-
Nature Biotechnology Jun 2022High-redox-potential reactive oxygen species and reactive nitrogen species (ROS/RNS), generated by NADPH oxidase-2 (NOX2), myeloperoxidase (MPO) and related enzymes, are...
High-redox-potential reactive oxygen species and reactive nitrogen species (ROS/RNS), generated by NADPH oxidase-2 (NOX2), myeloperoxidase (MPO) and related enzymes, are key effector molecules of innate immunity. High-redox-potential radicals are difficult to distinguish by imaging from less potent ROS/RNS functioning as background biological signaling molecules. Here we present 4-[F]fluoro-1-naphthol ([F]4FN), a redox-tuned radiopharmaceutical that selectively binds proteins and cells when oxidized by products of human MPO plus HO, but not HO alone, and can be detected using positron emission tomography (PET). Activating HL-60 neutrophil-like human cells with phorbol ester (PMA) caused [F]4FN retention five-fold over unstimulated cells. An MPO-specific inhibitor (4-ABAH) blocked cellular retention by more than 95%. [F]4FN PET/CT imaging discriminated inflammatory foci in vivo in three murine models of activated innate immunity: endotoxin-induced toxic shock, PMA-induced contact dermatitis and lipopolysaccharide-induced ankle arthritis. 4-ABAH and Cybb (Nox2) gene deletion strongly abrogated [F]4FN retention in vivo. Thus, [F]4FN shows promise as a robust reporter of innate immunity activation by PET/CT.
Topics: Animals; Humans; Hydrogen Peroxide; Immunity, Innate; Mice; NADPH Oxidases; Oxidation-Reduction; Positron Emission Tomography Computed Tomography; Reactive Oxygen Species
PubMed: 35190688
DOI: 10.1038/s41587-021-01169-y -
BMC Anesthesiology Jan 2020There is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested...
BACKGROUND
There is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested the hypotheses that 1) NET formation from neutrophils of septic patients is increased compared to healthy volunteers, both without stimulation and following incubation with mitochondrial DNA (mtDNA), a damage-associated molecular pattern, or phorbol 12-myristate 13-acetate (PMA; positive control) and 2) that serum nuclease activities are increased as well.
METHODS
Following ethic committee approval, we included 18 septic patients and 27 volunteers in this prospective observational trial. Blood was withdrawn and NET formation from neutrophils was analyzed in vitro without stimulation and following incubation with mtDNA (10 μg/well) or PMA (25 nmol). Furthermore, serum nuclease activity was assessed using gel electrophoresis.
RESULTS
In contrast to our hypothesis, in septic patients, unstimulated NET release from neutrophils was decreased by 46.3% (4.3% ± 1.8 SD vs. 8.2% ± 2.9, p ≤ 0.0001) and 48.1% (4.9% ± 2.5 vs. 9.4% ± 5.2, p = 0.002) after 2 and 4 h compared to volunteers. mtDNA further decreased NET formation in neutrophils from septic patients (4.7% ± 1.2 to 2.8% ± 0,8; p = 0.03), but did not alter NET formation in neutrophils from volunteers. Of note, using PMA, as positive control, we ensured that neutrophils were still able to form NETs, with NET formation increasing to 73.2% (±29.6) in septic patients and 91.7% (±7.1) in volunteers (p = 0.22). Additionally, we show that serum nuclease activity (range: 0-6) was decreased in septic patients by 39.6% (3 ± 2 vs 5 ± 0, median and ICR, p = 0.0001) compared to volunteers.
CONCLUSIONS
Unstimulated NET formation and nuclease activity are decreased in septic patients. mtDNA can further reduce NET formation in sepsis. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo.
TRIAL REGISTRATION
DRKS00007694, german clinical trials database (DRKS). Retrospectively registered 06.02.2015.
