Human Macrophage Cell Line Research Articles

Intracellular Methylglyoxal Accumulation in Classically Activated Mouse Macrophages is Mediated by HIF-1α.

Approximately one million cases of sepsis in the U.S.A. occur annually. The early phase of sepsis features dramatic changes in host metabolism and inflammation. While examining the effects of metabolic pathways on inflammation, we discovered that the highly reactive glycolytic metabolite, methylglyoxal (MG), accumulates intracellularly during classical activation of macrophages. Herein, we explored the role of glycolysis and the master regulator of glycolysis, Hypoxia-Inducing Factor-1α (HIF-1α), in inflammation and MG accumulation in mouse and human macrophages. To determine how HIF-1α regulates the inflammatory response of macrophages, we correlated HIF-1α stabilization with proinflammatory gene expression and MG-adduct accumulation in WT vs HIF1a-deficient macrophages treated with LPS or LPS+IFN-γ. A nearly complete loss of HIF-1α protein expression in response to the hypoxia mimetic, cobalt chloride, confirmed the phenotype of the HIF1a-deficient macrophages. Moreover, absence of HIF-1α was also associated with decreased MG accumulation. Increasing the glucose concentration in cultured macrophages was sufficient to cause accumulation of endogenous MG-adducts which correlated with increased Tnf and Il1b expression during classical activation. Use of the MG antagonist, aminoguanidine, led to a significant decrease in Tnf and Il1b expression in both mouse macrophages and in the THP-1 human macrophage cell line. Although off-target effects cannot be ruled out, these results are consistent with the possibility that MG regulates cytokine expression in classically activated macrophages. Collectively, this work suggests that HIF-1α stabilization is upstream of MG accumulation and that targeting the activity of HIF-1α in macrophages may be therapeutic during sepsis by limiting endogenous MG accumulation.

Inhibition of Mac-1 allows human macrophages to migrate against the direction of shear flow on ICAM-1.

All immune cells must transit from the blood to distal sites such as the lymph nodes, bone marrow, or sites of infection. Blood borne monocytes traffic to the site of inflammation by adhering to the endothelial surface and migrating along endothelial intracellular adhesion molecule 1 (ICAM-1) by their ligand's macrophage 1 antigen (Mac-1) and lymphocyte functional antigen 1 (LFA-1) to transmigrate through the endothelium. Poor patient prognoses in chronic inflammation and tumors have been attributed to the hyper recruitment of certain types of macrophages. Therefore, targeting the binding of ICAM-1 to its respective ligands provides a novel approach to targeting the recruitment of macrophages. To that end, we determined whether the loss of Mac-1 expression could induce this upstream migration behavior by using blocking antibodies against Mac-1 to examine the effects of hydrodynamic flow on the migration of the human macrophage cell line U-937 on ICAM-1 surfaces. Blocking Mac-1 on U-937 cells led to upstream migration against the direction of shear flow on ICAM-1 surfaces. In sum, the ability of macrophages to migrate upstream when Mac-1 is blocked represents a new avenue to precisely control the differentiation, migration, and trafficking of macrophages.

On probiotic integration in the management of inflammation and the maintenance of the intestinal epithelial barrier’s integrity

Inflammatory bowel disease epidemiology has grown dramatically in recent years, particularly in developed and developing Western countries. Many factors, including stress, diet, and medications, cause and exacerbate inflammatory conditions. Inflammation is closely related to the concept of intestinal barrier integrity. When integrity is compromised, toxins and pathogens can enter the bloodstream. In recent years, there has been a growing interest in using probiotic bacteria to prevent or treat a variety of pathologies, including inflammatory bowel disease. Some studies have looked at the effectiveness of multi-strain probiotic supplements in preventing intestinal barrier dysfunction in in vitro models of lipopolysaccharide-induced inflammation. To mimic the intestinal barrier, human colon adenocarcinoma cell lines were established in Transwell co-culture models. The epithelium permeability was assessed by measuring the transepithelial electrical resistance. The expression of individual proteins involved in barrier function was assessed. The immunomodulatory effects of probiotic formulations were studied in both human macrophage cell lines and ex vivo human peripheral blood mononuclear cell-derived macrophages. The intestinal epithelial layer was also interfaced with a human mast cell line. Selected probiotics have demonstrated high potential for use in maintaining intestinal barrier integrity and possessing anti-inflammatory properties.

