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Frontiers in Immunology 2020Neutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of...
BACKGROUND
Neutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of decondensed chromatin and about 30 enzymes and peptides. Some components, such as neutrophil elastase (NE) and myeloperoxidase (MPO), present antimicrobial but also cytotoxic properties, leading to tissue injury. Many inflammatory diseases are associated with NETs, and their final role has not been identified. Pulmonary surfactant is known to have immunoregulatory abilities that alter the function of adaptive and innate immune cells. The aim of this study was to investigate the hypothesis that natural surfactant preparations inhibit the formation of NETs.
METHODS
The effect of two natural surfactants (Alveofact and Curosurf) on spontaneous and phorbol-12-myristate-13-acetate-induced NET formation by neutrophils isolated by magnetic cell sorting from healthy individuals was examined. NETs were quantitatively detected by absorption and fluorometric-based assays for the NET-specific proteins (NE, MPO) and cell-free DNA. Immunofluorescence microscopy images were used for visualization.
RESULTS
Both surfactant preparations exerted a dose-dependent inhibitory effect on NET formation. Samples treated with higher concentrations and with 30 min pre-incubation prior to stimulation with phorbol-12-myristate-13-acetate had significantly lower levels of NET-specific proteins and cell-free DNA compared to untreated samples. Immunofluorescence microscopy confirmed these findings.
CONCLUSIONS
The described dose-dependent modulation of NET formation ex vivo suggests an interaction between exogenous surfactant supplementation and neutrophil granulocytes. The immunoregulatory effects of surfactant preparations should be considered for further examination of inflammatory diseases.
Topics: Biological Products; Cells, Cultured; Dose-Response Relationship, Drug; Extracellular Traps; Granulocytes; Humans; Immunomodulation; Leukocyte Elastase; Neutrophil Activation; Neutrophils; Phospholipids; Pulmonary Surfactants; Tetradecanoylphorbol Acetate
PubMed: 33574811
DOI: 10.3389/fimmu.2020.582895 -
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 -
Journal of Pharmacological Sciences Oct 2022Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases, and there are no effective drugs available so far. Lactucin and Lactucopicrin...
Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases, and there are no effective drugs available so far. Lactucin and Lactucopicrin belong to sesquiterpene lactones and are extracted from Cichorium glandulosum Boiss. et Huet (CG) possesses multiple biopharmacological activities. However, the therapeutic effects of both Lactucin and Lactucopicrin on many diseases and their underlying mechanisms remain largely unknown. Here, we analyzed the both natural compounds hypolipidemic effects on FFA-induced HepG2 cells and their potential mechanisms based on transcriptomics and experimental tests. Our results indicated that Lactucin (10 μM) and Lactucopicrin (20 μM) remarkably reduced TG accumulation. Transcriptomics analysis identified 1960, 1645, and 1791 differentially expressed genes (DEGs) and obtained 611 and 635 specific genes in different comparisons, respectively. The enrichment analysis and experimental validations (RT-qPCR and Western Blot) showed that their hypolipidemic activities were most probably exerted via regulating numerous key DEGs involved in lipid metabolism. Taken together, both Lactucin and Lactucopicrin may represent potent hepatoprotective agents. Both of them exhibited therapeutic effects against liver diseases such as NAFLD by regulating multi-gene and proteins like HADHA, ADAM17, SQSTM1, and GBA and modulating multi-pathways like fatty acid oxidation metabolic signaling.
Topics: Hep G2 Cells; Humans; Lactones; Lipid Metabolism; Non-alcoholic Fatty Liver Disease; Phorbols; Sesquiterpenes
PubMed: 36055749
DOI: 10.1016/j.jphs.2022.07.007 -
Biochemistry Jul 2019Bryostatin 1 is a natural macrolide shown to improve neuronal connections and enhance memory in mice. Its mechanism of action is largely attributed to the modulation of...
Bryostatin 1 is a natural macrolide shown to improve neuronal connections and enhance memory in mice. Its mechanism of action is largely attributed to the modulation of novel and conventional protein kinase Cs (PKCs) by binding to their regulatory C1 domains. Munc13-1 is a C1 domain-containing protein that shares common endogenous and exogenous activators with novel and conventional PKC subtypes. Given the essential role of Munc13-1 in the priming of synaptic vesicles and neuronal transmission overall, we explored the potential interaction between bryostatin 1 and Munc13-1. Our results indicate that in vitro bryostatin 1 binds to both the isolated C1 domain of Munc13-1 ( K = 8.07 ± 0.90 nM) and the full-length Munc13-1 protein ( K = 0.45 ± 0.04 nM). Furthermore, confocal microscopy and immunoblot analysis demonstrated that in intact HT22 cells bryostatin 1 mimics the actions of phorbol esters, a previously established class of Munc13-1 activators, and induces plasma membrane translocation of Munc13-1, a hallmark of its activation. Consistently, bryostatin 1 had no effect on the Munc13-1 construct that is insensitive to phorbol esters. Effects of bryostatin 1 on the other Munc13 family members, ubMunc13-2 and bMunc13-2, resembled those of Munc13-1 for translocation. Lastly, we observed an increased level of expression of Munc13-1 following a 24 h incubation with bryostatin 1 in both HT22 and primary mouse hippocampal cells. This study characterizes Munc13-1 as a molecular target of bryostatin 1. Considering the crucial role of Munc13-1 in neuronal function, these findings provide strong support for the potential role of Munc13s in the actions of bryostatin 1.
