ABSTRACT
Parkinson's Disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc). Apoptosis is thought to play a critical role in the progression of PD, and thus understanding the effects of antiapoptotic strategies is crucial for developing potential therapies. In this study, we developed a unique genetic model to selectively delete Casp3, the gene encoding the apoptotic protein caspase-3, in dopaminergic neurons (TH-C3KO) and investigated its effects in response to a subacute regime of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, which is known to trigger apoptotic loss of SNpc dopaminergic neurons. We found that Casp3 deletion did not protect the dopaminergic system in the long term. Instead, we observed a switch in the cell death pathway from apoptosis in wild-type mice to necrosis in TH-C3KO mice. Notably, we did not find any evidence of necroptosis in our model or in in vitro experiments using primary dopaminergic cultures exposed to 1-methyl-4-phenylpyridinium in the presence of pan-caspase/caspase-8 inhibitors. Furthermore, we detected an exacerbated microglial response in the ventral mesencephalon of TH-C3KO mice in response to MPTP, which mimicked the microglia neurodegenerative phenotype (MGnD). Under these conditions, it was evident the presence of numerous microglial phagocytic cups wrapping around apparently viable dopaminergic cell bodies that were inherently associated with galectin-3 expression. We provide evidence that microglia exhibit phagocytic activity towards both dead and stressed viable dopaminergic neurons through a galectin-3-dependent mechanism. Overall, our findings suggest that inhibiting apoptosis is not a beneficial strategy for treating PD. Instead, targeting galectin-3 and modulating microglial response may be more promising approaches for slowing PD progression.
Subject(s)
Apoptosis , Caspase 3 , Dopaminergic Neurons , Galectin 3 , Microglia , Necrosis , Phagocytosis , Animals , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/pathology , Microglia/metabolism , Microglia/pathology , Microglia/drug effects , Apoptosis/drug effects , Galectin 3/metabolism , Galectin 3/genetics , Caspase 3/metabolism , Mice , Phagocytosis/drug effects , Mice, Knockout , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Mice, Inbred C57BL , MaleABSTRACT
Antimicrobial peptides (AMPs) are a promising alternative to antibiotics in the fight against multi-drug resistant and immune system-evading bacterial infections. Protegrins are porcine cathelicidins which have been identified in porcine leukocytes. Protegrin-1 is the best characterized family member and has broad antibacterial activity by interacting and permeabilizing bacterial membranes. Many host defense peptides (HDPs) like LL-37 or chicken cathelicidin 2 (CATH-2) have also been shown to have protective biological functions during infections. In this regard, it is interesting to study if Protegrin-1 has the immune modulating potential to suppress unnecessary immune activation by neutralizing endotoxins or by influencing the macrophage functionality in addition to its direct antimicrobial properties. This study showed that Protegrin-1 neutralized lipopolysaccharide- (LPS) and bacteria-induced activation of RAW macrophages by binding and preventing LPS from cell surface attachment. Furthermore, the peptide treatment not only inhibited bacterial phagocytosis by murine and porcine macrophages but also interfered with cell surface and intracellular bacterial survival. Lastly, Protegrin-1 pre-treatment was shown to inhibit the amastigote survival in Leishmania infected macrophages. These experiments describe an extended potential of Protegrin-1's protective role during microbial infections and add to the research towards clinical application of cationic AMPs.
Subject(s)
Antimicrobial Cationic Peptides , Cathelicidins , Lipopolysaccharides , Macrophages , Phagocytosis , Animals , Mice , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Immunologic Factors/pharmacology , Lipopolysaccharides/metabolism , Macrophages/immunology , Macrophages/drug effects , Phagocytosis/drug effects , RAW 264.7 Cells , SwineABSTRACT
Macrophages are among the most important components of the innate immune system where the interaction of pathogens and their phagocytosis occur as the first barrier of immunity. When nanomaterials interact with the human body, they have to face macrophages as well. Thus, understanding of nanomaterials-macrophage interactions and underlying mechanisms is crucial. For this purpose, various methods are used. In this study, surface-enhanced Raman scattering (SERS) is proposed by studying lipopolysaccharide (LPS) induced macrophage polarization using gold nanoparticles (AuNPs) as an alternative to the current approaches. For this purpose, the murine macrophage cell line, RAW 264.7 cells, was polarized by LPS, and polarization mechanisms were characterized by nitrite release and reactive oxygen species (ROS) formation and monitored using SERS. The spectral changes were interpreted based on the molecular pathways induced by LPS. Furthermore, polarized macrophages by LPS were exposed to the toxic AuNPs doses to monitor the enhanced phagocytosis and related spectral changes. It was observed that LPS induced macrophage polarization and enhanced AuNP phagocytosis by activated macrophages elucidated clearly from SERS spectra in a label-free non-destructive manner.