Nanostructured Hybrid Polymer‐Lipid Drug Delivery Platforms for Rapamycin Repositioning in Anticancer Therapy

AbstractHere, hybrid polymer‐lipid nanoparticles are designed as colloidal carriers for Rapamycin, to improve the aqueous drug stability and to support the drug repositioning for cancer treatment, that is, against rhabdomyosarcoma (RMS). With this aim, Rapamycin – loaded hybrid nanoparticles are produced by using as nanoparticle core a graft copolymer obtained from the functionalization of the α,β‐poly(N‐2‐hydroxyethyl)‐DL‐aspartamide (PHEA) with Rhodamine B (RhB), Polylactic acid (PLA), the PHEA‐g‐RhB‐g‐PLA, and different phospholipids, that is, 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) coated, pegylated and Mannose/PEG, for the surface coating. The drug loading of these samples allows for controlled release, and improves drug stability at pH 5.5 and 7.4 compared to the free drug. Chemical‐physical characterization confirms the nanostructure size below 200 nm, ideal for systemic administration, and easy re‐dispersibility in aqueous media. Moreover, biological characterization to test the potential use as antitumor agent shows induction of cytotoxicity in human rhabdomyosarcoma (RD) and macrophage (RAW) cell lines in a time‐ and concentration – dependent manner, and stimulated autophagy, comparable to the free drug. The uptake study following the fluorescence of the copolymer reveals that the hybrid nanoparticles are internalized by both tested cell lines, with a significantly higher amounts of internalized particles in the case of surface mannosylated and/or pegylated systems.

Immunomodulatory of sesquiterpenoids and sesquiterpenoid dimers-based toll-like receptor 4 (TLR4) from Dysoxylum parasiticum stem bark

In recent decades, the interest in natural products with immunomodulatory properties has increased due to their therapeutic potential. These products have a wider range of pharmacological activities and demonstrate lower toxicity levels when compared to their synthetic counterparts. Therefore, this study aimed to investigate the immunomodulatory effects of sesquiterpenoids (SQs) and sesquiterpenoid dimers (SQDs) isolated from Dysoxylum parasiticum (Osbeck) Kosterm. stem bark on human and murine cells, particularly focusing on toll-like receptor 4 (TLR4). Utilizing the secreted alkaline phosphatase (SEAP) assay on engineered human and murine TLR4 of HEK-Blue cells, antagonist TLR4 compounds were identified, including SQs 6, 9, and 10, as well as SQDs 17 and 22. The results showed that 10-hydroxyl-15-oxo-α-cadinol (9) had a potent ability to reduce TLR4 activation induced by LPS stimulation, with minimal toxicity observed in both human and murine cells. The SEAP assay also revealed diverse immune regulatory effects for the same ligand. For instance, SQs 12, 14, and 16 transitioned from antagonism on human to murine TLR4. The SQs (4, 7, 11, and 15) and SQDs (18–20) offered partial antagonist effect exclusively on murine TLR4. Furthermore, these selected SQs and SQDs were assessed for their influence on the production of proinflammatory cytokines TNF-α, IL-1α, IL-1β, and IL-6 of the NF-κB signaling pathway in human and murine macrophage cell lines, showing a dose-dependent manner. Additionally, a brief discussion on the structure-activity relationship was presented.

Bovine DDX3X Restrains Bovine SP110c-Mediated Activation of Inflammasome in Macrophages.