Topics: Animals; Binding Sites; Bryostatins; Cell Line; Cells, Cultured; Mice; Models, Molecular; Molecular Docking Simulation; Nerve Tissue Proteins; Neurons; Phorbol Esters; Protein Binding
PubMed: 31243993
DOI: 10.1021/acs.biochem.9b00427 -
Journal of Innate Immunity 2022Previous research has indicated an intimate functional communication between mast cells (MCs) and neutrophils during inflammatory conditions, but the nature of such...
Previous research has indicated an intimate functional communication between mast cells (MCs) and neutrophils during inflammatory conditions, but the nature of such communication is not fully understood. Activated neutrophils are known to release DNA-containing extracellular traps (neutrophil extracellular traps [NETs]) and, based on the known ability of tryptase to interact with negatively charged polymers, we here hypothesized that tryptase might interact with NET-contained DNA and thereby regulate NET formation. In support of this, we showed that tryptase markedly enhances NET formation in phorbol myristate acetate-activated human neutrophils. Moreover, tryptase was found to bind vividly to the NETs, to cause proteolysis of core histones and to cause a reduction in the levels of citrullinated histone-3. Secretome analysis revealed that tryptase caused increased release of numerous neutrophil granule compounds, including gelatinase, lactoferrin, and myeloperoxidase. We also show that DNA can induce the tetrameric, active organization of tryptase, suggesting that NET-contained DNA can maintain tryptase activity in the extracellular milieu. In line with such a scenario, DNA-stabilized tryptase was shown to efficiently degrade numerous pro-inflammatory compounds. Finally, we showed that tryptase is associated with NET formation in vivo in a melanoma setting and that NET formation in vivo is attenuated in mice lacking tryptase expression. Altogether, these findings reveal that NET formation can be regulated by MC tryptase, thus introducing a novel mechanism of communication between MCs and neutrophils.
Topics: Animals; DNA; Extracellular Traps; Histones; Humans; Mice; Neutrophils; Tryptases
PubMed: 34937018
DOI: 10.1159/000520972 -
Microbiology (Reading, England) Mar 2022subspecies serovar Typhimurium (. Typhimurium) definitive phage type 104 (DT104), . Worthington, and produce ArtAB toxin, which catalyses ADP-ribosylation of...
subspecies serovar Typhimurium (. Typhimurium) definitive phage type 104 (DT104), . Worthington, and produce ArtAB toxin, which catalyses ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene () is encoded on a prophage in , and prophage induction by SOS-inducing agents is associated with increases in ArtAB production . However, little is known about the expression of . Here, we showed a significant increase in transcription of DT104 within macrophage-like RAW264.7 cells. Intracellular expression of ArtAB was also observed by immunofluorescence staining. The induced expression of in DT104 and was enhanced by treatment of RAW264.7 cells with phorbol 12-myristate 13-acetate (PMA), which stimulates the production of reactive oxygen species (ROS); however, such induction was not observed in . Worthington. Upregulation of , a major regulator of oxidative stress, and a repressor of prophage induction, was observed in . Worthington within RAW264.7 cells treated with PMA but not in the DT104 strain. Although the expression of was increased, was upregulated in which lacks the gene in the incomplete -encoded prophage. Taken together, oxidative stress plays a role in the production of toxins in macrophages, and high expression levels of and are responsible for the low expression of . Therefore, strain variation in the level of expression within macrophages could be explained by differences in the oxidative stress response of bacteria and might be reflected in its virulence.
Topics: Macrophages; Prophages; Salmonella typhimurium; Virulence
PubMed: 35333707
DOI: 10.1099/mic.0.001152 -
Bio-protocol May 2023T cells localized to the kidneys and vasculature/perivascular adipose tissue (PVAT) play an important role in hypertension and vascular injury. CD4, CD8, and γδ T-cell...
Determination of Interleukin-17A and Interferon-γ Production in γδ, CD4, and CD8 T Cells Isolated from Murine Lymphoid Organs, Perivascular Adipose Tissue, Kidney, and Lung.
