Human THP-1 Cells Research Articles

Long-Term Prevention of Arthroplasty Infections via Incorporation of Activated AgNbO3 Nanoparticles in PMMA Bone Cement.

We investigated the possibility of loading PMMA bone cement with antimicrobial nanostructured AgNbO3 particles to counter biofilm formation at the cement-tissue interface. We found that a formulation containing (1-4)% AgNbO3 showed high antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa while not showing any toxicity against THP1 human cell lines. In addition, loading the particles did not impact the mechanical properties of the cement. The results thus obtained illustrate the potential of the approach to replace the current technique of mixing cement with conventional antibiotics, which is associated with shortcomings such as efficacy loss from antibiotic depletion.

Octahydroindolizine alkaloid Homocrepidine A from Dendrobium crepidatum attenuate P. acnes-induced inflammatory in vitro and in vivo

Ethnopharmacological relevanceDendrobium crepidatum Lindl. ex Paxton is a perennial epiphyte of Dendrobium genus, distributed in southern China, and utilized as the traditional Chinese medicine “Shihu” in Yunnan Province. Due to its heat-clearing and detoxicating properties, it is formulated as the “XiaoCuoWan” as recorded in the China Pharmacopoeia, and specially used to treat chronic skin inflammatory diseases, such as acne. Aim of the studyThis research aimed to estimate impact of the octahydroindoline alkaloid Homocrepidine A (HCA), isolated from D. crepidatum, on acne inflammation using both human THP-1 cells and mouse models. Furthermore, the potential anti-inflammatory mechanism of HCA has been analyzed through molecular biology methods and computer simulation. Materials and methodsTHP-1 cells and mouse models induced by live Propionibacterium acnes (P. acnes) were employed to evaluate the anti-inflammatory properties of crude extract of D. crepidatum (DCE) and HCA. ELISA was utilized to detect the release of inflammatory cytokines in both cellular and murine ear tissues. RNAseq was used to screen the pathways associated with HCA-mediated inflammatory inhibition, while Western blot, RT-qPCR, and immunofluorescence were utilized to detect the expression of relevant proteins. Additionally, molecular docking simulations and cellular thermal shift assays were employed to confirm the target of HCA. ResultsOur research shows that DCE and HCA can effectively alleviate acne inflammation. HCA inhibits TLR2 expression by interacting with amino acid residues in the TIR domain of hTLR2, including Pro-681, Asn-688, Trp-684, and Ile-685. Moreover, HCA disrupts inflammatory signal transduction mediated by MAPK and NF-κB pathways through MyD88-dependent pathway. Additionally, HCA treatment facilitates Nrf2 nuclear translocation and upregulates HO-1 expression, thereby inhibiting NLRP3 inflammasomes activation. In vivo experiments further revealed that HCA markedly attenuated erythema and swelling caused by P. acnes in mice ears, while also decreasing the expression of pro-inflammatory cytokines IL-1β and IL-8. ConclusionsOur research highlights the protective effects of D. crepidatum and its bioactive compound HCA against acne inflammation, marking the first exploration of its potential in this context. The discoveries indicate that HCA treatment may represent a promising functional approach for acne therapy.

Identification of a functional missense variant in the matrix metallopeptidase 10 (MMP10) gene in two families with premature myocardial infarction

