Activated THP-1 Cells Research Articles

Napthalimide-based nuclease inhibitor: A multifunctional therapeutic material to bolster MRSA uptake by macrophage-like cells and mitigate pathogen adhesion on orthopaedic implant

The healthcare burden rendered by methicillin-resistant Staphylococcus aureus (MRSA) warrants the development of therapeutics that offer a distinct benefit in the clinics as compared to conventional antibiotics. The present study describes the potential of napthalimide-based synthetic ligands (C1-C3) as inhibitors of the staphylococcal nuclease known as micrococcal nuclease (MNase), a key virulence factor of the pathogen. Amongst the ligands, the most potent MNase inhibitor C1 rendered non-competitive inhibition, reduced MNase turnover number (Kcat) and catalytic efficiency (Kcat/Km) with an IC50 value of ~950 nM. CD spectroscopy suggested distortion of MNase conformation in presence of C1. Flow cytometry and confocal microscopy indicated that C1 restored the ability of activated THP-1 cells to engulf DNA-entrapped MRSA cells. Interestingly, C1 could inhibit MRSA adhesion onto collagen. For potential application, C1-loaded pluronic F-127 micellar nanocarrier (C1-PMC) was generated, wherein the anti-adhesion activity of the pluronic carrier (PMC) and C1 was harnessed in tandem to deter MRSA cell adhesion onto collagen. MRSA biofilm formation was hindered on C1-PMC-coated titanium (Ti) wire, while eluates from C1-PMC-coated Ti wires were non-toxic to HEK 293, MG-63 and THP-1 cells. The multifunctional C1 provides a blueprint for designing therapeutic materials that hold translational potential for mitigation of MRSA infections.

Extracellular vesicles secreted by the ТНР-1 cells influence on the inflammation gene expression in zebrafish

Extracellular vesicles secreted by immune cells may play a significant role in the initiation, maintenance, and progression of systemic inflammation. The aim of the study was to investigate the regulatory effect of extracellular vesicles (EVs) produced by activated monocyte-like THP-1 cells on expression levels of inflammatory genes in a zebrafish. Real-time PCR analysis was performed to investigate the relative expression levels of il-1β, il-6, tnf-α, ifn-γ, mpeg1.1, mpeg1.2, mpx, and il-10 genes in the brain, liver, and heart of zebrafish followed by intracelomic injection of EVs produced by THP-1 cells activated with tumor necrosis factor (TNF) and phorbol-12-myristate-13-acetate (PMA) at different concentrations. EVs, secreted by activated THP-1 cells with TNF at a concentration of 10 ng/mL and PMA at concentrations of 16 and 50 ng/mL, reduced the expression levels of il-1β, ifn-γ, tnf-α, mpx, mpeg1.1, mpeg1.2, and IL-10 genes in the brain, heart and liver of Danio rerio. Wherein, EVs secreted by THP-1 cells treated with TNF at doses of 10 and 20 ng/ml had opposite effects on the gene expression levels of il-1β in the brain, il-1β, il-10, and il-6 in the heart; on il-1β, il-10, mpx, and mpeg1.2 in the liver. EVs secreted by THP-1 cells treated with PMA at doses of 16 and 50 ng/ml had opposite effects on the expression levels of il-6 and il-10 genes in the heart and ifn-γ gene in the liver. EVs, produced by activated THP-1 cells have a systemic effect on Danio rerio manifested in a changing of the expression level of pro- and anti-inflammatory cytokine genes in the brain, liver, and heart. The qualitative composition of the EVs produced by activate THP-1 cells varies depending on the type and dose of the used stimulus, that reflects on strength and direction of the effects detected in vivo.