T cells localized to the kidneys and vasculature/perivascular adipose tissue (PVAT) play an important role in hypertension and vascular injury. CD4, CD8, and γδ T-cell subtypes are programmed to produce interleukin (IL)-17 or interferon-γ (IFNγ), and naïve T cells can be induced to produce IL-17 via the IL-23 receptor. Importantly, both IL-17 and IFNγ have been demonstrated to contribute to hypertension. Therefore, profiling cytokine-producing T-cell subtypes in tissues relevant to hypertension provides useful information regarding immune activation. Here, we describe a protocol to obtain single-cell suspensions from the spleen, mesenteric lymph nodes, mesenteric vessels and PVAT, lungs, and kidneys, and profile IL-17A- and IFNγ-producing T cells using flow cytometry. This protocol is different from cytokine assays such as ELISA or ELISpot in that no prior cell sorting is required, and various T-cell subsets can be identified and individually assessed for cytokine production simultaneously within an individual sample. This is advantageous as sample processing is kept to a minimum, yet many tissues and T-cell subsets can be screened for cytokine production in a single experiment. In brief, single-cell suspensions are activated in vitro with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and Golgi cytokine export is inhibited with monensin. Cells are then stained for viability and extracellular marker expression. They are then fixed and permeabilized with paraformaldehyde and saponin. Finally, antibodies against IL-17 and IFNγ are incubated with the cell suspensions to report cytokine production. T-cell cytokine production and marker expression is then determined by running samples on a flow cytometer. While other groups have published methods to perform T-cell intracellular cytokine staining for flow cytometry, this protocol is the first to describe a highly reproducible method to activate, phenotype, and determine cytokine production by CD4, CD8, and γδ T cells isolated from PVAT. Additionally, this protocol can be easily modified to investigate other intracellular and extracellular markers of interest, allowing for efficient T-cell phenotyping.
PubMed: 37251099
DOI: 10.21769/BioProtoc.4679 -
Innate Immunity Jul 2020In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET...
In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.
Topics: Adult; Amoxicillin; Animals; Anti-Bacterial Agents; Cells, Cultured; Chronic Disease; Extracellular Traps; Female; Humans; Immunity, Innate; Male; Metronidazole; Middle Aged; Neutrophils; Periodontitis
PubMed: 31757174
DOI: 10.1177/1753425919889392 -
Nutrients Jul 2022Regulatory T cells (Tregs) and CD4/CD25 T cells play an important role in the suppression of excessive immune responses, homeostasis of immune function, and oral...
Regulatory T cells (Tregs) and CD4/CD25 T cells play an important role in the suppression of excessive immune responses, homeostasis of immune function, and oral tolerance. In this study, we screened for food-derived polyphenols that induce Tregs in response to retinaldehyde dehydrogenase () activation using macrophage-like THP-1 cells. THP-1 cells were transfected with an EGFP reporter vector whose expression is regulated under the control of mouse promoter and named THP-1 (Raldh2p-EGFP) cells. The THP-1 (Raldh2p-EGFP) cells were treated with 33 polyphenols after inducing their differentiation into macrophage-like cells using phorbol 12-myristate 13-acetate. Of the 33 polyphenols, five (kaempferol, quercetin, morin, luteolin and fisetin) activated promoter activity, and both quercetin and luteolin activated the endogenous mRNA expression and enzymatic activity. Furthermore, these two polyphenols increased transforming growth factor beta 1 and forkhead box P3 mRNA expression, suggesting that they have Treg-inducing ability. Finally, we verified that these polyphenols could induce Tregs in vivo and consequently induce IgA production. Oral administration of quercetin and luteolin increased IgA production in feces of mice. Therefore, quercetin and luteolin can induce Tregs via activation and consequently increase IgA production, suggesting that they can enhance intestinal barrier function.
Topics: Aldehyde Oxidoreductases; Animals; Forkhead Transcription Factors; Immunoglobulin A; Luteolin; Mice; Polyphenols; Quercetin; RNA, Messenger; T-Lymphocytes, Regulatory
PubMed: 35889819
DOI: 10.3390/nu14142862 -
Immunologic Research Oct 2022Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators...
Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are essential for the innate immune responses against invading viruses as well as for sustained activation of macrophages. Tryptanthrin, a natural alkaloid, exhibits various bioactivities, including anti-microbial and anti-inflammatory effects. The aim of this study was to elucidate the effects of tryptanthrin on toll-like receptor 3 (TLR3)-mediated STAT1 activation in macrophages in vitro. Using phorbol myristate acetate (PMA)-differentiated THP-1 cells, we analyzed the protein level of phosphorylated-STAT1 (p-STAT1) upon stimulation with polyinosinic-polycytidylic acid (poly IC), a well-known TLR3 ligand, with and without tryptanthrin. We found that tryptanthrin decreased the protein level of p-STAT1 in a concentration-dependent manner after poly IC stimulation. On the other hand, tryptanthrin did not affect the levels of p-STAT1 upon stimulation with lipopolysaccharide from Escherichia coli. Consistently, tryptanthrin suppressed poly IC-induced mRNA expression of interferon (IFN)-stimulated genes which are regulated by STAT1. Moreover, tryptanthrin decreased the protein level of phosphorylated-IFN regulatory factor 3 and the subsequent IFN-β mRNA induction after poly IC stimulation. Tryptanthrin is a promising therapeutic agent for the aberrant activation of macrophages caused by viral infection.
Topics: Anti-Inflammatory Agents; Humans; Interferon Regulatory Factor-3; Interferon-beta; Janus Kinases; Ligands; Lipopolysaccharides; Poly I-C; Quinazolines; RNA, Messenger; STAT1 Transcription Factor; THP-1 Cells; Tetradecanoylphorbol Acetate; Toll-Like Receptor 3
PubMed: 35666435
DOI: 10.1007/s12026-022-09301-z