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Theranostics 2020Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by autoantibody production by hyper-activated B cells. Although mesenchymal stem...
Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by autoantibody production by hyper-activated B cells. Although mesenchymal stem cells (MSCs) ameliorate lupus symptoms by inhibiting T cells, whether they inhibit B cells has been controversial. Here we address this issue and reveal how to prime MSCs to inhibit B cells and improve the efficacy of MSCs in SLE. We examined the effect of MSCs on purified B cells and the therapeutic efficacy of MSCs in lupus-prone MRL. mice. We screened chemicals for their ability to activate MSCs to inhibit B cells. Mouse bone marrow-derived MSCs inhibited mouse B cells in a CXCL12-dependent manner, whereas human bone marrow-derived MSCs (hMSCs) did not inhibit human B (hB) cells. We used a chemical approach to overcome this hurdle and found that phorbol myristate acetate (PMA), phorbol 12,13-dibutyrate, and ingenol-3-angelate rendered hMSCs capable of inhibiting IgM production by hB cells. As to the mechanism, PMA-primed hMSCs attracted hB cells in a CXCL10-dependent manner and induced hB cell apoptosis in a PD-L1-dependent manner. Finally, we showed that PMA-primed hMSCs were better than naïve hMSCs at ameliorating SLE progression in MRL. mice. Taken together, our data demonstrate that phorbol esters might be good tool compounds to activate MSCs to inhibit B cells and suggest that our chemical approach might allow for improvements in the therapeutic efficacy of hMSCs in SLE.
Topics: Animals; Apoptosis; B-Lymphocytes; Cells, Cultured; Female; Humans; Lupus Erythematosus, Systemic; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C3H; Phorbol Esters; T-Lymphocytes
PubMed: 32929342
DOI: 10.7150/thno.46835 -
Phorbol myristate acetate induces differentiation of THP-1 cells in a nitric oxide-dependent manner.Nitric Oxide : Biology and Chemistry May 2021THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Differentiated...
INTRODUCTION
THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Differentiated THP-1 cells acquire macrophage-like characteristics including more adherence and altered cell function. Nitric oxide (NO), an intracellular messenger, is critical in regulating cell differentiation. Here we elucidated whether NO relates to PMA-induced monocyte-to-macrophage differentiation of THP-1 cells. The mutual regulation of calcium and NO was also investigated.
MATERIAL & METHODS
THP-1 cells were incubated with PMA for 24 h, followed by assay of adherence, morphological change, migration or IL-1β release. L-N-Nitroarginine methyl ester (l-NAME, a nitric oxide synthase inhibitor) or BAPTA-AM (a calcium chelator) was added before PMA stimulation, and levels of calcium and NO were measured. Furthermore, a selective inhibitor of inducible nitric oxide synthase (iNOS) activity was employed to study the role of iNOS.
RESULTS AND DISCUSSION
Effects of PMA on upregulation of adherence, lipopolysaccharide-triggered IL-1β, and migration ability of THP-1 cells were consistent with NO concentrations. Both l-NAME and BAPTA-AM mitigated effects of PMA on THP-1 cells differentiation. BAPTA-AM decreased levels of NO, while l-NAME had no effect on calcium levels. Of note, inhibition of iNOS activity decreased PMA-triggered upregulation of NO.
CONCLUSION
PMA induced differentiation of THP-1 cells partially in a NO-dependent manner. The calcium signaling may mediate PMA-triggered upregulation of NO.
Topics: Calcium; Calcium Signaling; Cell Differentiation; Cell Movement; Humans; Macrophages; Monocytes; Nitric Oxide; Nitric Oxide Synthase Type II; THP-1 Cells; Tetradecanoylphorbol Acetate; Up-Regulation
PubMed: 33667621
DOI: 10.1016/j.niox.2021.02.002 -
Epigenetics 2020The transcriptional program that dictates haematopoietic cell fate and differentiation requires an epigenetic regulatory and memory function, provided by a network of...