A positive family history is a major independent risk factor for atherosclerosis, and genetic variation is an important aspect of cardiovascular disease research. We identified a heterozygous missense variant p.L245P in the MMP10 gene in two families with premature myocardial infarction using whole-exome sequencing. The aim of this study was to investigate the consequences of this variant using in-silico and functional in-vitro assays. Molecular dynamics simulations were used to analyze protein interactions, calculate free binding energy, and measure the volume of the substrate-binding cleft of MMP10-TIMP1 models. The p.L245P variant showed an altered protein surface, different intra- and intermolecular interactions of MMP10-TIMP1, a lower total free binding energy between MMP10-TIMP1, and a volume-minimized substrate-binding cleft of MMP10 compared to the wild-type. For the functional assays, human THP-1 cells were transfected with plasmids containing MMP10 cDNA carrying the p.L245P and wild-type variant and differentiated into macrophages. Macrophage adhesion and migration assays were then conducted, and pro-inflammatory chemokine levels were evaluated. The p.L245P variant led to macrophages that were more adherent, less migratory, and secreted higher levels of the pro-inflammatory chemokines CXCL1 and CXCL8 than wild-type macrophages. Thus, the p.L245P variant in MMP10 may influence the pathogenesis of atherosclerosis in families with premature myocardial infarction by altering protein - protein interactions, macrophage adhesion and migration, and expression of pro-inflammatory chemokines, which may increase plaque rupture. These results could contribute to the development of selective MMP10 inhibitors and reduce the risk of atherosclerosis in families with a history of premature myocardial infarction.

Metabolic Reprogramming of Human Macrophages by Sickle Cell Hemoglobin

BACKGROUND: Sickle cell disease is caused by a single nucleotide mutation in the β-globin gene, resulting in the substitution of valine for glutamic acid, polymerization of hemoglobin and sickling of red blood cells (RBCs) under low oxygen conditions causing chronic hemolysis, and organs damage. SCA is characterized by the presence of cell-free plasma hemoglobin. Organ damage is associated with chronic inflammation and pro-inflammatory macrophages phenotype. Typically, anti-inflammatory response is elicited by the phagocytosis of senescent RBCs and cell-free hemoglobin. We have demonstrated that treatment of human macrophages with sickle cell hemoglobin (HbS) leads to lysosome accumulation, autophagy activation, and pro-inflammatory phenotype. The propensity of mutant HbS to polymerize under low pH may promote lysosomal HbS polymer accumulation, hindering degradation, and inducing macrophage differentiation towards a pro-inflammatory state. The differentiation of macrophages into specific phenotypes is associated with significant metabolic reprogramming. HYPOTHESIS: We hypothesize that the uptake of HbS by human macrophages induces their metabolic reprogramming via mitochondrial remodeling. METHODS: Human THP-1 cells were differentiated into macrophages with PMA and treated with either purified HbS or normal hemoglobin (HbA). Mitochondria were detected with MitoTrackerTM Green FM dye, and mitochondrial ROS (mtROS) level was detected by Mito SOX Red Kit. Transmission electron microscopy imaging was performed at Talos 200X transmission electron microscope. EPView (Olympus) software was used for the structure analysis. Total RNA was isolated by Trizol and sequenced on an Illumina® NextSeq 500. Comparisons between treatments were conducted using Gene Set Enrichment Analysis (GSEA). RESULTS: Immunofluorescent staining demonstrated accumulation of mitochondria after treatment with HbS compared to untreated (ctrl) or HbA-treated macrophages. Electron microscopy revealed an increase in the number and size of mitochondria. In 25 random microphotographs of cytoplasm, we detected 51 mitochondria in control, 79 in HbA treated and, 96 in HbS treated macrophages. The mitochondria volume and cristae size were significantly increased in macrophages treated with HbS. There was no increase in mtROS production in HbS treatment compared to ctrl. As expected, HbA treatment reduced mtROS production. The mitochondrial gene set (242 genes) was the second most enriched GSEA set in HbS-treated compared to HbA-treated macrophages. The larger mitochondrial size may be associated with a reduction of fission or an increase in fusion. We did not find differences in the expression of mitochondrial fission and fusion genes between HbS and HbA-treated macrophages. The increase in the mitochondrial size and cristae size may also be associated with increased oxidative phosphorylation. We found significant enrichment of genes responsible for oxidative phosphorylation in the HbS-treated macrophages. We did not find significant differences in the expression of glycolysis genes. CONCLUSION: Treatment of human macrophages with HbS significantly increases the number and volume of mitochondria, size of the cristae, and oxidative phosphorylation without upregulation of mtROS production and reduction of glycolysis. This work was supported by NIH Research Grants SC1HL150685, P50HL118006, and R01HL125005. We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Generation of a novel agonist of the human CD137 costimulatory pathway for cancer immunotherapy