Extracellular Vesicles Secreted by the Activated THP-1 Cells Influence the Inflammation Gene Expression in Zebrafish

Extracellular Vesicles Secreted by the Activated THP-1 Cells Influence the Inflammation Gene Expression in Zebrafish

Inhibition of staphylococcal nuclease by benzimidazole-based Ligand: Implications in DNA-Mediated entrapment and uptake of MRSA by Macrophage-like cells

The staphylococcal nuclease also referred as micrococcal nuclease (MNase) is a key drug target as the enzyme degrades the neutrophil extracellular trap (NET) and empowers the pathogen to subvert the host innate immune system. To this end, the current study presents a critical evaluation of MNase inhibition rendered by benzimidazole-based ligands (C1 and C2) and probes its therapeutic implications. A nuclease assay indicated that MNase inhibition rendered by C1 and C2 was ∼ 55 % and ∼ 72 %, respectively, at the highest tested concentration of 10 µM. Studies on enzyme kinetics revealed that C2 rendered non-competitive inhibition and significantly reduced MNase turnover number (Kcat) and catalytic efficiency (Kcat/Km) with an IC50 value of ∼ 1122 nM. In CD spectroscopy, a notable perturbation in the β-sheet content of MNase was observed in presence of C2. Fluorescence-microscope analysis indicated that MNase inhibition by C2 could restore entrapment of methicillin-resistant Staphylococcus aureus (MRSA) in calf-thymus DNA (CT-DNA). Flow cytometry and confocal microscope analysis revealed that uptake of DNA-entrapped MRSA by activated THP-1 cells was reinstated by MNase inhibition rendered by C2. Inhibition of nuclease by the non-toxic ligand C2 holds therapeutic prospect as it has the potential to bolster the DNA-mediated entrapment machinery and mitigate MRSA infections.

Djulis Hull Enhances the Efficacy of Ferric Citrate Supplementation via Restoring Normal Iron Efflux through the IL-6-Hepcidin-Ferroportin Pathway in High-Fat-Diet-Induced Obese Rats.

Obesity-related functional iron disorder remains a major nutritional challenge. We evaluated the effects of djulis hull (DH) on iron metabolism in 50% high-fat-diet-induced obese rats supplemented with ferric citrate (2 g iron/kg diet) for 12 weeks. DH supplementation (5, 10, 15% dry weight/kg diet) significantly increased serum and hepatic iron but decreased appetite hormones, body weight, hepcidin, and liver inflammation (all p < 0.05). The Spearman correlation showed that appetite hormones were negatively associated with iron but positively correlated with liver hepcidin (all p < 0.05). A Western blot analysis showed that DH significantly downregulated hepatic hepcidin through the IL-6-JAK-STAT3 and enhanced ferroportin (Fpn) via the Keap1-Nrf2 and PHD2-HIF-2α. An in vitro study revealed that major bioactive compounds of DH, hexacosanol, and squalene suppressed LPS-induced IL-6 and hepcidin but enhanced Fpn expression in activated THP-1 cells. In conclusion, DH may exert nutraceutical properties for the treatment of functional iron disorder and restoration of iron efflux may have beneficial effects on weight control.

Pharmacological Prevention of Ectopic Erythrophagocytosis by Cilostazol Mitigates Ferroptosis in NASH.

Hepatic iron overload (HIO) is a hallmark of nonalcoholic fatty liver disease (NAFLD) with a poor prognosis. Recently, the role of hepatic erythrophagocytosis in NAFLD is emerging as a cause of HIO. We undertook various assays using human NAFLD patient pathology samples and an in vivo nonalcoholic steatohepatitis (NASH) mouse model named STAMTM. To make the in vitro conditions comparable to those of the in vivo NASH model, red blood cells (RBCs) and platelets were suspended and subjected to metabolic and inflammatory stresses. An insert-coculture system, in which activated THP-1 cells and RBCs are separated from HepG2 cells by a porous membrane, was also employed. Through various analyses in this study, the effect of cilostazol was examined. The NAFLD activity score, including steatosis, ballooning degeneration, inflammation, and fibrosis, was increased in STAMTM mice. Importantly, hemolysis occurred in the serum of STAMTM mice. Although cilostazol did not improve lipid or glucose profiles, it ameliorated hepatic steatosis and inflammation in STAMTM mice. Platelets (PLTs) played an important role in increasing erythrophagocytosis in the NASH liver. Upregulated erythrophagocytosis drives cells into ferroptosis, resulting in liver cell death. Cilostazol inhibited the augmentation of PLT and RBC accumulation. Cilostazol prevented the PLT-induced increase in ectopic erythrophagocytosis in in vivo and in vitro NASH models. Cilostazol attenuated ferroptosis of hepatocytes and phagocytosis of RBCs by THP-1 cells. Augmentation of hepatic erythrophagocytosis by activated platelets in NASH exacerbates HIO. Cilostazol prevents ectopic erythrophagocytosis, mitigating HIO-mediated ferroptosis in NASH models.