The inflammasome is a vital part of the host's innate immunity activated by cellular infection or stress. Our previous research identified the bovine SP110c isoform (bSP110c) as a novel activator of the inflammasome that promoted the secretion of proinflammatory cytokines IL-1β and IL-18 in macrophages infected with Listeria monocytogenes or stimulated with lipopolysaccharide (LPS). However, the exact molecular mechanism for inhibiting bSP110c-induced inflammasome activation requires further clarification. Here, the researchers identified bovine DDX3X (bDDX3X) as an NLRP3-associated protein and an inhibitor of the bSP110c-induced inflammasome in the human THP1 macrophage cell line. Immunoprecipitation showed that bDDX3X interacted with the bSP110c CARD domain via its helicase domain. The co-expression of bSP110c and bDDX3X in THP1 macrophages significantly prevented the bSP110c-induced activation of inflammasomes. In addition, both bDDX3X and bSP110c interacted with bovine NLRP3 (bNLRP3), and bDDX3X enhanced the interaction between bSP110c and bNLRP3. The expression of bDDX3X in nigericin-stimulated THP1 macrophages significantly suppressed NLRP3 inflammasome activation, ASC speck formation, and pyroptosis. These findings demonstrate that bDDX3X negatively regulates the bSP110c-mediated inflammatory response by restricting the activation of the NLRP3 inflammasome. This discovery unveils a novel regulatory mechanism involving bDDX3X and bSP110c in coordinating inflammasome activation and subsequent cell-fate decisions in LPS-treated macrophages and, in turn, constitutes a step forward toward the implementation of marker-assisted selection in breeding programs aimed at utilizing cattle's immune defenses.

Phytochemical profile and determination of cytotoxicity, acute oral toxicity, genotoxicity, and mutagenicity of aqueous and ethanolic extracts of Pseudobombax marginatum (A. St.-Hil.) A. Robyns

ABSTRACT Pseudobombax marginatum, popularly known as “embiratanha,” is widely used by traditional communities as anti-inflammatory and analgesic agent. This study aimed to determine the phytochemical profile as well as cytotoxicity, acute oral toxicity, genotoxicity, and mutagenicity attributed to exposure to aqueous (AqEx) and ethanolic (EtEx) extracts of embiratanha bark. Phytochemical screening was conducted using thin-layer chromatography (TLC). Cell viability was analyzed using MTT assay with human mammary gland adenocarcinoma (MDA-MB-231) and macrophage (J774A.1) cell lines, exposed to concentrations of 12.5, 25, 50, or 100 µg/ml of either extract. For acute oral toxicity, comet assay and micronucleus (MN) tests, a single dose of 2,000 mg/kg of either extract was administered orally to Wistar rats. TLC analysis identified classes of metabolites in the extracts, including cinnamic acid derivatives, flavonoids, hydrolyzable tannins, condensed tannins, coumarins, and terpenes/steroids. In the cytotoxicity assay, the varying concentrations of extracts derived from embiratanha induced no significant alterations in the viability of MDA-MB-231 cells. The lowest concentration of EtEx significantly increased macrophage J774A.1 viability. However, the higher concentrations of AqEx markedly lowered macrophage J774A.1 viability. Animals exhibited no toxicity in the parameters analyzed in acute oral toxicity, comet assay, and MN tests. Further, EtEx promoted a significant reduction in DNA damage index and DNA damage frequency utilizing the comet assay, while the group treated with AqEx exhibited no marked differences. Thus, data demonstrated that AqEx or EtEx of embiratanha may be considered safe at a dose of 2,000 mg/kg orgally under our experimental conditions tested.

Spleen tyrosine kinase inhibitor R406 has both antiviral and anti-inflammatory effects on severe influenza A infection.