Abstract The use of immune checkpoint stimulators is an increasingly attractive therapeutic approach against cancer, and costimulatory pathway agonists are being developed for clinical use. Our laboratory has focused on the 4-1BB (CD137) pathway because of its critical role in CD8+ T cell expansion, survival, acquisition of effector function, and establishment of long-term memory. We previously reported the generation of a recombinant oligomeric form of the mouse ligand, muSA-4-1BBL, which, unlike the natural 4-1BB ligand, demonstrated robust costimulatory activity in soluble form. muSA-4-1BBL showed therapeutic efficacy as the immune adjuvant component of subunit vaccines in various mouse transplantable tumor models. In this study, we generated a human version of this ligand, huSA-4-1BBL, and characterized its structure and function. The molecule binds to 4-1BB on LPS-activated human THP-1 monocytes, but not ConA-stimulated mouse T cells expressing the 4-1BB receptor. Conversely, muSA-4-1BBL binds to ConA-stimulated mouse T cells, but not LPS-activated human THP-1 cells, demonstrating species-specific binding for both molecules. To assess the cancer immunotherapeutic efficacy of huSA-4-1BBL, we generated a transgenic mouse model where mouse 4-1BB was replaced with the human ortholog. The assessment of huSA-4-1BBL anti-tumor efficacy in this model will serve as a prelude for further development and translation of this novel agonist to the clinic for cancer immunotherapy.

Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

Disordered GPR43/NLRP3 expression in peripheral leukocytes of patients with atrial fibrillation is associated with intestinal short chain fatty acids levels

BackgroundAtrial fibrillation (AF) is associated with circulating inflammation. Short-chain fatty acids (SCFAs) derived from gut microbiota (GM) regulate leukocyte function and inhibit the release of inflammatory cytokines, which are partly mediated by the G-protein-coupled receptor 43 (GPR43) signaling. This study aimed to investigate the expression of GPR43/NOD-like receptors family pyrin domain containing 3 (NLRP3) in leukocytes and the interaction with intestinal SCFAs levels in AF patients.MethodsExpressions of GPR43 and NLRP3 mRNA in peripheral blood leukocytes from 23 AF patients and 25 non-AF controls were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expressions of leukocyte GPR43 and NLRP3 protein were evaluated by western blot analysis. The levels of plasma IL-1β were measured by enzyme-linked immunosorbent assay (ELISA). The fecal SCFAs levels based on GC/MS metabolome of corresponding 21 controls and 14 AF patients were acquired from our published dataset. To evaluate the expression of NLRP3 and GPR43 and the release of IL-1β, human THP-1 cells were stimulated with or without SCFAs (acetate, propionate, and butyrate), lipopolysaccharide (LPS), and nigericin in vitro, respectively.ResultsCompared to the controls, the mRNA expression in peripheral leukocytes was significantly reduced in AF patients (P = 0.011) coupled with the increase in downstream leukocyte NLRP3 mRNA expression (P = 0.007) and plasma IL-1β levels (P < 0.001), consistent with changes in GPR43 and NLRP3 protein expression. Furthermore, leukocyte GPR43 mRNA levels were positively correlated with fecal GM-derived acetic acid (P = 0.046) and negatively correlated with NLRP3 mRNA expression (P = 0.024). In contrast to the negative correlation between left atrial diameter (LAD) and GPR43 (P = 0.008), LAD was positively correlated with the leukocyte NLRP3 mRNA levels (P = 0.024). Subsequent mediation analysis showed that 68.88% of the total effect of intestinal acetic acid on AF might be mediated by leukocyte GPR43/NLRP3. The constructed GPR43–NLRP3 score might have a predictive potential for AF detection (AUC = 0.81, P < 0.001). Moreover, SCFAs treatment increased GPR43 expression and remarkably reduced LPS/nigericin-induced NLRP3 expression and IL-1β release in human THP-1 cells in vitro.ConclusionsDisrupted interactions between GPR43 and NLRP3 expression in peripheral blood leukocytes, associated with reduced intestinal GM-derived SCFAs, especially acetic acid, may be involved in AF development and left atrial enlargement by enhancing circulating inflammation.