Subject(s)
Gold , Lipopolysaccharides , Macrophages , Metal Nanoparticles , Phagocytosis , Reactive Oxygen Species , Spectrum Analysis, Raman , Lipopolysaccharides/pharmacology , Spectrum Analysis, Raman/methods , Animals , Mice , Gold/chemistry , Macrophages/cytology , Macrophages/immunology , Macrophages/drug effects , Metal Nanoparticles/chemistry , RAW 264.7 Cells , Phagocytosis/drug effects , Reactive Oxygen Species/metabolismABSTRACT
OBJECTIVE: To observe the effects and mechanisms of Maresin2 on the function of DCs(Dendritic cells). METHOD: The levels of IL-6, IL-12, TNF-α and IL-1ß secreted by BMDCs (Bone marrow-derived Dendritic cells) after Maresin2 treatment were detected by ELISA. At the same time, the expressions of costimulatory molecules CD40 and CD86 on the surface, the ability of phagocytosis of ovalbumin(OVA) antigen, and antigen presentation function in BMDCs were analyzed by flow cytometry. Finally, MAPK and NF-κB pathway signaling phosphorylation in Maresin2-treated BMDCs were detected by western blot. RESULTS: The secretion levels of IL-6, IL-12, TNF-α and IL-1ß were significantly decreased in the Maresin2 treatment group after LPS treatment (P < 0.05). The expression levels of CD86 and CD40 were significantly decreased after Maresin2 treatment (P < 0.05). Maresin2 enhanced the phagocytosis ability of ovalbumin(OVA) (P < 0.05), but the ability of antigen presentation of BMDCs with the treatment of Maresin2 changed slightly (P > 0.05). Phosphorylation of p38, JNK, p65, ikka/ß and ERK peaked at 15 min in the LPS group, while phosphorylation of p-p38 and p-ERK weakened 30 min and 60 min after treatment with Maresin2. CONCLUSIONS: Maresin2 inhibits inflammatory cytokine secretion but enhances phagocytosis via the MAPK/NF-κB pathway in BMDCs, which may contribute to negatively regulating inflammation.
Subject(s)
Cytokines , Dendritic Cells , NF-kappa B , Phagocytosis , Signal Transduction , Animals , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , NF-kappa B/metabolism , Mice , Cytokines/metabolism , Signal Transduction/drug effects , Phagocytosis/drug effects , Cells, Cultured , Ovalbumin/immunology , Lipopolysaccharides/pharmacology , Lipopolysaccharides/immunology , Mice, Inbred C57BL , Cell Differentiation/drug effects , CD40 Antigens/metabolism , Antigen Presentation/drug effects , Docosahexaenoic AcidsABSTRACT
Niacin was found in the lysolecithin model of multiple sclerosis (MS) to promote the phagocytic clearance of debris and enhance remyelination. Lysolecithin lesions have prominent microglia/macrophages but lack lymphocytes that populate plaques of MS or its experimental autoimmune encephalomyelitis (EAE) model. Thus, the current study assessed the efficacy of niacin in EAE. We found that niacin inconsistently affects EAE clinical score, and largely does not ameliorate neuropathology. In culture, niacin enhances phagocytosis by macrophages, but does not reduce T cell proliferation. We suggest that studies of niacin for potential remyelination in MS should include a therapeutic that targets adaptive immunity.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Mice, Inbred C57BL , Multiple Sclerosis , Niacin , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Animals , Niacin/therapeutic use , Female , Mice , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Phagocytosis/drug effects , Macrophages/drug effects , Macrophages/immunology , Cells, Cultured , Disease Models, Animal , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Leptomeningeal metastases (LMs) remain a devastating complication of non-small cell lung cancer (NSCLC), particularly following osimertinib resistance. We conducted single-cell RNA sequencing on cerebrospinal fluid (CSF) from EGFR-mutant NSCLC with central nervous system metastases. We found that macrophages of LMs displayed functional and phenotypic heterogeneity and enhanced immunosuppressive properties. A population of lipid-associated macrophages, namely RNASE1_M, were linked to osimertinib resistance and LM development, which was regulated by Midkine (MDK) from malignant epithelial cells. MDK exhibited significant elevation in both CSF and plasma among patients with LMs, with higher MDK levels correlating to poorer outcomes in an independent cohort. Moreover, MDK could promote macrophage M2 polarization with lipid metabolism and phagocytic function. Furthermore, malignant epithelial cells in CSF, particularly after resistance to osimertinib, potentially achieved immune evasion through CD47-SIRPA interactions with RNASE1_M. In conclusion, we revealed a specific subtype of macrophages linked to osimertinib resistance and LM development, providing a potential target to overcome LMs.