Topics: Adult; Aged; DNA, Mitochondrial; Deoxyribonucleases; Extracellular Traps; Female; Healthy Volunteers; Humans; Male; Middle Aged; Neutrophils; Retrospective Studies; Sepsis; Tetradecanoylphorbol Acetate; Young Adult
PubMed: 31931719
DOI: 10.1186/s12871-019-0911-7 -
Photochemistry and Photobiology Jan 2022Microvesicle particles (MVP) are bioactive subcellular particles which have been recently implicated in the keratinocyte response to many environmental stressors...
Microvesicle particles (MVP) are bioactive subcellular particles which have been recently implicated in the keratinocyte response to many environmental stressors including ultraviolet B radiation (UVB). Previous studies have demonstrated that UVB generates high levels of MVP in a process involving the platelet-activating factor receptor (PAFR) and the enzyme acid sphingomyelinase (aSMase). Yet the fluences of UVB needed to generate MVP are usually above those commonly encountered. Using models including human epithelial cell lines in vitro, human skin explants ex vivo and murine studies in vivo, the present studies indicate that pretreatment of epithelial cells/skin with PAFR agonist/phorbol ester can synergize with low fluences of UVB to generate high levels of MVP. These studies indicate the possibility that MVP could play a role in combinatorial pathologic processes involving UVB.
Topics: Animals; Cell Line; Cell-Derived Microparticles; Humans; Keratinocytes; Mice; Skin; Ultraviolet Rays
PubMed: 34324709
DOI: 10.1111/php.13495 -
Experimental and Therapeutic Medicine Jan 2020The aim of the present study was to explore the possible role of microRNA-144-5p (miR-144-5p) in rheumatoid arthritis (RA) and the associated mechanism. Following the...
The aim of the present study was to explore the possible role of microRNA-144-5p (miR-144-5p) in rheumatoid arthritis (RA) and the associated mechanism. Following the induction of THP-1 cell differentiation into macrophages by phorbol ester (100 ng/ml) treatment, an inflammatory model of RA was established by treating the THP-1 macrophages with 1 µg/ml lipopolysaccharide (LPS). The level of miR-144-5p was subsequently measured using reverse transcription-quantitative PCR, which found that the expression of miR-144-5p was significantly reduced in LPS-treated THP-1 macrophages. Bioinformatics analysis and a dual-luciferase reporter assay were used to predict and confirm TLR2 as a direct target of miR-144-5p, respectively. Toll-like receptor 2 (TLR2) was then validated as a target gene of miR-144-5p. The effects of miR-144-5p upregulation and TLR2 silencing on LPS-treated THP-1 macrophages were then determined by transfection with miR-144-5p mimic and TLR2-siRNA, respectively. Cell viability was subsequently measured using a Cell Counting Kit-8 assay, whilst the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8 secreted by THP-1 macrophages was measured using ELISA. Western blotting was performed to measure p65 phosphorylation (p-p65) in the NK-κB signaling pathway. It was found that miR-144-5p overexpression reduced macrophage cell viability, reduced the expression of TNF-α, IL-6 and IL-8, and reduced the expression of TLR2 and p-p65 compared with the control group. Likewise, TLR2 silencing also reduced macrophage cell viability and reduced the expression of TNF-α, IL-6 and IL-8 in THP-1 macrophages. In conclusion, the data from the present study suggested that miR-144-5p overexpression reduced THP-1 macrophage cell viability and inhibited the expression of TNF-α, IL-6 and IL-8 in cells, possibly by inhibiting the expression of TLR2 and suppressing the activation of NK-κB signaling. Therefore, miR-144-5p may serve as a novel therapeutic target for the treatment of RA.
PubMed: 31853295
DOI: 10.3892/etm.2019.8218 -
Journal of Biomolecular Structure &... 2022Munc13-1 is a presynaptic active zone protein that plays a critical role in priming the synaptic vesicle and releasing neurotransmitters in the brain. Munc13-1 acts as a...