Abstract 5766: Fungal genomes in drug discovery: Application of CETSA to study the mechanism of action of brefeldin A

Abstract Fungal genomes encode a vast collection of chemical diversity that has evolved over millions of years to engage specific targets and modulate diverse biological processes. These Genetically Encoded small Molecules (GEMs) constitute a largely untapped source of potential therapeutics. Although fungal GEMs such as cyclosporine, lovastatin, and penicillin have revolutionized modern medicine, these GEMs were discovered serendipitously through bioactivity-guided fractionation of fungal extracts. LifeMine is redefining GEM discovery through the deployment of proprietary genomic search algorithms that enable GEM target prediction a priori. Our strategy facilitates focused search-and-retrieval campaigns to rationally discover molecules selected by Nature to modulate the desired target protein. These GEM drug leads often exhibit unique structural and mechanistic features, necessitating comprehensive deconvolution of the human target profile and mechanism of action during the development process from GEM to clinical candidate. As a proof-of-concept study, we leveraged CETSA Explore to study the biological mechanism of the fungal polyketide lactone Brefeldin A (BFA) in human THP-1 cells. This GEM, originally isolated from the filamentous fungus Penicillium brefeldianis, inhibits protein trafficking between the endoplasmic reticulum and the Golgi complex, eliciting antiviral, antifungal, and anticancer activity. Our CETSA profiling study confirmed the Golgi-associated guanine nucleotide exchange factor GBF1 as the primary target of BFA, validating the established mechanism of this GEM as an inhibitor of GBF1-mediated activation of the small GTPase ARF1. These results demonstrate the utility of integrating the LifeMine GEM discovery engine with downstream proteomic profiling technologies to facilitate the mechanistic characterization of fungal GEMs en route to novel therapeutics. Citation Format: Tomas Friman, Benjamin M. Fontaine, Alexey Chernobrovkin, Daniel Martinez Molina, Victoria Brehmer, Stina Lundgren, Paola M. Castaldi, Gregory L. Verdine. Fungal genomes in drug discovery: Application of CETSA to study the mechanism of action of brefeldin A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5766.

Dielectric properties of human macrophages are altered by Mycobacterium tuberculosis infection.

The analysis of cell electrophysiology for pathogenic samples at BSL3 can be problematic. It is virtually impossible to isolate infected from uninfected without a label, for example green fluorescent protein, which can potentially alter the cell electrical properties. Furthermore, the measurement of highly pathogenic organismsoften requires equipment dedicated only for use with these organisms due to safety considerations. To address this, we have used dielectrophoresis to study the electrical properties of the human THP-1 cell line and monocyte-derived macrophages before and after infection with non-labelled Mycobacterium tuberculosis. Infection with these highly pathogenic bacilli resulted in changes including a raised surface conductance (associated with reduced zeta potential) and increased capacitance, suggesting an increase in surface roughness. We have also investigated the effect of fixation on THP-1 cells as a means to enable study on fixed samples in BSL1 or 2 laboratories, which suggests that the properties of these cells are largely unaffected by the fixation process. This advance results in a novel technique enabling the isolation of infected and non-infected cells in a sample without labelling.