The transcriptional program that dictates haematopoietic cell fate and differentiation requires an epigenetic regulatory and memory function, provided by a network of epigenetic factors that regulate DNA methylation, post-translational histone modifications and chromatin structure. Disturbed epigenetic regulation causes perturbations in the blood cell differentiation program that results in various types of haematopoietic disorders. Thus, accurate epigenetic regulation is essential for functional haematopoiesis. In this study, we used a CRISPR-Cas9 screening approach to identify new epigenetic regulators in myeloid differentiation. We designed a Chromatin-UMI CRISPR guide library targeting 1092 epigenetic regulators. Phorbol 12-myristate 13-acetate (PMA) treatment of the chronic myeloid leukaemia cell line K-562 was used as a megakaryocytic myeloid differentiation model. Both previously described developmental epigenetic regulators and novel factors were identified in our screen. In this study, we validated and characterized a role for the chromatin remodeller CHD2 in myeloid proliferation and megakaryocytic differentiation.
Topics: Cell Proliferation; Chromatin Assembly and Disassembly; DNA-Binding Proteins; Humans; K562 Cells; Megakaryocytes; Myelopoiesis; Tetradecanoylphorbol Acetate
PubMed: 31900031
DOI: 10.1080/15592294.2019.1710913 -
European Journal of Pharmacology Oct 2020In a cell line, stably expressing α-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced...
In a cell line, stably expressing α-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.
Topics: Adrenergic alpha-1 Receptor Agonists; Calcium; Cell Line; Endosomes; Humans; Luminescent Proteins; Methoxamine; Norepinephrine; Oxymetazoline; Phorbol Esters; Phosphorylation; Receptors, Adrenergic, alpha-1; Tetradecanoylphorbol Acetate; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins; trans-Golgi Network; Red Fluorescent Protein
PubMed: 32750368
DOI: 10.1016/j.ejphar.2020.173423 -
Cells Mar 2022Nociceptin and the nociceptin receptor (NOP) have been described as targets for treatment of pain and inflammation, whereas toll-like receptors (TLRs) play key roles in...
Nociceptin and the nociceptin receptor (NOP) have been described as targets for treatment of pain and inflammation, whereas toll-like receptors (TLRs) play key roles in inflammation and impact opioid receptors and endogenous opioids expression. In this study, interactions between the nociceptin and TLR systems were investigated. Human THP-1 cells were cultured with or without phorbol myristate acetate (PMA 5 ng/mL), agonists specific for TLR2 (lipoteichoic acid, LTA 10 µg/mL), TLR4 (lipopolysaccharide, LPS 100 ng/mL), TLR7 (imiquimod, IMQ 10 µg/mL), TLR9 (oligonucleotide (ODN) 2216 1 µM), PMA+TLR agonists, or nociceptin (0.01−100 nM). Prepronociceptin (ppNOC), NOP, and TLR mRNAs were quantified by RT-qPCR. Proteins were measured using flow cytometry. PMA upregulated ppNOC mRNA, intracellular nociceptin, and cell membrane NOP proteins (all p < 0.05). LTA and LPS prevented PMA’s upregulating effects on ppNOC mRNA and nociceptin protein (both p < 0.05). IMQ and ODN 2216 attenuated PMA’s effects on ppNOC mRNA. PMA, LPS, IMQ, and ODN 2216 increased NOP protein levels (all p < 0.05). PMA+TLR agonists had no effects on NOP compared to PMA controls. Nociceptin dose-dependently suppressed TLR2, TLR4, TLR7, and TLR9 proteins (all p < 0.01). Antagonistic effects observed between the nociceptin and TLR systems suggest that the nociceptin system plays an anti-inflammatory role in monocytes under inflammatory conditions.
Topics: Humans; Inflammation; Lipopolysaccharides; Opioid Peptides; RNA, Messenger; Tetradecanoylphorbol Acetate; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 7; Toll-Like Receptor 9; Toll-Like Receptors; Nociceptin
PubMed: 35406649
DOI: 10.3390/cells11071085 -
Molecules (Basel, Switzerland) Oct 2022is a xerophytic species widely used in Mexico as an ingredient in sweet food and fermented beverages; it is also used in traditional medicine to treat wound pain and...