Death due to severe influenza is usually a fatal complication of a dysregulated immune response more than the acute virulence of an infectious agent. Although spleen tyrosine kinase (SYK) as a critical immune signaling molecule and therapeutic target plays roles in airway inflammation and acute lung injury, the role of SYK in influenza virus infection is not clear. Here, we investigated the antiviral and anti-inflammatory effects of SYK inhibitor R406 on influenza infection through a coculture model of human alveolar epithelial (A549) and macrophage (THP-1) cell lines and mouse model. The results showed that R406 treatment increased the viability of A549 and decreased the pathogenicity and mortality of lethal influenza virus in mice with influenza A infection, decreased levels of intracellular signaling molecules under the condition of inflammation during influenza virus infection. Combination therapy with oseltamivir further ameliorated histopathological damage in the lungs of mice and further delayed the initial time to death compared with R406 treatment alone. This study demonstrated that phosphorylation of SYK is involved in the pathogenesis of influenza, and R406 has antiviral and anti-inflammatory effects on the treatment of the disease, which may be realized through multiple pathways, including the already reported SYK/STAT/IFNs-mediated antiviral pathway, as well as TNF-α/SYK- and SYK/Akt-based immunomodulation pathway.

Proteome profile of Leishmania donovani Centrin1−/− parasite-infected human macrophage cell line and its implications in determining possible mechanisms of protective immunity

Our developed cell division-specific ‘centrin’ gene deleted Leishmania donovani (LdCen1−/−) the causative parasite of the fatal visceral-leishmaniasis (VL), exhibits a selective growth arrest at the intracellular stage and is anticipated as a live attenuated vaccine candidate against VL. LdCen1−/− immunization in animals has shown increased IFN-γ secreting CD4+ and CD8+ T cells along with protection conferred by a protective proinflammatory immune response. A label-free proteomics approach has been employed to understand the physiology of infection and predict disease interceptors during Leishmania-host interactions. Proteomic modulation after infection of human macrophage cell lines suggested elevated annexin A6, implying involvement in various biological processes such as membrane repair, transport, actin dynamics, cell proliferation, survival, differentiation, and inflammation, thereby potentiating its immunological protective capacity. Additionally, S100A8 and S100A9 proteins, known for maintaining homeostatic balance in regulating the inflammatory response, have been upregulated after infection. The inhibitory clade of serpins, known to inhibit cysteine proteases (CPs), was upregulated in host cells after 48 h of infection. This is reflected in the diminished expression of CPs in the parasites during infection. Such proteome analysis confirms LdCen1−/− efficacy as a vaccine candidate and predicts potential markers in future vaccine development strategies against infectious diseases.

Oxidized low-density lipoprotein changes the inflammatory status and metabolomics profiles in human and mouse macrophages and microglia

The conjunctiva of primary open angle glaucoma patients showed high level of oxidized low-density lipoprotein (ox-LDL), which is associated with the inflammatory response. Microglia and macrophages are the immune cells involved in retinal ganglion cell survival regulation; yet, their roles of the ox-LDL-induced inflammation in glaucoma remain elusive. Here we aimed to investigate the lipid uptake, inflammatory cytokine expression, and metabolomics profiles of human and murine-derived microglial and macrophage cell lines treated with ox-LDL. Under the same ox-LDL concentration, macrophages exhibited higher lipid uptake and expression of pro-inflammatory cytokines as compared to microglia. The ox-LDL increased the levels of fatty acid metabolites in macrophages and sphingomyelin metabolites in microglia. In summary, this study revealed the heterogeneity in the inflammatory capacity and metabolic profiles of macrophages and microglia under the stimulation of ox-LDL.