The MYC-NFATC2 axis maintains the cell cycle and mitochondrial function in acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) is a clonal haematological malignancy affecting the myeloid lineage, with generally poor patient outcomes owing to the lack of targeted therapies. The histone lysine demethylase 4A (KDM4A) has been established as a novel therapeutic target in AML, due to its selective oncogenic role within leukaemic cells. We identify that the transcription factor nuclear factor of activated T cells 2 (NFATC2) is a novel binding and transcriptional target of KDM4A in the human AML THP-1 cell line. Furthermore, cytogenetically diverse AML cell lines, including THP-1, were dependent on NFATC2 for colony formation in vitro, highlighting a putative novel mechanism of AML oncogenesis. Our study demonstrates that NFATC2 maintenance of cell cycle progression in human AML cells was driven primarily by CCND1. Through RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq), NFATc2 was shown to bind to the promoter region of genes involved in oxidative phosphorylation and subsequently regulate their gene expression in THP-1 cells. Furthermore, our data show that NFATC2 shares transcriptional targets with the transcription factor c-MYC, with MYC knockdown phenocopying NFATC2 knockdown. These data suggest a newly identified co-ordinated role for NFATC2 and MYC in the maintenance of THP-1 cell function, indicative of a potential means of therapeutic targeting in human AML.

Effect of n-Hexane Extract from Tanacetum argenteum (Lam.) Willd. subsp. argenteum on the Secretion of Proinflammatory Cytokines in THP-1 Cell Line

Inflammation is an initial biological process that involves the activation of the immune system in response to injury, infection or exposure to toxic agents. During this process, cytokines, small proteins produced by immune cells, play a vital role in regulating the immune response. Inflammatory cytokines, including interleukins, tumor necrosis factor-α, nitric oxide, and interferon-gamma, initiate the immune response and promote inflammation. Natural products are frequently a source of potential anti-inflammatory compounds, and screening natural products can lead to the discovery of novel bioactive compounds. The present study aimed to investigate the effects of n-hexane extract from Tanacetum argenteum subsp. argenteum on the lipopolysaccharide-induced inflammatory response in human macrophages THP-1 cell. Cells were incubated with different concentrations of n-hexane extract, and the inhibitor effects of the extract exposure on various cytokine secretions were determined. The findings demonstrated that n-hexane extract dramatically decreased the levels of interleukin‑6, interleukin‑1β, and tumor necrosis factor‑α in differentiated THP-1 cells, indicating the remarkable anti-inflammatory potential of the extract. Furthermore, in silico analysis of toxicogenomic data revealed the interactions between the active component of the n-hexane extract and interleukin-6, interleukin-1β, and tumor necrosis factor.

A66 EFFECTS OF HYMENOLEPIS DIMINUTA EXTRACELLULAR VESICLES ON MOUSE MACROPHAGES AND THEIR MITOCHONDRIA

Abstract Background In recent years, extracellular vesicles (EVs) have emerged as important means of intercellular communication and immune modulation between helminth and host. Earlier work revealed that excretory/secretory products from the rat tapeworm Hymenolepis diminuta (HES) have anti-inflammatory properties when applied to macrophages. In this study, we have investigated the immunomodulatory properties of H. diminuta EVs (HEVs) on macrophages. Aims 1. To isolate and characterize bacterial contaminant-free H. diminuta EVs (Hd-EVs). 2. To determine the effects of Hd-EVs on murine macrophage metabolism and LPS-evoked cytokine production. Methods Adult H. diminuta were cultured for 6h in serum-free RMPI and Hd-EVs isolated from the conditioned medium using an exoEasy kit. Hd-EV protein content was measured using a micro-BCA kit, and size and quantity were measured using Nanosight tracking analysis. Murine bone marrow-derived macrophages or the human macrophage THP-1 cell line were treated with LPS (10 ng/ml) ± a crude H. diminuta extract (HdE), HES, or Hd-EVs for 24h. TNF-α levels in the culture medium was measured by ELISA. Macrophage oxidative phosphorylation (OX.PHOS) and glycolysis were measured in real-time using an Agilent Seahorse XF Analyzer. Results Analysis of 14 Hd-EV samples revealed 4.1 X 109 - 5.48 X 1010 particles/mL, with Hd-EVs predominantly 100-350 nm in diameter and with a protein content of 64.9-181.6 ng/mL. HdE and HES reduced TNF-α production in LPS-activated macrophages by 39-76% (n=11), while Hd-EVs did not lower TNF-α production. Metabolic analysis suggests that HES and Hd-EVs can up-regulate macrophage glycolysis, while only Hd-EVs exposed macrophages showed increased OX.PHOS (n=6). Conclusions We have successfully isolated and begun to characterize EVs from adult H. diminuta. Despite the known abilities of HES, we unexpectedly noted that exposure to Hd-EVs did not result in a statistically significant decrease in macrophage TNF-α release. However, the increase in OX.PHOS macrophages treated with Hd-EVs is something to examine further. The next steps are to determine the contents and immunomodulatory activity of the Hd-EVs, thus advancing knowledge of how helminths can manipulate their hosts. Funding Agencies NSERC Clinical Practice

