying mechanism is not well defined. Blunted immune response and enhanced production of pro-inflammatory cytokines (cytokine storm) are two main features observed in patients infected with SARS-CoV-2. Analysis based on network pharmacology has revealed that both QPD and XBD played an important role in the regulation of host immunity. We therefore investigated the role of QPD and XBD in the modulation of innate immunity , focusing on the type 1 interferon (IFN) signaling pathway in A549 cells and pro-inflammatory cytokine production in macrophages. A549 cells were treated with QPD or XBD and the production of endogenous IFNα and IFNβ as well as the expression levels of some interferon-stimulated genes (ISGs) were detected by reverse transcriptase-quantitative PCR (RT-qPCR). Macrophages derived from THP-1 cells were treated with QPD or XBD and their pro-inflammatory cytokine expression levels were measured by RT-qPCR, 6 h post LPS stimulation. In addition, the expression levels of some pro-inflammatory cytokines were further analyzed by ELISA. The effect of QPD and XBD on the NF-κB signaling pathway and the pinocytosis activity of THP-1-derived macrophages were evaluated by Western blot and neutral red uptake assay, respectively. Although QPD and XBD showed very little effect on the type 1 IFN signaling pathway in A549 cells, either QPD or XBD markedly inhibited the production of pro-inflammatory markers including interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1, and chemokine ligand 10 in THP-1-derived M1 macrophages. In addition, the phosphorylation of IκBα and NF-κB p65 during the process of macrophage polarization was significantly suppressed following QPD or XBD treatment. QPD and XBD also suppressed the pinocytosis activity of macrophages. QPD and XBD have been shown to have robust anti-inflammatory activities . Our study demonstrated that both QPD and XBD decreased pro-inflammatory cytokine expression, inhibited the activation of the NF-κB signaling pathway, and blunted pinocytosis activity in THP-1-derived macrophages.
PubMed: 34764867
DOI: 10.3389/fphar.2021.722126 -
Pharmaceuticals (Basel, Switzerland) Dec 2022Plants have been considered for many years as an important source of medicine to treat different diseases. L. (Asteraceae, Compositae) is known for its diuretic,...
Plants have been considered for many years as an important source of medicine to treat different diseases. L. (Asteraceae, Compositae) is known for its diuretic, anti-inflammatory, and sedative effects. It is also used in the treatment of several ailments, such as cancer. In order to evaluate the anticancer and immunomodulatory activities, crude ethanol extract was prepared from the aerial part of and then fractionated using solvents with different polarities. As well, the chemical composition of extract and fractions were identified using LC-MS analysis. The antitumor effect of was assessed in both in vitro and in vivo models. Apoptosis induction was measured in vitro using a caspase-3 activity kit. Lymphocyte proliferation and phagocytosis and pinocytosis induction were used to quantify the effect of the plant extract and fractions on acquired and innate immunity, respectively. The effect of extract, and fractions on the levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10) in murine lymphocytes was determined using a mouse-uncoated TH1/TH2 ELISA kit. Results showed that ethanol extract had the highest antiproliferative activity (IC₅₀ = 2.5 mg mL) against EMT6/P cell lines, while the aqueous and chloroform fractions had the highest apoptotic activity with 2.2 and 1.7 folds, respectively. On the other hand, the -hexane fraction was the most effective in stimulating lymphocyte proliferation, whereas ethanol extract, aq. Methanol and aqueous fractions exhibited the highest phagocytic activity. As well, extract and fractions were able to modulate the expression of IL-2, IL-4, and IFN-γ. A remarkable decrease in tumor size was accomplished following the treatment of tumor-bearing mice with extract and fractions. Both aq. Methanol and chloroform fractions showed the highest percentage change in tumor size with -58 and -55%, respectively. As well, tumor-bearing mice treated with chloroform fraction demonstrated a high curable percentage with a value of 57.1%. Anyway, extract and fractions exhibited no toxic impact on the liver or kidney functions of the mice-treated groups. These findings may confirm that has favorable anticancer and immunomodulatory effects. However, additional studies are required to fully understand the mechanisms of action of this plant and the signaling pathways involved in its effects. Moreover, more testing is needed to have better insight into the apoptotic pathway and to know the exact concentration of active compounds.