Subject(s)
Acrylamides , Aniline Compounds , Carcinoma, Non-Small-Cell Lung , Drug Resistance, Neoplasm , Lung Neoplasms , Macrophages , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Humans , Lung Neoplasms/pathology , Lung Neoplasms/drug therapy , Aniline Compounds/pharmacology , Aniline Compounds/therapeutic use , Acrylamides/pharmacology , Acrylamides/therapeutic use , Macrophages/metabolism , Macrophages/drug effects , Animals , Mice , Cell Line, Tumor , Female , Meningeal Carcinomatosis/drug therapy , Meningeal Carcinomatosis/pathology , Meningeal Carcinomatosis/secondary , Lipid Metabolism/drug effects , CD47 Antigen/metabolism , CD47 Antigen/genetics , Male , Phagocytosis/drug effects , ErbB Receptors/metabolism , ErbB Receptors/genetics , Indoles , PyrimidinesABSTRACT
BACKGROUND: Atherosclerotic (AS) plaques require a dense necrotic core and a robust fibrous cap to maintain stability. While previous studies have indicated that the traditional Chinese medicine Huang Lian Jie Du Decoction (HLJDD) possesses the capability to stabilize AS plaques, the underlying mechanisms remain obscure. This study aims to delve deeper into the potential mechanisms by which HLJDD improves AS through an integrated research strategy. METHODS: Leveraging an AS model in ApoE-/- mice exposed to a high-fat diet (HFD), we scrutinized the therapeutic effects of HLJDD using microscopic observations, oil red O staining, HE staining and Masson staining. Employing comprehensive techniques of network pharmacology, bioinformatics, and molecular docking, we elucidated the mechanism by which HLJDD stabilizes AS plaques. In vitro experiments, utilizing ox-LDL-induced macrophages and apoptotic vascular smooth muscle cells (VSMCs), assessed the impact of HLJDD on efferocytosis and the role of SLC2A1. RESULTS: In vivo experiments showcased the efficacy of HLJDD in reducing the quantity of aortic plaques, diminishing lipid deposition, and enhancing plaque stability in AS mice. Employing network pharmacology and machine learning, we pinpointed SLC2A1 as a crucial regulatory target. Molecular docking further validated the binding of HLJDD components with SLC2A1. The experiments demonstrated a dose-dependent upregulation in SLC2A1 expression by HLJDD, amplifying efferocytosis. Importantly, this effect was reversed by the SLC2A1 inhibitor STF-31, highlighting the pivotal role of SLC2A1 as a target. CONCLUSION: The HLJDD can modulate macrophage efferocytosis by enhancing the expression levels of SLC2A1, thereby improving the stability of atherosclerotic plaques.
Subject(s)
Drugs, Chinese Herbal , Glucose Transporter Type 1 , Macrophages , Plaque, Atherosclerotic , Animals , Plaque, Atherosclerotic/drug therapy , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Mice , Male , Macrophages/drug effects , Macrophages/metabolism , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 1/genetics , Diet, High-Fat , Mice, Inbred C57BL , Phagocytosis/drug effects , Humans , Molecular Docking Simulation , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Atherosclerosis/drug therapy , Atherosclerosis/metabolism , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Disease Models, Animal , Apoptosis/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Lipoproteins, LDL/metabolism , RAW 264.7 Cells , Mice, Knockout, ApoE , EfferocytosisABSTRACT
Multinucleated microglia have been observed in contexts associated with infection, inflammation, and aging. Though commonly linked to pathological conditions, the larger cell size of multinucleated microglia might enhance their phagocytic functions, potentially aiding in the clearance of brain debris and suggesting a reassessment of their pathological significance. To assess the phagocytic capacity of multinucleated microglia and its implications for brain debris clearance, we induced their formation by inhibiting Pyk2 activity using the pharmacological inhibitor PF-431396, which triggers cytokinesis regression. Multinucleated microglia demonstrate enhanced phagocytic function, as evidenced by their increased capacity to engulf ß-amyloid (Aß) oligomers. Concurrently, the phosphorylation of Pyk2, induced by Aß peptide, was diminished upon treatment with a Pyk2 inhibitor (Pyk2-Inh, PF-431396). Furthermore, the increased expression of Lamp1, a lysosomal marker, with Pyk2-inh treatment, suggests an enhancement in proteolytic activity. In vivo, we generated an acute Alzheimer's disease (AD) model by infusing Aß into the brains of Iba-1 EGFP transgenic (Tg) mice. The administration of the Pyk2-Inh led to an increased migration of microglia toward amyloid deposits in the brains of Iba-1 EGFP Tg mice, accompanied by morphological activation, suggesting a heightened affinity for Aß. In human microglia, lipopolysaccharide (LPS)-induced inflammatory responses showed that inhibition of Pyk2 signaling significantly reduced the transcription and protein expression of pro-inflammatory markers. These results suggest that Pyk2 inhibition can modulate microglial functions, potentially reducing neuroinflammation and aiding in the clearance of neurodegenerative disease markers. This highlights Pyk2 as a promising target for therapeutic intervention in neurodegenerative diseases.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Disease Models, Animal , Focal Adhesion Kinase 2 , Mice, Transgenic , Microglia , Phagocytosis , Focal Adhesion Kinase 2/metabolism , Focal Adhesion Kinase 2/antagonists & inhibitors , Animals , Amyloid beta-Peptides/metabolism , Microglia/drug effects , Microglia/metabolism , Mice , Phagocytosis/drug effects , Phagocytosis/physiology , Alzheimer Disease/pathology , Alzheimer Disease/metabolism , Humans , Mice, Inbred C57BLABSTRACT