The Impact of Lens Epithelium-Derived Growth Factor p75 Dimerization on Its Tethering Function.

The transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75) plays an important role in the biology of the cell and in several human diseases, including MLL-rearranged acute leukemia, autoimmunity, and HIV-1 infection. In both health and disease, LEDGF/p75 functions as a chromatin tether that interacts with proteins such as MLL1 and HIV-1 integrase via its integrase-binding domain (IBD) and with chromatin through its N-terminal PWWP domain. Recently, dimerization of LEDGF/p75 was shown, mediated by a network of electrostatic contacts between amino acids from the IBD and the C-terminal α6-helix. Here, we investigated the functional impact of LEDGF/p75 variants on the dimerization using biochemical and cellular interaction assays. The data demonstrate that the C-terminal α6-helix folds back in cis on the IBD of monomeric LEDGF/p75. We discovered that the presence of DNA stimulates LEDGF/p75 dimerization. LEDGF/p75 dimerization enhances binding to MLL1 but not to HIV-1 integrase, a finding that was observed in vitro and validated in cell culture. Whereas HIV-1 replication was not dependent on LEDGF/p75 dimerization, colony formation of MLLr-dependent human leukemic THP-1 cells was. In conclusion, our data indicate that intricate changes in the quaternary structure of LEDGF/p75 modulate its tethering function.

Antimicrobial Effect of Cannabidiol on Intracellular Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), has killed nearly one billion people during the last two centuries. Nowadays, TB remains a major global health problem ranked among the top 10 causes of death worldwide. One of the main challenges in developing new strategies to fight TB is focused on reducing the duration and complexity of drug regimens. Cannabidiol (CBD) is the main nonpsychoactive ingredient extracted from the Cannabis sativa L. plant, which has been shown to be biologically active against bacteria. The purpose of this work was to investigate the antimicrobial effect of CBD on M. tuberculosis intracellular infection. Materials and Methods: To assess the minimum inhibitory concentration (MIC) of CBD on mycobacterial strains, the MTT assay was performed on Mycobacterium smegmatis, and the Colony-Forming Unit (CFU) assay was conducted on MtbH37Rv. Additionally, the cytotoxic effect of CBD on THP-1 cells was assessed by MTT assay. Moreover, macrophages derived from the THP-1 cell were infected with MtbH37Rv (multiplicity of infection 1:10) to evaluate the intracellular activity of CBD by determining the CFU/mL. Results: Antimicrobial activity against M. smegmatis (MIC=100 μM) and MtbH37Rv (MIC=25 μM) cultures was exhibited by CBD. Furthermore, the effect of CBD was also evaluated on MtbH37Rv infected macrophage cells. Interestingly, a reduction in viable intracellular MtbH37Rv bacteria was observed after 24 h of treatment. Moreover, CBD exhibited a safe profile toward human THP-1 cells, since it showed no toxicity (CC50=1075 μM) at a concentration of antibacterial effect (selectivity index 43). Conclusion: These results extend the knowledge regarding the antimicrobial activity of CBD and demonstrate its ability to kill the human intracellular pathogen M. tuberculosis.