CircRNA_0075723 protects against pneumonia-induced sepsis through inhibiting macrophage pyroptosis by sponging miR-155-5p and regulating SHIP1 expression.

Circular RNAs (circRNAs) have been linked to regulate macrophage polarization and subsequent inflammation in sepsis. However, the underlying mechanism and the function of circRNAs in macrophage pyroptosis in pneumonia-induced sepsis are still unknown. In this study, we screened the differentially expressed circRNAs among the healthy individuals, pneumonia patients without sepsis and pneumonia-induced sepsis patients in the plasma by RNA sequencing (RNA-seq). Then we evaluated macrophage pyroptosis in sepsis patients and in vitro LPS/nigericin activated THP-1 cells. The lentiviral recombinant vector for circ_0075723 overexpression (OE-circ_0075723) and circ_0075723 silence (sh-circ_0075723) were constructed and transfected into THP-1 cells to explore the potential mechanism of circ_0075723 involved in LPS/nigericin induced macrophage pyroptosis. We found circ_0075723, a novel circRNA that was significantly downregulated in pneumonia-induced sepsis patients compared to pneumonia patients without sepsis and healthy individuals. Meanwhile, pneumonia-induced sepsis patients exhibited activation of NLRP3 inflammasome and production of the pyroptosis-associated pro-inflammatory cytokines IL-1β and IL-18. circ_0075723 inhibited macrophage pyroptosis via sponging miR-155-5p which promoted SHIP1 expression directly. Besides, we found that circ_0075723 in macrophages promoted VE-cadherin expression in endothelial cells through inhibiting the release of NLRP3 inflammasome-related cytokines, IL-1β and IL-18, and protects endothelial cell integrity. Our findings propose a unique approach wherein circ_0075723 suppresses macrophage pyroptosis and inflammation in pneumonia-induced sepsis via sponging with miR-155-5p and promoting SHIP1 expression. These findings indicate that circRNAs could be used as possible potential diagnostic and therapeutic targets for pneumonia-induced sepsis.

Identification of Small Molecule Inhibitors against Mycobacteria in Activated Macrophages.

Mycobacterial pathogens are intrinsically resistant to many available antibiotics, making treatment extremely challenging, especially in immunocompromised individuals and patients with underlying and chronic lung conditions. Even with lengthy therapy and the use of a combination of antibiotics, clinical success for non-tuberculous mycobacteria (NTM) is achieved in fewer than half of the cases. The need for novel antibiotics that are effective against NTM is urgent. To identify such new compounds, a whole cell high-throughput screen (HTS) was performed in this study. Compounds from the Chembridge DIVERSet library were tested for their ability to inhibit intracellular survival of M. avium subsp. hominissuis (MAH) expressing dtTomato protein, using fluorescence as a readout. Fifty-eight compounds were identified to significantly inhibit fluorescent readings of MAH. In subsequent assays, it was found that treatment of MAH-infected THP-1 macrophages with 27 of 58 hit compounds led to a significant reduction in intracellular viable bacteria, while 19 compounds decreased M. abscessus subsp. abscessus (Mab) survival rates within phagocytic cells. In addition, the hit compounds were tested in M. tuberculosis H37Ra (Mtb) and 14 compounds were found to exhibit activity in activated THP-1 cells. While the majority of compounds displayed inhibitory activity against both replicating (extracellular) and non-replicating (intracellular) forms of bacteria, a set of compounds appeared to be effective exclusively against intracellular bacteria. The efficacy of these compounds was examined in combination with current antibiotics and survival of both NTM and Mtb were evaluated within phagocytic cells. In time-kill dynamic studies, it was found that co-treatment promoted increased bacterial clearance when compared with the antibiotic or compound group alone. This study describes promising anti-NTM and anti-Mtb compounds with potential novel mechanisms of action that target intracellular bacteria in activated macrophages.

Remnant Cholesterol and Dyslipidemia Are Risk Factors for Guillain-Barré Syndrome and Severe Guillain-Barré Syndrome by Promoting Monocyte Activation.