This study was conducted to evaluate the potential anti-inflammatory and immune-enhancement properties of lipids derived from Aptocyclus ventricosus eggs on RAW264.7 cells. Firstly, we determined the fatty acid compositions of A. ventricosus lipids by performing gas chromatography analysis. The results showed that A. ventricosus lipids contained saturated fatty acids (24.37%), monounsaturated fatty acids (20.90%), and polyunsaturated fatty acids (54.73%). They also contained notably high levels of DHA (25.91%) and EPA (22.05%) among the total fatty acids. Our results for the immune-associated biomarkers showed that A. ventricosus lipids had immune-enhancing effects on RAW264.7 cells. At the maximum dose of 300 µg/mL, A. ventricosus lipids generated NO (119.53%) and showed greater phagocytosis (63.69%) ability as compared with untreated cells. A. ventricosus lipids also upregulated the expression of iNOS, IL-1ß, IL-6, and TNF-α genes and effectively upregulated the phosphorylation of MAPK (JNK, p38, and ERK) and NF-κB p65, indicating that these lipids could activate the MAPK and NF-κB pathways to stimulate macrophages in the immune system. Besides their immune-enhancing abilities, A. ventricosus lipids significantly inhibited LPS-induced RAW264.7 inflammatory responses via the NF-κB and MAPK pathways. The results indicated that these lipids significantly reduced LPS-induced NO production, showing a decrease from 86.95% to 38.89%. Additionally, these lipids downregulated the expression of genes associated with the immune response and strongly suppressed the CD86 molecule on the cell surface, which reduced from 39.25% to 33.80%. Collectively, these findings imply that lipids extracted from A. ventricosus eggs might have biological immunoregulatory effects. Thus, they might be considered promising immunomodulatory drugs and functional foods.
Subject(s)
NF-kappa B , Signal Transduction , Animals , Mice , RAW 264.7 Cells , NF-kappa B/metabolism , Signal Transduction/drug effects , Lipids , Macrophages/drug effects , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/isolation & purification , MAP Kinase Signaling System/drug effects , Eggs , Phagocytosis/drug effects , Fatty Acids/pharmacology , Nitric Oxide/metabolism , Cytokines/metabolismABSTRACT
Phagocytic cells of the mammalian innate immune system play a critical role in protecting the body from bacterial infections. The multiple facets of this encounter (chemotaxis, phagocytosis, destruction, evasion and pathogenicity) are largely recapitulated in the phagocytic amoeba Dictyostelium discoideum. Here we identified a new chemical compound (K14; ZINC19168591) which inhibited intracellular destruction of ingested K. pneumoniae in D. discoideum cells. Concomitantly, K14 reduced proteolytic activity in D. discoideum phagosomes. In kil1 KO cells, K14 lost its ability to inhibit phagosomal proteolysis and to inhibit intra-phagosomal bacterial destruction, suggesting that K14 inhibits a Kil1-dependent protease involved in bacterial destruction. These observations stress the key role that proteases play in bacterial destruction. They also reveal an unsuspected link between Kil1 and phagosomal proteases. K14 can be used in the future as a tool to probe the role of different proteases in phagosomal physiology and in the destruction of ingested bacteria.
Subject(s)
Dictyostelium , Peptide Hydrolases , Phagosomes , Dictyostelium/enzymology , Phagosomes/metabolism , Peptide Hydrolases/metabolism , Phagocytosis/drug effects , Proteolysis/drug effects , Protozoan Proteins/metabolism , Protozoan Proteins/geneticsABSTRACT
Microglia, traditionally regarded as innate immune cells in the brain, drive neuroinflammation and synaptic dysfunctions in the early phases of Alzheimer disease (AD), acting upstream to Aß accumulation. Colony stimulating factor 1-receptor (CSF-1R) is predominantly expressed on microglia and its levels are significantly increased in neurodegenerative diseases, possibly contributing to the chronic inflammatory microglial response. On the other hand, CSF-1R inhibitors confer neuroprotection in preclinical models of neurodegenerative diseases. Here, we determined the effects of the CSF-1R inhibitor PLX3397 on the Aß-mediated synaptic alterations in ex vivo hippocampal slices. Electrophysiological findings show that PLX3397 rescues LTP impairment and neurotransmission changes induced by Aß. In addition, using confocal imaging experiments, we demonstrate that PLX3397 stimulates a microglial transition toward a phagocytic phenotype, which in turn promotes the clearance of Aß from glutamatergic terminals. We believe that the selective pruning of Aß-loaded synaptic terminals might contribute to the restoration of LTP and excitatory transmission alterations observed upon acute PLX3397 treatment. This result is in accordance with the mechanism proposed for CSF1R inhibitors, that is to eliminate responsive microglia and replace it with newly generated, homeostatic microglia, capable of promoting brain repair. Overall, our findings identify a connection between the rapid microglia adjustments and the early synaptic alterations observed in AD, possibly highlighting a novel disease-modifying target.