STAT3-Mediated Ferroptosis is Involved in Sepsis-Associated Acute Respiratory Distress Syndrome.

Sepsis-induced acute respiratory distress syndrome (ARDS) poses a grave danger to life, resulting from sepsis-induced multi-organ failure. Although ferroptosis, a form of iron-dependent lipid peroxidative cell death, has been associated with sepsis-induced ARDS, the specific mechanisms are not fully understood. In this study, we utilized WGCNA, PPI, friends analysis, and six machine learning techniques (Lasso, SVM, RFB, XGBoost, AdaBoost, and LightGBM) to pinpoint STAT3 as a potential diagnostic marker. A significant increase in monocyte and neutrophil levels was observed in patients with sepsis-induced ARDS, as revealed by immune infiltration analyses, when compared to controls. Moreover, there was a positive correlation between STAT3 expression and the level of infiltration. Single-cell analysis uncovered a notable disparity in B-cell expression between sepsis and sepsis-induced ARDS. Furthermore, in vitro experiments using LPS-treated human bronchial epithelial cells (BEAS-2B) and THP1 cells demonstrated a significant increase in STAT3 phosphorylation expression. Additionally, the inhibition of STAT3 phosphorylation by Stattic effectively prevented LPS-induced ferroptosis in both BEAS-2B and THP1 cells. This indicates that the activation of STAT3 phosphorylation promotes ferroptosis in human bronchial epithelial cells in response to LPS. In summary, this research has discovered and confirmed STAT3 as a potential biomarker for the diagnosis and treatment of sepsis-induced ARDS.

Soluble urate-induced effects on cytokine production in vitro – Assessment of methodologies and cell types

BackgroundHyperuricemia has been shown to be an inducer of pro-inflammatory mediators by human primary monocytes. To study the deleterious effects of hyperuricemia, a reliable and stable in vitro model using soluble urate is needed. One recent report showed different urate-dissolving methods resulted in either pro-inflammatory or anti-inflammatory properties. The aim of this study was to compare the effect of two methods of dissolving urate on both primary human peripheral blood mononuclear cells (PBMCs) and THP-1 cells. The two methods tested were ‘pre-warming’ and ‘dissolving with NaOH’. MethodsPrimary human PBMCs and THP-1 cells were exposed to urate solutions, prepared using the two methodologies: pre-warming and dissolving with NaOH. Afterwards, cells were stimulated with various stimuli, followed by the measurement of the inflammatory mediators IL-1β, IL-6, IL-1Ra, TNF, IL-8, and MCP-1. ResultsIn PBMCs, we observed an overall pro-inflammatory effect of urate, both in the pre-warming and the NaOH dissolving method. A similar pro-inflammatory effect was seen in THP-1 cells for both dissolving methods after restimulation. However, THP-1 cells exhibited pro-inflammatory profile with exposure to urate alone without restimulation. We did not find MSU crystals in our cellular assays. ConclusionsOverall, the urate dissolving methods do not have critical impact on its inflammatory properties. Soluble urate prepared using either of the two methods showed mostly pro-inflammatory effects on human primary PBMCs and monocytic cell line THP-1. However, human primary PBMCs and the THP-1 differ in their response to soluble urate without restimulation.

HDAC11 Regulates Palmitate-induced NLRP3 Inflammasome Activation by Inducing YAP Expression in THP-1 Cells and PBMCs.