Munc13-1 is a presynaptic active zone protein that plays a critical role in priming the synaptic vesicle and releasing neurotransmitters in the brain. Munc13-1 acts as a scaffold and is activated when diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane. Our previous studies showed that bryostatin 1 activated the Munc13-1, but resveratrol inhibited the phorbol ester-induced Munc13-1 activity. To gain structural insights into the binding of the ligand into Munc13-1 C1 in the membrane, we conducted 1.0 μs molecular dynamics (MD) simulation on Munc13-1 C1-ligand-lipid ternary system using phorbol 13-acetate, bryostatin 1 and resveratrol as ligands. Munc13-1 C1 shows higher conformational stability and less mobility along membrane with phorbol 13-acetate and bryostatin 1 than with resveratrol. Bryostatin 1 and phorbol ester remained in the protein active site, but resveratrol moved out of Munc13-1 C1 during the MD simulation. While bryostatin 1-bound Munc13-1 C1 showed two different positioning in the membrane, phorbol 13-acetate and resveratrol-bound Munc13-1 C1 only showed one positioning. Phorbol 13-acetate formed hydrogen bond with Ala-574 and Gly-589. Bryostatin 1 had more hydrogen bonds with Trp-588 and Arg-592 than with other residues. Resveratrol formed hydrogen bond with Ile-590. This study suggests that different ligands control Munc13-1 C1's mobility and positioning in the membrane differently. Ligand also has a critical role in the interaction between Munc13-1 C1 and lipid membrane. Our results provide structural basis of the pharmacological activity of the ligands and highlight the importance of membrane in Munc13-1 activity.Communicated by Ramaswamy H. Sarma.
Topics: Molecular Dynamics Simulation; Ligands; Resveratrol; Phorbol Esters; Lipids
PubMed: 34779746
DOI: 10.1080/07391102.2021.2001375 -
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 -
Scientific Reports Feb 2023Macrophages (MQs) pro-inflammatory phenotype is triggered by gliadin peptides. Akkermansia muciniphila (A. muciniphila) showed to enhance the anti-inflammatory phenotype...
Macrophages (MQs) pro-inflammatory phenotype is triggered by gliadin peptides. Akkermansia muciniphila (A. muciniphila) showed to enhance the anti-inflammatory phenotype of MQs. This study aimed to investigate the anti-inflammatory effects of A. muciniphila, on gliadin stimulated THP-1 derived macrophages. THP-1 cell line monocytes were differentiated into MQs by phorbol 12-myristate 13-acetate (PMA). MQs were treated with A. muciniphila before and after stimulation with gliadin (pre- and post-treat). CD11b, as a marker of macrophage differentiation, and CD206 and CD80, as M1 and M2 markers, were evaluated by flow cytometry technique. The mRNA expression of TGF-β, IL-6, and IL-10 and protein levels of IL-10 and TNF-α were measured by RT-PCR and ELISA techniques, respectively. Results show an increased percentage of M1 phenotype and release of proinflammatory cytokines (like TNF-α and IL-6) by macrophages upon incubation with gliadin. Pre- and post-treatment of gliadin-stimulated macrophages with A. muciniphila induced M2 phenotype associated with decreased proinflammatory (IL-6, TNF-α) and increased anti-inflammatory (IL-10, TGF-β) cytokines expression relative to the group that was treated with gliadin alone. This study suggests the potential beneficial effect of A. muciniphila on gliadin-stimulated MQs and the importance of future studies focusing on their exact mechanism of action on these cells.