is a xerophytic species widely used in Mexico as an ingredient in sweet food and fermented beverages; it is also used in traditional medicine to treat wound pain and rheumatic damage, and as a remedy for psoriasis. Among the various extracts and extract fractions that have been evaluated for their anti-inflammatory effects, the acetonic extract (AaAc) and its acetonic (F-Ac) and methanolic (F-MeOH) fractions were the most active in a xylene-induced ear edema model in mice, when orally administered. Four fractions resulting from chemically resolving F-Ac (F1-F4) were locally applied to mice with phorbol 12-myristate 13-acetate (TPA)-induced ear inflammation; F1 inhibited inflammation by 70% and was further evaluated in a carrageenan-induced mono-arthritis model. When administered at doses of 12.5, 25, and 50 mg/kg, F1 reduced articular edema and the spleen index. In addition, it modulated spleen and joint cytokine levels and decreased pain. According to a GC-MS analysis, the main components of F1 are fatty-acid derivatives: palmitic acid methyl ester, palmitic acid ethyl ester, octadecenoic acid methyl ester, linoleic acid ethyl ester, and oleic acid ethyl ester.
Topics: Mice; Animals; Agave; Anti-Inflammatory Agents; Plant Extracts; Fatty Acids; Edema; Carrageenan; Pain; Esters; Phytotherapy
PubMed: 36364031
DOI: 10.3390/molecules27217204 -
Neuroscience Letters Oct 2021An insult can trigger a protective response or even cell death depending on different factors that include the duration and magnitude of the event and the ability of the...
An insult can trigger a protective response or even cell death depending on different factors that include the duration and magnitude of the event and the ability of the cell to activate protective intracellular signals, including inflammatory cytokines. Our previous work showed that the treatment of Lister Hooded rat retinal cell cultures with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, increases the survival of retinal ganglion cells (RGCs) kept in culture for 48 h after axotomy. Here we aim to analyze how PMA modulates the levels of TNF-α and IL-1β (both key inflammatory mediators) and the impact of this modulation on RGCs survival. We hypothesize that the increase in RGCs survival mediated by PMA treatment depends upon modulation of the levels of IL-1β and TNF-α. The effect of PMA treatment was assayed on cell viability, caspase 3 activation, TNF-α and IL-1β release and TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) levels. PMA treatment increases IL-1β and TNF-α levels in 15 min in culture and increases the release of both cytokines after 30 min and 24 h, respectively. Both IL-1β and TNF-α levels decrease after 48 h of PMA treatment. PMA treatment also induces an increase in TNFRII levels while decreasing TNFRI after 24 h. PMA also inhibited caspase-3 activation, and decreased ROS production and EthD-1/calcein ratio in retinal cell cultures leading to an increase in cell viability. The neutralization of IL-1β (anti-IL1β 0,1ng/mL), the neutralization of TNF-α (anti-TNF-α 0,1ng/mL) and the TNF-α inhibition using a recombinant soluble TNFRII abolished PMA effect on RGCs survival. These data suggest that PMA treatment induces IL1β and TNF-α release and modulation of TNFRI/TNFRII expression promoting RGCs survival after axotomy.
Topics: Animals; Animals, Newborn; Axotomy; Cell Survival; Cells, Cultured; Female; Interleukin-1beta; Male; Primary Cell Culture; Protein Kinase C; Rats; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Retinal Ganglion Cells; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha
PubMed: 34437989
DOI: 10.1016/j.neulet.2021.136197 -
Diabetes Mar 2021Low-grade persistent inflammation is a feature of diabetes-driven vascular complications, in particular activation of the Nod-like receptor family pyrin domain...
Low-grade persistent inflammation is a feature of diabetes-driven vascular complications, in particular activation of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome to trigger the maturation and release of the inflammatory cytokine interleukin-1β (IL-1β). We investigated whether inhibiting the NLRP3 inflammasome, through the use of the specific small-molecule NLRP3 inhibitor MCC950, could reduce inflammation, improve vascular function, and protect against diabetes-associated atherosclerosis in the streptozotocin-induced diabetic apolipoprotein E-knockout mouse. Diabetes led to an approximately fourfold increase in atherosclerotic lesions throughout the aorta, which were significantly attenuated with MCC950 ( < 0.001). This reduction in lesions was associated with decreased monocyte-macrophage content, reduced necrotic core, attenuated inflammatory gene expression (IL-1β, tumor necrosis factor-α, intracellular adhesion molecule 1, and MCP-1; < 0.05), and reduced oxidative stress, while maintaining fibrous cap thickness. Additionally, vascular function was improved in diabetic vessels of mice treated with MCC950 ( < 0.05). In a range of cell lines (murine bone marrow-derived macrophages, human monocytic THP-1 cells, phorbol 12-myristate 13-acetate-differentiated human macrophages, and aortic smooth muscle cells from humans with diabetes), MCC950 significantly reduced IL-1β and/or caspase-1 secretion and attenuated leukocyte-smooth muscle cell interactions under high glucose or lipopolysaccharide conditions. In summary, MCC950 reduces plaque development, promotes plaque stability, and improves vascular function, suggesting that targeting NLRP3-mediated inflammation is a novel therapeutic strategy to improve diabetes-associated vascular disease.