Histone deacetylase 11 (HDAC11) has been implicated in the pathogenesis of metabolic diseases characterized by chronic low-grade inflammation, such as obesity. However, the influence of HDAC11 on inflammation and the specific effect of HDAC11 on the palmitic acid (PA)-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation are poorly understood. The effect of PA treatment on HDAC11 activity and the NLRP3 inflammasome was investigated in human peripheral blood mononuclear cells and THP-1 cells. The PA-induced responses of key markers of NLRP3 inflammasome activation, including NLRP3 gene expression, caspase-1 p10 activation, cleaved IL-1β production, and extracellular IL-1β release, were assessed as well. The role of HDAC11 was explored using a specific inhibitor of HDAC11 and by knockdown using small interfering (si)HDAC11 RNA. The relationship between HDAC11 and yes-associated protein (YAP) in the PA-induced NLRP3 inflammasome was investigated in THP-1 cells with HDAC11 or YAP knockdown. Following PA treatment, HDAC11 activity and protein levels increased significantly, concomitant with activation of the NLRP3 inflammasome. Notably, PA-induced the upregulation of NLRP3, caspase-1 p10 activation, the production of cleaved IL-1β, and the release of IL-1β into the extracellular space, all of which were attenuated by FT895 treatment and by HDAC11 knockdown. In THP-1 cells, PA induced the expression of YAP and its interaction with NLRP3, resulting in NLRP3 inflammasome activation, whereas both were inhibited by FT895 and siHDAC11 RNA. These findings demonstrate a pivotal role for HDAC11 in the PA-induced activation of the NLRP3 inflammasome. HDAC11 inhibition thus represents a promising therapeutic strategy for mitigating NLRP3 inflammasome-related inflammation in the context of obesity.

The influence of cigR gene on the pathogenicity of Salmonella paratyphi A in vitro and in vivo.

Salmonella Paratyphi A is the causative agent of paratyphoid fever A which is a serious threat to human health in many countries. The cigR gene located in Salmonella pathogenicity island 3 is a type III secretion system 2 effector gene. However, the influence of cigR gene on the pathogenicity of Salmonella Paratyphi A remains unclear. Here, a cigR gene deletion mutant of Salmonella Paratyphi A was constructed and its pathogenic changes were also evaluated. It was found that both the growth and biochemical features have not changed after the loss of cigR, but the absence of cigR significantly enhanced the replication and/or survival ability in phorbol-12-myristate-13-acetate (PMA)-differentiated human macrophage THP-1 cells and in mouse; the proliferative activity and apoptosis of PMA-differentiated THP-1 cell were significantly decreased and increased, respectively, after the lack of cigR gene; and the mutant showed increased virulence to a mouse infection model by decreased half-lethal dose (LD50) value and enhanced the proliferation ratio of bacteria in vivo. These results demonstrated that CigR is an anti-virulence factor and plays an important role in the pathogenicity of Salmonella Paratyphi A.

POLARIZED MACROPHAGE-DERIVED EXTRACELLULAR VESICLES AS POTENTIAL MEDIATORS OF TENDON REPAIR

Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells.For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed.We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored.

Exploring hydrophilic 2,2-di(indol-3-yl)ethanamine derivatives against Leishmania infantum.

Herein we report the design and the synthesis of a library of new and more hydrophilic bisindole analogues based on our previously identified antileishmanial compound URB1483 that failed the preliminary in vivo test. The novel bisindoles were phenotypically screened for efficacy against Leishmania infantum promastigotes and simultaneously for toxicity on human macrophage-like THP-1 cells. Among the less toxic compounds, eight bisindoles showed IC50 below 10 μM. The most selective compound 1h (selectivity index = 10.1, comparable to miltefosine) and the most potent compound 2c (IC50 = 2.7 μM) were tested for their efficacy on L. infantum intracellular amastigotes. The compounds also demonstrated their efficacy in the in vitro infection model, showing IC50 of 11.1 and 6.8 μM for 1h and 2c, respectively. Moreover, 1h showed a better toxicity profile than the commercial drug miltefosine. For all these reasons, 1h could be a possible new starting point for hydrophilic antileishmanial agents with low cytotoxicity on human macrophage-like cells.