Topics: Interleukin-10; Gliadin; Tumor Necrosis Factor-alpha; Interleukin-6; Macrophages; Cytokines; Anti-Inflammatory Agents; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta
PubMed: 36828897
DOI: 10.1038/s41598-023-30266-y -
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 -
Frontiers in Immunology 2021Besides being a key effector arm of innate immunity, a plethora of non-canonical functions of complement has recently been emerging. Factor H (FH), the main regulator of... (Comparative Study)
Comparative Study
Besides being a key effector arm of innate immunity, a plethora of non-canonical functions of complement has recently been emerging. Factor H (FH), the main regulator of the alternative pathway of complement activation, has been reported to bind to various immune cells and regulate their functions, beyond its role in modulating complement activation. In this study we investigated the effect of FH, its alternative splice product FH-like protein 1 (FHL-1), the FH-related (FHR) proteins FHR-1 and FHR-5, and the recently developed artificial complement inhibitor mini-FH, on two key innate immune cells, monocytes and neutrophilic granulocytes. We found that, similar to FH, the other factor H family proteins FHL-1, FHR-1 and FHR-5, as well as the recombinant mini-FH, are able to bind to both monocytes and neutrophils. As a functional outcome, immobilized FH and FHR-1 inhibited PMA-induced NET formation, but increased the adherence and IL-8 production of neutrophils. FHL-1 increased only the adherence of the cells, while FHR-5 was ineffective in altering these functions. The adherence of monocytes was increased on FH, recombinant mini-FH and FHL-1 covered surfaces and, except for FHL-1, the same molecules also enhanced secretion of the inflammatory cytokines IL-1β and TNFα. When monocytes were stimulated with LPS in the presence of immobilized FH family proteins, FH, FHL-1 and mini-FH enhanced whereas FHR-1 and FHR-5 decreased the secretion of TNFα; FHL-1 and mini-FH also enhanced IL-10 release compared to the effect of LPS alone. Our results reveal heterogeneous effects of FH and FH family members on monocytes and neutrophils, altering key features involved in pathogen killing, and also demonstrate that FH-based complement inhibitors, such as mini-FH, may have effects beyond their function of inhibiting complement activation. Thus, our data provide new insight into the non-canonical functions of FH, FHL-1, FHR-1 and FHR-5 that might be exploited during protection against infections and in vaccine development.
Topics: Animals; Blood Proteins; Cell Shape; Complement Factor H; Complement Inactivating Agents; Complement System Proteins; Cytokines; Extracellular Traps; Humans; Inflammation Mediators; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Monocytes; Muscle Proteins; Neutrophils; Phenotype; Sf9 Cells; Signal Transduction; Tetradecanoylphorbol Acetate; U937 Cells
PubMed: 34671340
DOI: 10.3389/fimmu.2021.660852 -
Clinical Interventions in Aging 2022The aim of the paper is to establish and quantify the relation between healthy ageing and the innate and adaptive immune parameters as indicators of age-related diseases.
PURPOSE
The aim of the paper is to establish and quantify the relation between healthy ageing and the innate and adaptive immune parameters as indicators of age-related diseases.
PATIENTS
In order to observe the immunological changes that occur according to age, several humoral and cellular immune parameters were investigated for 288 healthy donors (30-80 years). Subjects' selection was done using clinical, biochemical and immunological parameters of inclusion/exclusion criteria from SENIEUR protocol.
RESULTS
Age-related changes were observed for both humoral and cellular immune parameters. Lymphocyte immunophenotyping revealed several significant differences in the distribution of cells, both intra- and inter-age groups, namely decreased values of T-CD3, T-CD8 and NK cells, and elevated values for T-CD4, T-CD4/T-CD8 ratio and B cells. The percentages of unstimulated neutrophils that show basal oxidative activity and the intensity of this activity had an increasing tendency age-related. The percentage of N-Formyl-Methionyl-Leucyl-Phenylalanine stimulated neutrophils clearly decreases with age, and is associated with an increasing intensity of oxidative activity. Our data also have shown an increased percentage of oxidative neutrophils after phorbol 12-myristate 13-acetate stimulation and an elevated oxidative activity with age.
CONCLUSION
Overall healthy ageing is governed by some immune-related deregulations that account for immune exhaustion due to numerous developed immune processes during a life-time and the age-related diseases.
Topics: Acetates; Aged; Aged, 80 and over; Healthy Aging; Humans; Myristates; N-Formylmethionine Leucyl-Phenylalanine; Tetradecanoylphorbol Acetate
PubMed: 36247200
DOI: 10.2147/CIA.S375926