Topics: Animals; Apolipoproteins E; Atherosclerosis; Blood Glucose; Cells, Cultured; Fluorescent Antibody Technique; Glucose; Humans; Immunohistochemistry; Inflammasomes; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Smooth Muscle; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; THP-1 Cells; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha
PubMed: 33323396
DOI: 10.2337/db20-0357 -
Veterinary Sciences Sep 2021L. has gained importance as a source of seed oil for biodiesel production. The meal contained about 60% protein with a good balance of essential amino acids, containing... (Review)
Review
L. has gained importance as a source of seed oil for biodiesel production. The meal contained about 60% protein with a good balance of essential amino acids, containing various bioactive compounds, including saponins, phytic acids, trypsin inhibitors, lectins, phenolics, and flavonoids, which render it as a potential biofeed for animal production. The meal demonstrated various biological activities, including antioxidant, antibacterial, and anti-inflammatory effects which enhance its property as a bio-feed. The levels of these bioactive compounds in the seeds are dependent on the genotypes. The possessed different varieties which are either toxic or non-toxic according to the presence of phorbol esters. The presence of phorbol esters in the meal confirmed the toxic variety of resulting in the limited application of meal as a biofeed. The meal devoid of phorbol esters could be applied as a biofeed in the animal production industry, and for the toxic varieties, various techniques such as physicochemical and biological treatments have been introduced to the industry to remove the phorbol esters from meal. Several studies employing various cells and animals confirmed the toxicity of the phorbol esters. The molecular mechanism of action of phorbol esters is through up-regulation of PKC-β II gene, overexpression of down-stream proto-oncogenes resulted in inflammation and oxidative stress ending by apoptotic cell death. Despite the presence of valuable bioactive compounds in the meal, its nutritional application is not recommended unless the phorbol esters are completely removed.
PubMed: 34564573
DOI: 10.3390/vetsci8090179 -
Clinical and Experimental Medicine May 2022Neutrophils (PMNs) contain and release a powerful arsenal of mediators, including several granular enzymes, reactive oxygen species (ROS) and neutrophil extracellular...
Neutrophils (PMNs) contain and release a powerful arsenal of mediators, including several granular enzymes, reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). Although airway neutrophilia is associated with severity, poor response to glucocorticoids and exacerbations, the pathophysiological role of neutrophils in asthma remains poorly understood. Twenty-four patients with asthma and 22 healthy controls (HCs) were prospectively recruited. Highly purified peripheral blood neutrophils (> 99%) were evaluated for ROS production and activation status upon stimulation with lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Plasma levels of myeloperoxidase (MPO), CXCL8, matrix metalloproteinase-9 (MMP-9), granulocyte-monocyte colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF-A) were measured by ELISA. Plasma concentrations of citrullinated histone H3 (CitH3) and circulating free DNA (dsDNA) were evaluated as NET biomarkers. Activated PMNs from asthmatics displayed reduced ROS production and activation status compared to HCs. Plasma levels of MPO, MMP-9 and CXCL8 were increased in asthmatics compared to HCs. CitH3 and dsDNA plasma levels were increased in asthmatics compared to controls and the CitH3 concentrations were inversely correlated to the % decrease in FEV/FVC in asthmatics. These findings indicate that neutrophils and their mediators could have an active role in asthma pathophysiology.
Topics: Asthma; Biomarkers; Extracellular Traps; Histones; Humans; Matrix Metalloproteinase 9; Neutrophils; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; Vascular Endothelial Growth Factor A
PubMed: 34342773
DOI: 10.1007/s10238-021-00750-8