Dexamethasone protects against asthma via regulating Hif-1α-glycolysis-lactate axis and protein lactylation

PurposeAsthma can not be eradicated till now and its control primarily relies on the application of corticosteroids. Recently, glycolytic reprogramming has been reportedly contributed to asthma, this study aimed to reveal whether the effect of corticosteroids on asthma control is related to their regulation of glycolysis and glycolysis-dependent protein lactylation. MethodsOvalbumin (OVA) aeroallergen was used to challenge mice and stimulate human macrophage cell line THP-1 following dexamethasone (DEX) treatment. Airway hyperresponsiveness, airway inflammation, the expressions of key glycolytic enzymes and pyroptosis markers, the level of lactic acid, real-time glycolysis and oxidative phosphorylation (OXPHOS), and protein lactylation were analyzed. ResultsDEX significantly attenuated OVA-induced eosinophilic airway inflammation, including airway hyperresponsiveness, leukocyte infiltration, goblet cell hyperplasia, Th2 cytokines production and pyroptosis markers expression. Meanwhile, OVA-induced Hif-1α-glycolysis axis was substantially downregulated by DEX, which resulted in low level of lactic acid. Besides, key glycolytic enzymes in the lungs of asthmatic mice were notably co-localized with F4/80-positive macrophages, indicating metabolic shift to glycolysis in lung macrophages during asthma. This was confirmed in OVA-stimulated THP-1 cells that DEX treatment resulted in reductions in pyroptosis, glycolysis and lactic acid level. Finally, protein lactylation was found significantly increased in the lungs of asthmatic mice and OVA-stimulated THP-1 cells, which were both inhibited by DEX. ConclusionOur present study revealed that the effect of DEX on asthma control was associated with its suppressing of Hif-1α-glycolysis-lactateaxis and subsequent protein lactylation, which may open new avenues for the therapy of eosinophilic asthma.

IL-6 facilitates cross-talk between epithelial cells and tumor- associated macrophages in Helicobacter pylori-linked gastric carcinogenesis

PurposeHelicobacter pylori (H. pylori) is a significant risk factor for development of gastric cancer (GC), one of the deadliest malignancies in the world. However, the mechanism by which H. pylori induces gastric oncogenesis remains unclear. Here, we investigated the function of IL-6 in gastric oncogenesis and macrophage-epithelial cell interactions. MethodsWe analyzed publicly available datasets to investigate the expression of IL-6 and infiltration of M2 macrophages in GC tissues, and determine the inter-cellular communication in the context of IL-6. Human gastric epithelial and macrophage cell lines (GES-1 and THP-1-derived macrophages, respectively) were used in mono- and co-culture experiments to investigate autocrine-and paracrine induction of IL-6 expression in response to H. pylori or IL-6 stimulation. ResultsWe found that IL-6 is highly expressed in GC and modulates survival. M2 macrophage infiltration is predominant in GC and drives an IL-6 mediated communication with gastric epithelium cells. In vitro, IL-6 triggers its own expression in GES-1 and THP-1-derived macrophages cells. In addition, these cell lines are able to upregulate each other's IL-6 levels in an autocrine fashion, which is enhanced by H. pylori stimulation. ConclusionThis study indicates that IL-6 in the tumor microenvironment is essential for intercellular communication. We show that H. pylori enhances an IL-6-driven autocrine and paracrine positive feedback loop between macrophages and gastric epithelial cells, which may contribute to gastric carcinogenesis.