Subject(s)
Aminopyridines , Amyloid beta-Peptides , Hippocampus , Long-Term Potentiation , Microglia , Animals , Microglia/drug effects , Microglia/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Amyloid beta-Peptides/metabolism , Long-Term Potentiation/drug effects , Male , Aminopyridines/pharmacology , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Pyrroles/pharmacology , Mice , Phagocytosis/drug effects , Synaptic Transmission/drug effects , Mice, Inbred C57BL , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Glutamic Acid/metabolismABSTRACT
Coriander (Coriandrum sativum L.) is a member of the Umbelliferae/Apiaceae family and one of the well-known essential oil-containing plants, in which the seeds are used in traditional medicine, and as flavoring in food preparation. Knowing the diverse chemical components of different parts of the plant, this work aims to investigate the antioxidant, the anti-inflammatory, and the immunostimulatory modulator effects of the Jordanian C. sativum's seed extracted essential oil (JCEO). Coriander oil extract was prepared by hydro-distillation method using the Clevenger apparatus. Different concentrations of coriander oil were examined by using DPPH radical scavenging assay, MTT assay, pro-inflammatory cytokine (Tumor Necrosis Factor-TNF-alpha) production in RAW264.7 murine macrophages in addition, scratch-wound assessment, NO level examination, Th1/Th2 assay, phagocytosis assay, and fluorescence imaging using DAPI stain were conducted. JCEO had a potential metabolic enhancer effect at a concentration of 0.3 mg/mL on cell viability with anti-inflammatory activities via increasing cytokines like IL-10, IL-4, and limiting NO, INF-γ, and TNF-α release into cell supernatant. Antioxidant activity was seen significantly at higher concentrations of JCEO reaching 98.7% when using 100mg/mL and minimally reaching 50% at 12.5mg/mL of the essential oil. Treated macrophages were able to attain full scratch closure after 48-hrs at concentrations below 0.3mg/mL. The seed-extracted JCEO showed significant free radical scavenging activity even at lower dilutions. It also significantly induced an anti-inflammatory effect via an increase in the release of cytokines but reduced the LPS-induced NO and TNF-α production at 0.16-0.3mg/mL. In summary, coriander essential oil demonstrated antioxidant, anti-inflammatory, and immunostimulatory effects, showcasing its therapeutic potential at specific concentrations. The findings underscore its safety and metabolic enhancement properties, emphasizing its promising role in promoting cellular health.
Subject(s)
Anti-Inflammatory Agents , Antioxidants , Coriandrum , Macrophages , Oils, Volatile , Seeds , Animals , Mice , Oils, Volatile/pharmacology , Oils, Volatile/chemistry , Seeds/chemistry , Antioxidants/pharmacology , Coriandrum/chemistry , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Macrophages/drug effects , Macrophages/metabolism , RAW 264.7 Cells , Tumor Necrosis Factor-alpha/metabolism , Cell Survival/drug effects , Nitric Oxide/metabolism , Phagocytosis/drug effects , Cytokines/metabolism , JordanABSTRACT
BACKGROUND: Organophosphate esters (OPEs) are flame retardants and plasticizers used in consumer products. OPEs are found ubiquitously throughout the environment with high concentrations in indoor house dust. Exposure to individual OPEs is associated with immune dysfunction, particularly in macrophages. However, OPEs exist as complex mixtures and the effects of environmentally relevant mixtures on the immune system have not been investigated. OBJECTIVES: The objectives of this study were to evaluate the toxicity of an environmentally relevant mixture of OPEs that models Canadian house dust on macrophages using phenotypic and functional assessments in vitro. METHODS: High-content live-cell fluorescent imaging for phenotypic biomarkers of toxicity in THP-1 macrophages treated with the OPE mixture was undertaken. We used confocal microscopy and cholesterol analysis to validate and expand on the observed OPE-induced lipid phenotype. Then, we used flow cytometry and live-cell imaging to conduct functional tests and uncover mechanisms of OPE-induced phagocytic suppression. Finally, we validated our THP-1 findings in human primary peripheral blood mononuclear cells (hPBMC) derived macrophages. RESULTS: Exposure to non-cytotoxic dilutions of the OPE mixture resulted in higher oxidative stress and disrupted lysosome and lipid homeostasis in THP-1 and primary macrophages. We further observed that phagocytosis of apoptotic cells in THP-1 and primary macrophages was lower in OPE-exposed cells vs. controls. In THP-1 macrophages, phagocytosis of both Gram-positive and Gram-negative bacteria was also lower in OPE-exposed cells vs. controls. Additionally, the OPE mixture altered the expression of phagocytic receptors linked to the recognition of phosphatidylserine and pathogen-associated molecular patterns. DISCUSSION: The results of this in vitro study suggested that exposure to an environmentally relevant mixture of OPEs resulted in higher lipid retention in macrophages and poor efferocytic response. These effects could translate to enhanced foam cell generation resulting in higher cardiovascular mortality. Furthermore, bacterial phagocytosis was lower in OPE-exposed macrophages in an in vitro setting, which may indicate the potential for reduced bacterial clearance in models of infections. Taken together, our data provide strong evidence that mixtures of OPEs can influence the biology of macrophages and offer new mechanistic insights into the impact of OPE mixtures on the immune system. https://doi.org/10.1289/EHP13869.