BackgroundGuillain–Barré syndrome (GBS) is the most common severe acute paralytic neuropathy, with a mortality rate of 5% and permanent sequelae rate of 10%. Currently, the cause of GBS remains unclear. Therefore, we sought to determine potential predictors for GBS and its severity.MethodsA case–control study was performed at Tiantan Hospital in Beijing from January 2017 to December 2021. Laboratory and clinical characteristics were assessed in recruited GBS patients and healthy control individuals (matched by sex and age). The potential risk factors for GBS and severe GBS were assessed using a logistic regression analysis. The mRNA levels of toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2) and nuclear factor κB (NF-κB) in GBS patients and control PBMCs were detected by fluorescence quantitative PCR. THP-1 cells were costimulated with LPS and free cholesterol to demonstrate the effect of free cholesterol on monocyte activation.ResultsA total of 147 GBS patients and 153 healthy individuals were included in the study. Logistic regression analyses showed that preceding infection, alcohol consumption, remnant cholesterol, homocysteine and the dyslipidemia index were correlated with a higher risk of GBS. In contrast, increased HDL cholesterol was correlated with a lower risk of GBS. Moreover, remnant cholesterol and the dyslipidemia index were significantly correlated with severe GBS. The mRNA levels of TLR4, TLR2 and NF-κB in the PBMCs of GBS patients were significantly higher than those of healthy individuals. LPS activated THP-1 cells, and free cholesterol treatment increased the expression of TLR4, TLR2, NF-κB and IL-1β mRNA in LPS-activated THP-1 cells.ConclusionDyslipidemia was correlated with the risk of GBS and severe GBS. Remnant cholesterol may promote the activation of monocytes in GBS patients. It may be valuable to control lipid levels in the prevention of GBS and severe GBS.

Abstract 2558: Determining the expression of RNA binding protein Rbm3 in tumor cells and immune cells in the tumor microenvironment in prostate cancer

Abstract Prostate cancer is the most common cancer among men. Currently, targeting the AR pathway, chemotherapy or immune-based therapies are major options but only provide a modest improvement in overall survival. RNA binding proteins have been shown to regulate AR expression in the progression of PCa. We and others have demonstrated that RNA binding protein RBM3 is upregulated in various solid tumors including PCa. We have also shown that RBM3 binds to 3’UTR of mRNAs of tumor-promoting factors and increases their mRNA stability and translation. However, the role of RBM3 in PCa is not well evaluated. We first analyzed the expression levels of RBM3 in the TCGA database. There was a significant increase in RBM3 expression even in cancers with a Gleason score of 6 upto Gleason 10 cancers. To confirm this we performed RT-PCR analyses of a prostate cancer cDNA panel. There was a significant increase in RBM3 expression in the PCa tissues compared to normal control. We used LNCaP and its derivative cell line C4-2B which show features of progressed disease such as metastatic capability and hormone independence. RT-PCR and western blot analyses demonstrated significantly higher RBM3 levels in C4-2B cells as compared to LNCaP cells suggesting a role for RBM3 in tumor progression. The tumor microenvironment also plays a very important role in prognosis of PCa. Specifically, tumor-associated macrophages (TAMs) have been shown to increase metastatic potential and increase tumor angiogenesis. To determine the levels of RBM3 expression and its effects on macrophage infiltration, we mined the TCGA database using TIMER2.0 software. There was a positive correlation of RBM3 expression with infiltration of both M1 and M2 macrophages. To further study the effect of interactions between PCa cells and TAMs, we used immortalized THP1 monocytes, which can be activated to M1 and M2 macrophages. We observed that just converting the THP1 cells to M1 or M2 macrophages increased RBM3 expression in both cell types. Also, when M1 and M2 macrophages were treated with conditioned media from LNCaP or C4-2B cells, there was an induction in the expression of RBM3. Similarly, when conditioned media from M0, M1, M2 activated THP1 cells were applied to LNCaP and C4-2B cells, there was an increase in RBM3 expression in the PCa cells. This suggests a positive cross-talk between the macrophages and PCa cells. We evaluated the cytokine profile in the conditioned media from M1 and M2 macrophages and determined that while M1 macrophages had increased levels of CXCL10, M2 macrophages showed higher levels of IL4 and IL10. Moreover, PCa cells have higher levels of CXCR3, the receptor for CXCL10. Together, these data suggest that crosstalk between TAMs and cancer cells in the PCA microenvironment plays a significant role in increasing RBM3 expression, which in turn enhances global translation of disease progression related genes. Citation Format: Afreen Asif Ali Sayed, David Standing, Prasad Dandawate, Shahid Umar, Roy Jensen, Rahul Parikh, John Taylor, Shrikant Anant. Determining the expression of RNA binding protein Rbm3 in tumor cells and immune cells in the tumor microenvironment in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2558.