PubMed: 36558955
DOI: 10.3390/ph15121504 -
Frontiers in Cellular and Infection... 2023is the main causative agent of hospital-acquired diarrhea and the potentially lethal disease, infection. The cornerstone of the current therapy is the use of...
is the main causative agent of hospital-acquired diarrhea and the potentially lethal disease, infection. The cornerstone of the current therapy is the use of antibiotics, which is not fully effective. The molecular mechanisms, inflammatory conditions and host-immune responses that could benefit the persistence or elimination of remain unclear. Macrophages perform different ways of endocytosis as part of their immune surveillance functions and platelets, classically known for their coagulatory role, are also important modulators of the immune system. The aim of this study was to evaluate the endocytosis of vegetative by human macrophages and the involvement of platelets in this process. Our results showed that both macrophages and platelets interact with live and heat-killed . Furthermore, platelets form complexes with human monocytes in healthy donor's fresh blood and the presence of increased these cell-cell interactions. Using flow cytometry and confocal microscopy, we show that macrophages can internalize and that platelets improve this uptake. By using inhibitors of different endocytic pathways, we demonstrate that macropinocytosis is the route of entry of into the cell. Taken together, our findings are the first evidence for the internalization of vegetative non-toxigenic and hypervirulent by human macrophages and highlight the role of platelets in innate immunity during infection. Deciphering the crosstalk of with immune cells could provide new tools for understanding the pathogenesis of infection and for the development of host-directed therapies.
Topics: Humans; Clostridioides difficile; Clostridioides; Blood Platelets; Macrophages; Pinocytosis
PubMed: 38249298
DOI: 10.3389/fcimb.2023.1252509 -
Journal of Extracellular Vesicles Jun 2021Oncogenic RAS impacts communication between cancer cells and their microenvironment, but it is unclear how this process influences cellular interactions with...
Oncogenic RAS impacts communication between cancer cells and their microenvironment, but it is unclear how this process influences cellular interactions with extracellular vesicles (EVs). This is important as intercellular EV trafficking plays a key role in cancer invasion and metastasis. Here we report that overexpression of mutant RAS drives the EV internalization switch from endocytosis (in non-transformed cells) to macropinocytosis (in cancer cells) resulting in enhanced EV uptake. This process depends on the surface proteoglycan, fibronectin and EV engulfment mechanism regulated by CRAF. Both mutant RAS and activated CRAF expression is associated with formation of membrane ruffles to which they colocalize along with actin, sodium-hydrogen exchangers (NHEs) and phosphorylated myosin phosphatase (pMYPT). RAS-transformed cells internalize EVs in the vicinity of ruffled structures followed by apparent trafficking to lysosome and degradation. NHE inhibitor (EIPA) suppresses RAS-driven EV uptake, along with adhesion-independent clonal growth and experimental metastasis in mice. Thus, EV uptake may represent a targetable step in progression of RAS-driven cancers.
Topics: Animals; Biological Transport; Cell Communication; Cell Line, Tumor; Cell Transformation, Neoplastic; Endocytosis; Extracellular Vesicles; Genes, ras; Humans; Mice; Mice, SCID; Neoplasm Metastasis; Neoplastic Processes; Pinocytosis; Proto-Oncogene Proteins c-raf; Tumor Microenvironment; ras Proteins
PubMed: 34136107
DOI: 10.1002/jev2.12091 -
Scientific Reports Apr 2021The ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet. This metabolic shift is mainly...
The ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet. This metabolic shift is mainly driven by transcriptional regulation of genes for tetrahymanol synthesis (TS) and sterol bioconversion (SB). The mechanistic details of sterol uptake, intracellular trafficking and the associated gene expression changes are unknown. By following cholesterol incorporation over time in a conditional phagocytosis-deficient mutant, we found that although phagocytosis is the main sterol intake route, a secondary endocytic pathway exists. Different expression patterns for TS and SB genes were associated with these entry mechanisms. Squalene synthase was down-regulated by a massive cholesterol intake only attainable by phagocytosis-proficient cells, whereas C22-sterol desaturase required ten times less cholesterol and was up-regulated in both wild-type and mutant cells. These patterns are suggestive of at least two different signaling pathways. Sterol trafficking beyond phagosomes and esterification was impaired by the NPC1 inhibitor U18666A. NPC1 is a protein that mediates cholesterol export from late endosomes/lysosomes in mammalian cells. U18666A also produced a delay in the transcriptional response to cholesterol, suggesting that the regulatory signals are triggered between lysosomes and the endoplasmic reticulum. These findings could hint at partial conservation of sterol homeostasis between eukaryote lineages.
Topics: Animals; Biological Transport; Cholesterol; Endoplasmic Reticulum; Endosomes; Gene Expression Regulation; Homeostasis; Humans; Phagocytosis; Pinocytosis; Protozoan Proteins; Signal Transduction; Sterols; Tetrahymena thermophila
PubMed: 33907281
DOI: 10.1038/s41598-021-88737-z -
The EMBO Journal Oct 2020Genetic variation in LRRK2 associates with the susceptibility to Parkinson's disease, Crohn's disease, and mycobacteria infection. High expression of LRRK2 and its...
Genetic variation in LRRK2 associates with the susceptibility to Parkinson's disease, Crohn's disease, and mycobacteria infection. High expression of LRRK2 and its substrate Rab10 occurs in phagocytic cells in the immune system. In mouse and human primary macrophages, dendritic cells, and microglia-like cells, we find that Rab10 specifically regulates a specialized form of endocytosis known as macropinocytosis, without affecting phagocytosis or clathrin-mediated endocytosis. LRRK2 phosphorylates cytoplasmic PI(3,4,5)P3-positive GTP-Rab10, before EEA1 and Rab5 recruitment to early macropinosomes occurs. Macropinosome cargo in macrophages includes CCR5, CD11b, and MHCII, and LRRK2-phosphorylation of Rab10 potently blocks EHBP1L1-mediated recycling tubules and cargo turnover. EHBP1L1 overexpression competitively inhibits LRRK2-phosphorylation of Rab10, mimicking the effects of LRRK2 kinase inhibition in promoting cargo recycling. Both Rab10 knockdown and LRRK2 kinase inhibition potently suppress the maturation of macropinosome-derived CCR5-loaded signaling endosomes that are critical for CCL5-induced immunological responses that include Akt activation and chemotaxis. These data support a novel signaling axis in the endolysosomal system whereby LRRK2-mediated Rab10 phosphorylation stalls vesicle fast recycling to promote PI3K-Akt immunological responses.
Topics: Animals; Carrier Proteins; Cell Membrane; Chemokine CCL5; Chemotaxis; Dendritic Cells; Endosomes; Female; Gene Knockdown Techniques; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Macrophages; Male; Mass Spectrometry; Mice; Mice, Transgenic; Microglia; Monocytes; Mutation; Phagocytes; Phosphorylation; Pinocytosis; Protein Binding; Proto-Oncogene Proteins c-akt; Signal Transduction; rab GTP-Binding Proteins
PubMed: 32853409
DOI: 10.15252/embj.2020104862 -
Biology Open Nov 2023Autotaxin, encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid, thereby regulating many biologic functions. We previously reported that Enpp2...