Subject(s)
Esters , Macrophages , Organophosphates , Macrophages/drug effects , Humans , Organophosphates/toxicity , Flame Retardants/toxicity , Oxidative Stress/drug effects , Phenotype , Dust , THP-1 Cells , Phagocytosis/drug effectsABSTRACT
Background: Myocardial infarction (MI) is characterized by irreversible cardiomyocyte death resulting from an inadequate supply of oxygenated blood to the myocardium. Recent studies have indicated that ferroptosis, a form of regulated cell death, exacerbates myocardial injury during MI. Concurrently, the upregulation of CD47 on the surface of damaged myocardium following MI impairs the clearance of dead cells by macrophages, thereby hindering efferocytosis. In this context, simultaneously inhibiting ferroptosis and enhancing efferocytosis may represent a promising strategy to mitigate myocardial damage post-MI. Methods: In this study, we engineered platelet membrane-coated hollow mesoporous silicon nanoparticles (HMSN) to serve as a drug delivery system, encapsulating ferroptosis inhibitor, Ferrostatin-1, along with an anti-CD47 antibody. We aimed to assess the potential of these nanoparticles (designated as Fer-aCD47@PHMSN) to specifically target the site of MI and evaluate their efficacy in reducing cardiomyocyte death and inflammation. Results: The platelet membrane coating on the nanoparticles significantly enhanced their ability to successfully target the site of myocardial infarction (MI). Our findings demonstrate that treatment with Fer-aCD47@PHMSN resulted in a 38.5% reduction in cardiomyocyte ferroptosis under hypoxia, indicated by decreased lipid peroxidation and increased in vitro. Additionally, Fer-aCD47@PHMSN improved cardiomyocyte efferocytosis by approximately 15% in vitro. In MI mice treated with Fer-aCD47@PHMSN, we observed a substantial reduction in cardiomyocyte death (nearly 30%), decreased inflammation, and significant improvement in cardiac function. Conclusion: Our results demonstrated that the cooperation between the two agents induced anti-ferroptosis effects and enhanced dead cardiomyocyte clearance by macrophage as well as anti-inflammation effects. Thus, our nanoparticle Fer-aCD47@PHMSN provides a new therapeutic strategy for targeted therapy of MI.
Subject(s)
CD47 Antigen , Ferroptosis , Myocardial Infarction , Myocytes, Cardiac , Nanoparticles , Myocardial Infarction/drug therapy , Myocardial Infarction/metabolism , Ferroptosis/drug effects , Animals , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Nanoparticles/chemistry , Mice , CD47 Antigen/metabolism , Phagocytosis/drug effects , Cyclohexylamines/pharmacology , Cyclohexylamines/chemistry , Male , Phenylenediamines/pharmacology , Phenylenediamines/chemistry , Macrophages/drug effects , Blood Platelets/drug effects , Mice, Inbred C57BL , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Drug Carriers/chemistry , Humans , EfferocytosisABSTRACT
Alzheimer's disease (AD) is a progressive neurodegenerative disease accompanied by irreversible cognitive impairment. A deleterious feedback loop between oxidative stress and neuroinflammation in early AD exacerbates AD-related pathology. Platycodon grandiflorum root extract (PGE) has antioxidant and anti-inflammatory effects in several organs. However, the mechanisms underlying the effects of PGE in the brain remain unclear, particularly regarding its impact on oxidative/inflammatory damage and Aß deposition. Thus, we aim to identify the mechanism through which PGE inhibits Aß deposition and oxidative stress in the brain by conducting biochemical and histological analyses. First, to explore the antioxidant mechanism of PGE in the brain, we induced oxidative stress in mice injected with scopolamine and investigated the effect of PGE on cognitive decline and oxidative damage. We also assessed the effect of PGE on reactive oxygen species (ROS) generation and the expressions of antioxidant enzymes and neurotrophic factor in H2O2- and Aß-treated HT22 hippocampal cells. Next, we investigated whether PGE, which showed antioxidant effects, could reduce Aß deposition by mitigating neuroinflammation, especially microglial phagocytosis. We directly verified the effect of PGE on microglial phagocytosis, microglial activation markers, and pro-inflammatory cytokines in Aß-treated BV2 microglial cells. Moreover, we examined the effect of PGE on neuroinflammation, inducing microglial responses in Aß-overexpressing 5XFAD transgenic mice. PGE exerts antioxidant effects in the brain, enhances microglial phagocytosis of Aß, and inhibits neuroinflammation and Aß deposition, ultimately preventing neuronal cell death in AD. Taken together, our findings indicate that the therapeutic potential of PGE in AD is mediated by its targeting of multiple pathological processes.