Time-Dependent Anti-inflammatory Effects of a Lipid Extract from Macrocystis pyrifera on Toll-Like Receptor 2 Signaling in Human THP-1 Monocytes

AbstractSeaweeds reportedly contain anti-inflammatory compounds; however, little is known about the therapeutic potential of Macrocystis pyrifera. This study investigated the anti-inflammatory properties of a methanol:chloroform extract, chromatographic fractions, and fatty acids identified from M. pyrifera (Linnaeus) C. Agardh. In human THP-1 monocytes stimulated with the Toll-like receptor 2 agonist lipoteichoic acid, the extract decreased mRNA and protein levels of interleukin-1β, interleukin-8, and monocyte chemoattractant protein-1 to varied degrees at nontoxic concentrations. The greatest anti-inflammatory effects were elicited when the extract was applied between 6 h prior to, and 6 h after, the stimuli. Reduced levels of nuclear factor kappa-light-chain-enhancer of activated B cells signaling proteins were observed in extract-treated cells, with a significant decrease in the myeloid differentiation factor 88 protein abundance relative to stimulated THP-1 cells. Chromatographic fractionation of the extract yielded 40 fractions, of which fraction F25 exhibited the greatest inhibition of monocyte chemoattractant protein-1 production in activated THP-1 cells. Fatty acids abundant within the extract and F25 were identified then tested, individually and in combination, for their anti-inflammatory effects. Myristic acid, palmitoleic acid, and α-linolenic acid, but not the fatty acid combination, inhibited lipoteichoic acid-stimulated monocyte chemoattractant protein-1 production without compromising THP-1 cell viability. These findings indicate that the fatty acid-rich extract and fraction from M. pyrifera provide anti-inflammatory and cytoprotective effects that may be beneficial for use as a therapeutic.

Modulatory Effects of Fractalkine on Inflammatory Response and Iron Metabolism of Lipopolysaccharide and Lipoteichoic Acid-Activated THP-1 Macrophages.

Fractalkine (CX3CL1) acts as a chemokine as well as a regulator of iron metabolism. Fractalkine binds CX3CR1, the fractalkine receptor on the surface of monocytes/macrophages regulating different intracellular signalling pathways such as mitogen-activated protein kinase (MAPK), phospholipase C (PLC) and NFκB contributing to the production of pro-inflammatory cytokine synthesis, and the regulation of cell growth, differentiation, proliferation and metabolism. In this study, we focused on the modulatory effects of fractalkine on the immune response and on the iron metabolism of Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (LPS) and Staphylococcus aureus lipoteichoic acid (LTA) activated THP-1 cells to get a deeper insight into the role of soluble fractalkine in the regulation of the innate immune system. Pro-inflammatory cytokine secretions of the fractalkine-treated, LPS/LTA-treated, and co-treated THP-1 cells were determined using ELISArray and ELISA measurements. We analysed the protein expression levels of signalling molecules regulated by CX3CR1 as well as hepcidin, the major iron regulatory hormone, the iron transporters, the iron storage proteins and mitochondrial iron utilization. The results showed that fractalkine treatment alone did not affect the pro-inflammatory cytokine secretion, but it was proposed to act as a regulator of the iron metabolism of THP-1 cells. In the case of two different LPS and one type of LTA with fractalkine co-treatments, fractalkine was able to alter the levels of signalling proteins (NFκB, PSTAT3, Nrf2/Keap-1) regulating the expression of pro-inflammatory cytokines as well as hepcidin, and the iron storage and utilization of the THP-1 cells.