The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis

ObjectiveThe serine protease inhibitor SerpinB3 has been described as critical mediator of liver fibrosis and it has been recently proposed as an additional hepatokine involved in NASH development and insulin resistance. Protease Activated Receptor 2 has been identified as a novel regulator of hepatic metabolism. A targeted therapeutic strategy for NASH has been investigated, using 1-Piperidine Propionic Acid (1-PPA), since this compound has been recently proposed as both Protease Activated Receptor 2 and SerpinB3 inhibitor. MethodsThe effect of SerpinB3 on inflammation and fibrosis genes was assessed in human macrophage and stellate cell lines. Transgenic mice, either overexpressing SerpinB3 or carrying Serpinb3 deletion and their relative wild type strains, were used in experimental NASH models. Subgroups of SerpinB3 transgenic mice and their controls were also injected with 1-PPA to assess the efficacy of this compound in NASH inhibition. Results1-PPA did not present significant cell and organ toxicity and was able to inhibit SerpinB3 and PAR2 in a dose-dependent manner. This effect was associated to a parallel reduction of the synthesis of the molecules induced by endogenous SerpinB3 or by its paracrine effects both in vitro and in vivo, leading to inhibition of lipid accumulation, inflammation and fibrosis in experimental NASH. At mechanistic level, the antiprotease activity of SerpinB3 was found essential for PAR2 activation, determining upregulation of the CCAAT Enhancer Binding Protein beta (C/EBP-β), another pivotal regulator of metabolism, inflammation and fibrosis, which in turn determined SerpinB3 synthesis. Conclusions1-PPA treatment was able to inhibit the PAR2 - C/EBP-β - SerpinB3 axis and to protect from NASH development and progression, supporting the potential use of a similar approach for a targeted therapy of NASH.

HDAC6/aggresome processing pathway importance for inflammasome formation is context-dependent

The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend the cell and promote the secretion of pro-inflammatory cytokines, including IL-1β and IL-18. Previous research has shown that in immortalized bone marrow-derived macrophages (iBMDMs) inflammasome assembly is dependent on the deacetylase HDAC6 and the aggresome processing pathway (APP), a cellular pathway involved in the disposal of misfolded proteins. Here we used primary BMDMs from mice in which HDAC6 is ablated or impaired and found that inflammasome activation was largely normal. We also used human peripheral blood mononuclear cells and monocytes cell lines expressing a synthetic protein blocking the HDAC6-ubiquitin interaction and impairing the APP and found that inflammasome activation was moderately affected. Finally, we used a novel HDAC6 degrader and showed that inflammasome activation was partially impaired in human macrophage cell lines with depleted HDAC6. Our results therefore show that HDAC6 importance in inflammasome activation is context dependent.

Promoter methylation and increased expression of PD-L1 in patients with active tuberculosis.

The PD-1/PD-L1 pathway plays a pivotal role in T cell activity and is involved in the pathophysiology of Mycobacterium tuberculosis (MTB) infection. DNA methylation is a mechanism that modulates PD-L1 expression in cancer cells. However, its effect on PD-L1 expression in macrophages after MTB infection remains unknown. We prospectively enrolled patients with active tuberculosis (TB) and non-TB subjects. The expression of PD-L1 and methylation-related genes in peripheral blood mononuclear cells (PBMCs) were investigated and their correlation with disease severity and treatment outcomes were examined. PD-L1 promoter methylation status was evaluated using bisulfite sequencing. Immunohistochemistry (IHC) and immunofluorescence (IF) staining were used to visualize PD-L1- and TET-1-expressing cells in lung tissues from patients with TB and in macrophage cell lines with MTB-related stimulation. In total, 80 patients with active TB and 40 non-TB subjects were enrolled in the analysis. Patients with active TB had significantly higher expression of PD-L1, DNMT3b, TET1, TET2, and lower expression of DNMT1, compared to that in the non-TB subjects. The expression of PD-L1 and TET-1 was significantly associated with 1-month smear and culture non-conversion. IHC and IF staining demonstrated the co-localization of PD-L1- and TET-1-expressing macrophages in patients with pulmonary TB and in human macrophage cell lines after MTB-related stimulation. DNMT inhibition and TET-1 knockdown in human macrophages increased and decreased PD-L1 expression, respectively. Overall, PD-L1 expression is increased in patients with active TB and is correlated with treatment outcomes. DNA methylation is involved in modulating PD-L1 expression in human macrophages.