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Antioxidants , Microglia , Neuroinflammatory Diseases , Oxidative Stress , Plant Extracts , Plant Roots , Platycodon , Animals , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Oxidative Stress/drug effects , Plant Extracts/pharmacology , Mice , Platycodon/chemistry , Amyloid beta-Peptides/metabolism , Male , Plant Roots/chemistry , Microglia/drug effects , Microglia/metabolism , Antioxidants/pharmacology , Neuroinflammatory Diseases/drug therapy , Neuroinflammatory Diseases/metabolism , Cell Line , Mice, Inbred C57BL , Reactive Oxygen Species/metabolism , Disease Models, Animal , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/isolation & purification , Hippocampus/drug effects , Hippocampus/metabolism , Phagocytosis/drug effects , Neuroprotective Agents/pharmacology , Mice, Transgenic , Brain/drug effects , Brain/metabolism , Brain/pathologyABSTRACT
Polysaccharides have garnered significant attention as potential nanoparticle carriers for targeted tumor therapy due to their excellent biodegradability and biocompatibility. Polyguluronic acid (PG) is a homogeneous acidic polysaccharide fragment derived from alginate, which is found in brown algae, possesses excellent bioactivities, unique properties. This study explored the immunomodulatory activity of PG and developed PG-based nanogels through modified disulfide bonds and Ca2+ dual crosslinking. We characterized their structure, assessed their drug-loading and release properties, and ultimately validated both the safety of the nanocarrier and the in vitro anti-tumor efficacy of the encapsulated drug. Results indicated that PG significantly enhanced the proliferative activity and phagocytosis of RAW264.7 cells while promoting reactive oxygen species (ROS) production and cytokine secretion. The study identified TLR4 as the primary receptor for PG recognition in RAW264.7 cells. Furthermore, PG-based drug-carrying nanogels were prepared, exhibiting uniform sizes of about 184â¯nm and demonstrating exceptional encapsulation efficiency (82.15 ± 0.82â¯%) and drug loading capacity (8.12 ± 0.08â¯%). In vitro release experiments showed that these nanogels could responsively release drugs under conditions of high glutathione (GSH) reduction, facilitating drug accumulation at tumor sites and enhancing therapeutic efficacy. This research not only expands the application of PG in drug delivery systems but also provides valuable insights into leveraging natural immunomodulatory polysaccharides as carriers for targeted drug delivery.
Subject(s)
Drug Delivery Systems , Polysaccharides , Mice , Animals , RAW 264.7 Cells , Polysaccharides/chemistry , Polysaccharides/pharmacology , Reactive Oxygen Species/metabolism , Drug Liberation , Cell Proliferation/drug effects , Drug Carriers/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Particle Size , Alginates/chemistry , Phagocytosis/drug effects , Nanogels/chemistry , Cell Survival/drug effects , Nanoparticles/chemistryABSTRACT
The inhibitors, CK-666 and CK-869, are widely used to probe the function of Arp2/3 complex mediated actin nucleation in vitro and in cells. However, in mammals, the Arp2/3 complex consists of 8 iso-complexes, as three of its subunits (Arp3, ArpC1, ArpC5) are encoded by two different genes. Here, we used recombinant Arp2/3 with defined composition to assess the activity of CK-666 and CK-869 against iso-complexes. We demonstrate that both inhibitors prevent linear actin filament formation when ArpC1A- or ArpC1B-containing complexes are activated by SPIN90. In contrast, inhibition of actin branching depends on iso-complex composition. Both drugs prevent actin branch formation by complexes containing ArpC1A, but only CK-869 can inhibit ArpC1B-containing complexes. Consistent with this, in bone marrow-derived macrophages which express low levels of ArpC1A, CK-869 but not CK-666, impacted phagocytosis and cell migration. CK-869 also only inhibits Arp3- but not Arp3B-containing iso-complexes. Our findings have important implications for the interpretation of results using CK-666 and CK-869, given that the relative expression levels of ArpC1 and Arp3 isoforms in cells and tissues remains largely unknown.