A high-fat diet has negative effects on tendon resident cells in an in vivo rat model

BackgroundTendinopathy is a major complication of diet-induced obesity. However, the effects of a high-fat diet (HFD) on tendon have not been well characterised. We aimed to determine: [1] the impact of a HFD on tendon properties and gene expression; and [2] whether dietary transition to a control diet (CD) could restore normal tendon health.MethodsSprague–Dawley rats were randomised into three groups from weaning and fed either a: CD, HFD or HFD for 12 weeks and then CD thereafter (HF-CD). Biomechanical, histological and structural evaluation of the Achilles tendon was performed at 17 and 27 weeks of age. Tail tenocytes were isolated with growth rate and collagen production determined. Tenocytes and activated THP-1 cells were exposed to conditioned media (CM) of visceral adipose tissue explants, and gene expression was analysed.ResultsThere were no differences in the biomechanical, histological or structural tendon properties between groups. However, tenocyte growth and collagen production were increased in the HFD group at 27 weeks. There was lower SOX-9 expression in the HFD and HF-CD groups at 17 weeks and higher expression of collagen-Iα1 and matrix metalloproteinase-13 in the HFD group at 27 weeks. THP-1 cells exposed to adipose tissue CM from animals fed a HFD or HF-CD had lower expression of Il-10 and higher expression of Il-1β.ConclusionsIn this rodent model, a HFD negatively altered tendon cell characteristics. Dietary intervention restored some gene expression changes; however, adipose tissue secretions from the HF-CD group promoted an increased inflammatory state in macrophages. These changes may predispose tendon to injury and adverse events later in life.

Attenuation of NLRP3 Inflammasome Activation by Indirubin-Derived PROTAC Targeting HDAC6.

Histone deacetylase 6 (HDAC6) is a potential therapeutic target for treating several diseases. A recent study revealed that HDAC6 is important for NLRP3 inflammasome activation, suggesting that targeting HDAC6 could be useful for treating many inflammatory disorders. Using the proteolysis targeting chimera (PROTAC) strategy, we herein report an HDAC6 degrader with low cytotoxicity by tethering a selective HDAC6 inhibitor derived from a natural product, indirubin, with pomalidomide, a CRBN E3 ligand. Our HDAC6 degrader efficiently and selectively decreased HDAC6 levels in several cell lines, including activated THP-1 cells. Application of this HDAC6 degrader attenuated NLRP3 inflammasome activation in LPS-induced mice, which for the first time demonstrates that HDAC6 PROTAC could be a novel strategy to treat NLRP3 inflammasome-associated diseases.

Pre-Clinical Characterization of a Novel Fusion Protein (AT-03), with Pan-Amyloid Binding and Removal

Background: Amyloidosis is characterized by extracellular deposition of protein fibrils that can involve any organ or tissue in the body. It is a severe, progressive and often lethal disorder. There are approximately seventeen different systemic amyloid diseases, each associated with the deposition of a specific precursor protein. Toxic amyloid deposits result in significant organ dysfunction and increased morbidity and mortality. Reduction and removal of tissue amyloid deposits could restore organ function, improve quality of life and increase life expectancy for patients. We have developed and characterized a fusion protein consisting of serum amyloid protein (SAP) linked to a single chain human IgG1 Fc domain, designated AT-03. SAP is a pentameric serum protein that is ubiquitously associated with amyloid deposits. The high affinity and specificity for amyloid resulted in the use of radio-iodinated SAP as an imaging agent for the non-invasive detection of systemic amyloid. The SAP domain of the fusion protein targets the amyloid deposits delivering Fc domains to stimulate amyloid removal via macrophage mediated phagocytosis.Methods: Binding of AT-03 to ATTR and AL fibrils and extracts was assessed by ELISA. Ex vivo phagocytosis of amyloid substrates was studied using activated human THP-1 cells. The biodistribution of AT-03 in murine models of AA, AApoA2 and AL amyloidosis was evaluated immunohistochemically or by SPECT imaging and microautoradiography. Clearance of AA amyloidosis in response to AT-03 therapy was accessed in a mouse model using histological scoring of amyloid burden.Results: AT-03 bound amyloid AL and ATTR substrates with sub nanomolar EC50 values. Systemic administration of AT-03 resulted in specific binding to diverse forms of amyloid in the heart, kidney, liver and/or spleen in multiple animal models. Opsonization of amyloid deposits with AT-03 in vitro resulted in a dose dependent phagocytosis by activated THP-1 cells. A single IV dose of AT-03 resulted in significant reduction of splenic AA amyloid within 14 days post-injection.Conclusions: We have developed a novel fusion protein with sub nanomolar pan amyloid reactivity that specifically binds diverse forms of amyloid in multiple organs and tissues in vivo and can effectuate amyloid removal. The data indicates that AT-03 may serve as a novel therapeutic for the removal of systemic amyloid deposits of diverse types. AT-03 is currently being developed for clinical evaluation. DisclosuresSirac: Attralus, Inc: Patents & Royalties, Research Funding. Jaccard: Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Abbvie: Honoraria. Bridoux: AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy. Bell: Attralus Inc: Current Employment. Guthrie: Attralus Inc: Current Employment. Selvarajah: Attralus Inc: Current Employment. Kennel: Attralus Inc: Current holder of stock options in a privately-held company, Patents & Royalties: Inventor and patent holder. Wall: Attralus Inc: Current holder of stock options in a privately-held company, Patents & Royalties: Inventor and patent holder, Research Funding.