RNA-seq analysis reveals modulation of inflammatory pathways by an enriched-triterpene natural extract in mouse and human macrophage cell lines

Chronic inflammation is crucial in developing insulin resistance and type 2 diabetes. Previous studies have shown that a leaf extract of Eucalyptus tereticornis, with ursolic acid (UA), oleanolic acid (OA), and ursolic acid lactone (UAL) as the main molecules (78 %) mixed with unknown minor metabolites (22 %), provided superior anti-inflammatory, hypoglycemic, and hypolipidemic effects than reconstituted triterpenoid mixtures in macrophage cell lines and a pre-diabetic mouse model. Further identification of the molecular mechanisms of action of this mixture of triterpenes is required. This study aims to analyse the RNA expression profiles of mouse and human macrophage cell lines treated with the natural extract and its components. Activated macrophage cell lines were treated with the natural extract, UA, OA, UAL or a triterpene mixture (M1). RNA was extracted and sequenced using the DNBseq platform and the EnrichR software to perform gene enrichment analysis using the Gene Ontology database, Kyoto Encyclopedia of Genes and Genomes, and Reactome. To conduct clustering analysis, we standardised the normalised counts of each gene and applied k-means clustering. The combination of molecules in the natural extract has an additive or synergic effect that affects the expression of up-regulated genes by macrophage activation. Triterpenes (M1) regulated 76 % of human and 68 % of mouse genes, while uncharacterised minority molecules could regulate 24 % of human and 32 % of mouse genes. The extract inhibited the expression of many cytokines (IL6, IL1, OSM), chemokines (CXCL3), inflammatory mediators (MMP8 and MMP13) and the JAK-STAT signalling pathway in both models. The natural extract has a more powerful immunomodulatory effect than the triterpene mixture, increasing the number of genes regulated in mouse and human models. Our study shows that Eucalyptus tereticornis extract is a promising option for breaking the link between inflammation and insulin resistance.

In vitro antileishmanial activity of thioridazine on amphotericin B unresponsive/ sensitive Leishmania donovani promastigotes and intracellular amastigotes

The recent increase in the drug (liposomal amphotericin-B) unresponsive cases becomes hostile for the visceral leishmaniasis (VL) elimination target. The quest for new antileishmanial drugs is on the way and may demand more time. Meanwhile, drug repurposing is a quite promising option to explore further. We made such an attempt with thioridazine (TRZ), a first-line antipsychotic drug, which was reported for antimicrobial activity. In this study, we evaluated the drug activity of TRZ against amphotericin-B (Amp-B) sensitive and unresponsive Leishmania donovani promastigotes, as well as intracellular amastigotes (drug sensitive). We observed a potent antileishmanial activity of TRZ with significantly low half maximal inhibitory concentrations (IC50) on both the variants of promastigotes (0.61 ± 0.15 μM). These concentrations are comparable to the previously reported IC50 concentration of the current antileishmanial drug (Amp-B) against L. donovani. Light microscopy reveals the perturbations in promastigote morphology upon TRZ treatment. The in vitro studies on human macrophage cell lines determine the 50% cytotoxicity concentration (CC50) of TRZ on host cells as 20.046 μM and a half maximal effective concentration (EC50) as 0.91 μM during L. donovani infection, in turn selectivity index (SI) was calculated as 22.03 μM. Altogether, the results demonstrate that TRZ has the potential for drug repurposing and further studies on animal models could provide better insights for VL treatment.

Understanding the modulations of glycero-lysophospholipids in an elastase-induced murine emphysema model

BackgroundIncreasing evidence indicates that bioactive lipid mediators are involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Recently, glycero-lysophospholipids, such as lysophosphatidic acid (LysoPA) and lysophosphatidylserine (LysoPS), have been recognized as significant inflammation-related lipid mediators. However, their association with COPD remains unclear. MethodsWe used an elastase-induced murine emphysema model to analyze the levels of lysophospholipids and diacyl-phospholipids in the lungs. Additionally, we assessed the expression of LysoPS-related genes and published data on smokers. ResultsIn the early phase of an elastase-induced murine emphysema model, the levels of LysoPS and its precursor (phosphatidylserine [PS]) were significantly reduced, without significant modulations in other glycero-lysophospholipids. Additionally, there was an upregulation in the expression of lysoPS receptors, specifically GPR34, observed in the lungs of a cigarette smoke-exposed mouse model and the alveolar macrophages of human smokers. Elastase stimulation induces GPR34 expression in a human macrophage cell line in vitro. ConclusionsElastase-induced lung emphysema affects the LysoPS/PS-GPR34 axis, and cigarette smoking or elastase upregulates GPR34 expression in alveolar macrophages. This novel association may serve as a potential pharmacological target for COPD treatment.