Autotaxin, encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid, thereby regulating many biologic functions. We previously reported that Enpp2 mRNA was abundantly expressed in yolk sac visceral endoderm (VE) cells and that Enpp2-/- mice were lethal at embryonic day 9.5 owing to angiogenic defects in the yolk sac. Enpp2-/- mice showed lysosome fragmentation in VE cells and embryonic abnormalities including allantois malformation, neural tube defects, no axial turning, and head cavity formation. However, whether the defects in endocytic vesicle formation affect membrane trafficking in VE cells remained to be directly examined. In this study, we found that pinocytosis, transcytosis, and secretion of angiogenic factors such as vascular endothelial growth factor and transforming growth factor β1 were impaired in Enpp2-/- VE cells. Moreover, pharmacologic inhibition of membrane trafficking phenocopied the defects of Enpp2-/- mice. These findings demonstrate that Enpp2 promotes endocytosis and secretion of angiogenic factors in VE cells, thereby regulating angiogenesis/vasculogenesis and embryonic development.
Topics: Animals; Female; Mice; Pregnancy; Cell Differentiation; Embryonic Development; Endoderm; Vascular Endothelial Growth Factor A; Yolk Sac; Phosphoric Diester Hydrolases
PubMed: 37795611
DOI: 10.1242/bio.060081 -
Vascular Pharmacology Jun 2021Smooth muscle cells contribute significantly to lipid-laden foam cells in atherosclerotic plaques. However, the underlying mechanisms transforming smooth muscle cells...
OBJECTIVE
Smooth muscle cells contribute significantly to lipid-laden foam cells in atherosclerotic plaques. However, the underlying mechanisms transforming smooth muscle cells into foam cells are poorly understood. The purpose of this study was to gain insight into the molecular mechanisms regulating smooth muscle foam cell formation.
APPROACH AND RESULTS
Using human coronary artery smooth muscle cells we found that the transcriptional co-activator MRTFA promotes lipid accumulation via several mechanisms, including direct transcriptional control of LDL receptor, enhanced fluid-phase pinocytosis and reduced lipid efflux. Inhibition of MRTF activity with CCG1423 and CCG203971 significantly reduced lipid accumulation. Furthermore, we demonstrate enhanced MRTFA expression in vascular remodeling of human vessels.
CONCLUSIONS
This study demonstrates a novel role for MRTFA as an important regulator of lipid homeostasis in vascular smooth muscle cells. Thus, MRTFA could potentially be a new therapeutic target for inhibition of vascular lipid accumulation.
Topics: Cell Transdifferentiation; Coronary Vessels; Foam Cells; HEK293 Cells; Humans; Lipid Metabolism; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Pinocytosis; Receptors, LDL; Trans-Activators; Up-Regulation; Vascular Remodeling
PubMed: 33516965
DOI: 10.1016/j.vph.2021.106837 -
Frontiers in Immunology 2022Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in...
Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism . C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.
Topics: Dictyostelium; Chemotaxis; Pinocytosis; Phagocytosis; Folic Acid
PubMed: 36524124
DOI: 10.3389/fimmu.2022.1075386 -
Nature Dec 2019Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express...
Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express oncogenic RAS are able to internalize and degrade extracellular protein via a fluid-phase uptake mechanism termed macropinocytosis. There is increasing recognition of the role of this RAS-dependent process in the generation of free amino acids that can be used to support tumour cell growth under nutrient-limiting conditions. However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic RAS. Here we identify vacuolar ATPase (V-ATPase) as an essential regulator of RAS-induced macropinocytosis. Oncogenic RAS promotes the translocation of V-ATPase from intracellular membranes to the plasma membrane via a pathway that requires the activation of protein kinase A by a bicarbonate-dependent soluble adenylate cyclase. Accumulation of V-ATPase at the plasma membrane is necessary for the cholesterol-dependent plasma-membrane association of RAC1, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations establish a link between V-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of RAS-mutant tumour cells.
Topics: Animals; Bicarbonates; Carcinogenesis; Cell Line, Tumor; Cell Membrane; Cholesterol; Cyclic AMP-Dependent Protein Kinases; Female; Humans; Mice; Mice, Nude; Neoplasms; Oncogene Protein p21(ras); Pinocytosis; Signal Transduction; Sodium-Bicarbonate Symporters; Vacuolar Proton-Translocating ATPases
PubMed: 31827278
DOI: 10.1038/s41586-019-1831-x