Effects of E-Cigarette Flavoring Chemicals on Human Macrophages and Bronchial Epithelial Cells.

E-cigarettes utilize a wide range of flavoring chemicals with respiratory health effects that are not well understood. In this study, we used pulmonary-associated cell lines to assess the in vitro cytotoxic effects of 30 flavoring chemicals. Human bronchial epithelial cells (BEAS-2B) and both naïve and activated macrophages (THP-1) were treated with 10, 100, and 1000 µM of flavoring chemicals and analyzed for changes in viability, cell membrane damage, reactive oxygen species (ROS) production, and inflammatory cytokine release. Viability was unaffected for all chemicals at the 10 and 100 µM concentrations. At 1000 µM, the greatest reductions in viability were seen with decanal, hexanal, nonanal, cinnamaldehyde, eugenol, vanillin, alpha-pinene, and limonene. High amounts of ROS were elicited by vanillin, ethyl maltol, and the diketones (2,3-pentanedione, 2,3-heptanedione, and 2,3-hexanedione) from both cell lines. Naïve THP-1 cells produced significantly elevated levels of IL-1β, IL-8, and TNF-α when exposed to ethyl maltol and hexanal. Activated THP-1 cells released increased IL-1β and TNF-α when exposed to ethyl maltol, but many flavoring chemicals had an apparent suppressive effect on inflammatory cytokines released by activated macrophages, some with varying degrees of accompanying cytotoxicity. The diketones, L-carvone, and linalool suppressed cytokine release in the absence of cytotoxicity. These findings provide insight into lung cell cytotoxicity and inflammatory cytokine release in response to flavorings commonly used in e-cigarettes.

Apatinib triggers autophagic and apoptotic cell death via VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling in lung cancer

BackgroundRecently, a variety of clinical trials have shown that apatinib, a small-molecule anti-angiogenic drug, exerts promising inhibitory effects on multiple solid tumors, including non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism of apatinib on NSCLC remains unclear.MethodsMTT, EdU, AO/EB staining, TUNEL staining, flow cytometry, colony formation assays were performed to investigate the effects of apatinib on cell proliferation, cell cycle distribution, apoptosis and cancer stem like properties. Wound healing and transwell assays were conducted to explore the role of apatinib on migration and invasion. The regulation of apatinib on VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling were detected. Furthermore, we collected conditioned medium (CM) from A549 and H1299 cells to stimulate phorbol myristate acetate (PMA)-activated THP-1 cells, and examined the effect of apatinib on PD-L1 expression in macrophages. The Jurkat T cells and NSCLC cells co-culture model was used to assess the effect of apatinib on T cells activation. Subcutaneous tumor formation models were established to evaluate the effects of apatinib in vivo. Histochemical, immunohistochemical staining and ELISA assay were used to examine the levels of signaling molecules in tumors.ResultsWe showed that apatinib inhibited cell proliferation and promoted apoptosis in NSCLC cells in vitro. Apatinib induced cell cycle arrest at G1 phase and suppressed the expression of Cyclin D1 and CDK4. Moreover, apatinib upregulated Cleaved Caspase 3, Cleaved Caspase 9 and Bax, and downregulated Bcl-2 in NSCLC cells. The colony formation ability and the number of CD133 positive cells were significantly decreased by apatinib, suggesting that apatinib inhibited the malignant and stem-like features of NSCLC cells. Mechanistically, apatinib inhibited PD-L1 and c-Myc expression by targeting VEGFR2/STAT3 signaling. Apatinib also inhibited PD-L1 expression in THP-1 derived macrophages stimulated by CM from NSCLC cells. Furthermore, apatinib pretreatment increased CD69 expression and IFN-γ secretion in stimulated Jurkat T cells co-cultured with NSCLC cells. Apatinib also promoted ROS production and inhibited Nrf2 and p62 expression, leading to the autophagic and apoptotic cell death in NSCLC. Moreover, apatinib significantly inhibited tumor growth in vivo.ConclusionOur data indicated that apatinib induced autophagy and apoptosis in NSCLC via regulating VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling.