Examining the immunotoxicity of oil sands process affected waters using a human macrophage cell line

Oil sands process affected water (OSPW) is produced during the surface mining of the oil sands bitumen deposits in Northern Alberta. OSPW contains variable quantities of organic and inorganic components causing toxic effects on living organisms. Advanced Oxidation Processes (AOPs) are widely used to degrade toxic organic components from OSPW including naphthenic acids (NAs). However, there is no established biological procedure to assess the effectiveness of the remediation processes. Our previous study showed that human macrophage cells (THP-1) can be used as a bioindicator system to evaluate the effectiveness of OSPW treatments through examining the proinflammatory gene transcription levels. In the present study, we investigated the immunotoxicological changes in THP-1 cells following exposure to untreated and AOP-treated OSPW. Specifically, using proinflammatory cytokine protein secretion assays we showed that AOP treatment significantly abrogates the ability of OSPW to induce the secretion of IL-1β, IL-6, IL-8, TNF-α, IL-1Ra and MCP-1. By measuring transcriptional activity as well as surface protein expression levels, we also showed that two select immune cell surface markers, CD40 and CD54, were significantly elevated following OSPW exposure. However, AOP treatments abolished the immunostimulatory properties of OSPW to enhance the surface expression of these immune proteins. Finally, a transcriptome-based approach was used to examine the proinflammatory effects of OSPW as well as the abrogation of immunotoxicity following AOP treatments. Overall, this research shows how a human macrophage cell-based biomonitoring system serves as an effective in vitro tool to study the immunotoxicity of OSPW samples before and after targeted remediation strategies.

Echinococcus granulosus cyst fluid inhibits KDM6B-mediated demethylation of trimethylated histone H3 lysine 27 and interleukin-1β production in macrophages

BackgroundEchinococcus granulosus can manipulate its host's immune response to ensure its own survival. However, the effect of histone modifications on the regulation of the NOD-like receptor protein 3 (NLRP3) inflammasome and downstream interleukin-1β (IL-1β) production in response to the parasite is not fully understood.MethodsWe evaluated IL-1β secretion through enzyme-linked immunosorbent assay and assessed reactive oxygen species levels using the dichlorodihydrofluorescein diacetate probe. Western blotting and quantitative real-time polymerase chain reaction were performed to examine the expression of NLRP3 and IL-1β in mouse peritoneal macrophages and Tohoku Hospital Pediatrics-1 cells, a human macrophage cell line. The presence of trimethylated histone H3 lysine 27 (H3K27me3) modification on NLRP3 and IL-1β promoters was studied by chromatin immunoprecipitation.ResultsTreatment with E. granulosus cyst fluid (EgCF) considerably reduced IL-1β secretion in mouse and human macrophages, although reactive oxygen species production increased. EgCF also suppressed the expression of NLRP3 and IL-1β. Mechanistically, EgCF prompted the enrichment of repressive H3K27me3 modification on the promoters of both NLRP3 and IL-1β in macrophages. Notably, the presence of EgCF led to a significant reduction in the expression of the H3K27me3 demethylase KDM6B.ConclusionsOur study revealed that EgCF inhibits KDM6B expression and H3K27me3 demethylation, resulting in the transcriptional inhibition of NLRP3 and IL-1β. These results provide new insights into the immune evasion mechanisms of E. granulosus.Graphical