Subject(s)
Actin-Related Protein 2-3 Complex , Actin-Related Protein 2-3 Complex/metabolism , Animals , Mice , Macrophages/metabolism , Macrophages/drug effects , Cell Movement/drug effects , Phagocytosis/drug effects , Humans , Actins/metabolism , Actin Cytoskeleton/metabolism , Protein Isoforms/metabolismABSTRACT
Glycinamide ribonucleotide formyltransferase (GARFT) is an important enzyme in the folate metabolism pathway, and chemical drugs targeting GARFT have been used in tumor treatments over the past few decades. The development of novel antimetabolism drugs that target GARFT with improved performance and superior activity remains an attractive strategy. Herein, we proposed a targeted double-template molecularly imprinted polymer (MIP) for enhancing macrophage phagocytosis and synergistic antimetabolic therapy. The double-template MIP was prepared by imprinting the exposed peptide segment of the extracellular domain of CD47 and the active center of GARFT. Owing to the imprinted cavities on the surface of MIP, it can actively target cancer cells and mask the "do not eat me" signal upon binding to CD47 thereby blocking the CD47-SIRPα pathway and ultimately enhancing phagocytosis by macrophages. In addition, MIP can specifically bind to the active center of GARFT upon entry into the cells, thereby inhibiting its catalytic activity and ultimately interfering with the normal expression of DNA. A series of cell experiments demonstrated that MIP can effectively target CD47 overexpressed 4T1 cancer cells and inhibit the growth of 4T1 cells. The enhanced phagocytosis ability of macrophages-RAW264.7 cells was also clearly observed by confocal imaging experiments. In vivo experiments also showed that the MIP exhibited a satisfactory tumor inhibition effect. Therefore, this study provides a new idea for the application of molecular imprinting technology to antimetabolic therapy in conjunction with macrophage-mediated immunotherapy.
Subject(s)
CD47 Antigen , Macrophages , Molecularly Imprinted Polymers , Phagocytosis , CD47 Antigen/metabolism , CD47 Antigen/chemistry , Phagocytosis/drug effects , Animals , Mice , Macrophages/drug effects , Macrophages/metabolism , RAW 264.7 Cells , Molecularly Imprinted Polymers/chemistry , Cell Line, Tumor , Female , Mice, Inbred BALB C , Humans , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacologyABSTRACT
In this study, we examined how systemic inflammation affects repair of brain injury. To this end, we created a brain-injury model by stereotaxic injection of ATP, a damage-associated molecular pattern component, into the striatum of mice. Systemic inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS-ip). An analysis of magnetic resonance images showed that LPS-ip reduced the initial brain injury but slowed injury repair. An immunostaining analysis using the neuronal marker, NeuN, showed that LPS-ip delayed removal of dead/dying neurons, despite the fact that LPS-ip enhanced infiltration of monocytes, which serve to phagocytize dead cells/debris. Notably, infiltrating monocytes showed a widely scattered distribution. Bulk RNAseq analyses showed that LPS-ip decreased expression of genes associated with phagocytosis, with PCR and immunostaining of injured brains confirming reduced levels of Cd68 and Clec7a, markers of phagocytic activity, in monocytes. Collectively, these results suggest that systemic inflammation affects properties of blood monocytes as well as brain cells, resulting in delay in clearing damaged cells and activating repair processes.
Subject(s)
Brain , Inflammation , Lipopolysaccharides , Mice, Inbred C57BL , Monocytes , Phagocytosis , Animals , Phagocytosis/drug effects , Monocytes/metabolism , Inflammation/pathology , Brain/pathology , Male , Lipopolysaccharides/pharmacology , Brain Injuries/pathology , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Neurons/metabolism , Neurons/pathology , Neurons/drug effects , Lectins, C-Type/metabolism , Wound Healing , Mice , Adenosine Triphosphate/metabolism , CD68 MoleculeABSTRACT
This study investigated the toxic effects of Bisphenol A (BPA) on the Pacific abalone (Haliotis discus hannai) using in vitro assays with primary cultured hemocytes. The abalone hemocytes were exposed to BPA concentrations up to 100 µM to assess cytotoxicity. Subsequently, hemocytes were exposed to sublethal BPA concentrations (LC20 = 2.3 µM and LC50 = 5.8 µM) for 48 h, and we evaluated the cellular immune responses of hemocytes via flow cytometry. Results showed no significant differences between LC20 and control groups, but LC50 exposure significantly reduced phagocytosis and oxidative capacities while increasing nitric oxide production. These findings suggest that BPA exposure negatively affects the immune system of the Pacific abalone, which makes them more susceptible to infections and other stressors in their natural environment. The study also implies that in vitro assays utilizing primary cultured abalone hemocytes may serve as effective proxies for quantifying the cytotoxic effects of chemical pollutants.