Nicotinamide Mononucleotide Alleviates LPS-Induced Inflammation and Oxidative Stress via Decreasing COX-2 Expression in Macrophages.

Macrophage activation is an important process in controlling infection, but persistent macrophage activation leads to chronic inflammation and diseases, such as tumor progression, insulin resistance and atherosclerosis. Characterizing metabolic signatures of macrophage activation is important for developing new approaches for macrophage inactivation. Herein, we performed metabolomic analysis on lipopolysaccharide (LPS)-activated macrophages and identified the associated changes in metabolites. Notably, the cellular Nicotinamide adenine dinucleotide+ levels were decreased while NADPH was increased, proposing that NAD+ restoration can inhibit macrophage activation. Indeed, supplementation of nicotinamide mononucleotide (NMN) increased cellular NAD+ levels and decreased cytokine productions in LPS-activated cells. Quantitative proteomics identified that nicotinamide mononucleotide downregulated the expressions of LPS-responsive proteins, in which cyclooxygenase-2 (COX-2) expression was significantly decreased in NMN-treated cells. Consequently, the cellular levels of prostaglandin E2 (PGE2) was also decreased, indicating that NMN inactivated macrophages via COX-2-PGE2 pathway, which was validated in activated THP-1 cells and mouse peritoneal macrophages. In conclusion, the present study identified the metabolic characteristics of activated macrophages and revealed that NMN replenishment is an efficient approach for controlling macrophage activation.

Impact of cigarette versus electronic cigarette aerosol conditioned media on aortic endothelial cells in a microfluidic cardiovascular model

Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0–1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.

Chemical Investigation of Marine-Derived Fungus Aspergillus flavipes for Potential Anti-Inflammatory Agents.

The marine fungus, Aspergillus flavipes (MTCC 5220), was isolated from the pneumatophore of a mangrove plant Acanthus ilicifolius found in Goa, India. The crude extract of A. flavipes was found to show anti-inflammatory activity. It blocked interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-activated THP-1 cells with IC50 of 2.69±0.5 μM and 6.64±0.4 μM, respectively. The chemical investigation led to the isolation of optically inactive 4β-[(1E)-propen-1-yl]cyclopentane-1β,2β-diol (1) along with a new optically active diastereoisomeric compound, 4β-[(1E)-propen-1-yl]cyclopentane-1β,2α-diol (2). In addition, the fungus also produced known compounds (+)-terrein (3), butyrolactone I (4) and butyrolactone II (5) in high yields. Among these, (+)-terrein (3) exhibited IL-6 and TNF-α inhibition activity with IC50 of 8.5±0.68 μM and 15.76±0.18 μM, respectively, while butyrolactone I (4) exhibited IC50 of 12.03±0.85 μM (IL-6) and 43.29±0.76 μM (TNF-α) inhibition activity with low toxicity to host cells in LPS stimulated THP-1 cells. This is the first report of the isolation and characterization of 4β-[(1E)-propen-1-yl]cyclopentane-1β,2α-diol (2). The structures of all the isolated compounds were elucidated on the basis of extensive detailed NMR spectroscopic data. Anti-inflammatory activity of the fungi A. flavipes is presented here for the first time, which was due to (+)-terrein and butyrolactone I, as the major constituents and they can be further explored in the therapeutic area.