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Exposure to mineral fibers represents an occupational and environmental hazard since particulate inhalation leads to several health disorders. However, few data are available on the effect of fibers with high solubility like natural epsomite, a water‐soluble fiber with an inhalable size that allows it to penetrate biological systems, with regard to the respiratory tract. This study evaluated the natural (fibrous epsomite) and synthetic (Epsom salt) magnesium sulfate pathogenicity. Investigations have been performed through morpho‐functional and biochemical analyses, in an in vitro cell model that usually grows as monocytes, but that under appropriate conditions differentiates into macrophages. These latter, known as alveolar macrophages, if referred to lungs, represent the first line of defense against harmful inhaled stimuli. Morphological observations reveal that, if Epsom salt induces osmotic stress on cell culture, natural epsomite fibers lead to cellular alterations including thickening of the nuclear envelope and degenerated mitochondria. Moreover, the insoluble fraction (impurities) internalized by cells induces diffuse damage characterized at the highest dosage and exposure time by secondary necrosis or necrotic cell death features. Biochemical analyses confirm this mineral behavior that involves MAPK pathway activation, resulting in many different cellular responses ranging from proliferation control to cell death. Epsom salt leads to MAPK/ERK activation, a marker predictive of overall survival. Unlike, natural epsomite induces upregulation of MAPK/p38 protein involved in the phosphorylation of downstream targets driving necrotic cell death. These findings demonstrate natural epsomite toxicity on U937 cell culture, making the inhalation of these fibers potentially hazardous for human health. Research Highlights: Natural epsomite and synthetic Epsom salt effects have been evaluated in U937 cell model.Epsom salt induces an osmotic cellular stress.Natural epsomite fibers lead to cellular damage and can be considered potentially dangerous for human health. [ABSTRACT FROM AUTHOR]

Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy. [ABSTRACT FROM AUTHOR]

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

Background & aimsThe mechanisms by which macrophages regulate intestinal epithelial cell (IEC) barrier properties are poorly understood. Protein tyrosine phosphatase non-receptor type 2 (PTPN2) protects the IEC barrier from inflammation-induced disruption and regulates macrophage functions. We investigated whether PTPN2 controls interactions between IECs and macrophages to maintain intestinal barrier function.MethodsHuman IEC (Caco-2BBe/HT-29.cl19a cells) and mouse enteroid monolayers were cocultured with human macrophages (THP-1, U937, primary monocyte-derived macrophages from patients with inflammatory bowel disease [IBD]) or mouse macrophages, respectively. We assessed barrier function (transepithelial electrical resistance [TEER] and permeability to 4-kDa fluorescently labeled dextran or 70-kDa rhodamine B-dextran) and macrophage polarization. We analyzed intestinal tissues from mice with myeloid cell-specific deletion of PTPN2 (Ptpn2-LysMCre mice) and mice without disruption of Ptpn2 (controls); some mice were given injections of a neutralizing antibody against interleukin (IL) 6. Proteins were knocked down in macrophages and/or IECs with small hairpin RNAs.ResultsKnockdown of PTPN2 in either macrophages and/or IECs increased the permeability of IEC monolayers, had a synergistic effect when knocked down from both cell types, and increased the development of inflammatory macrophages in macrophage-IEC cocultures. Colon lamina propria from Ptpn2-LysMCre mice had significant increases in inflammatory macrophages; these mice had increased in vivo and ex vivo colon permeability to 4-kDa fluorescently labeled dextran and reduced ex vivo colon TEER. Nanostring analysis showed significant increases in the expression of IL6 in colon macrophages from Ptpn2-LysMCre mice. An IL6-blocking antibody reversed the effects of PTPN2-deficient macrophages, reducing the permeability of IEC monolayers in culture and in Ptpn2-LysMCre mice. Macrophages from patients with IBD carrying a single-nucleotide polymorphism associated with the disease (PTPN2 rs1893217) had the same features of PTPN2-deficient macrophages from mice, including reduced TEER and increased permeability in cocultures with human IEC or mouse enteroid monolayers, which were restored by anti-IL6.ConclusionsPTPN2 is required for interactions between macrophages and IECs; loss of PTPN2 from either cell type results in intestinal barrier defects, and loss from both cell types has a synergistic effect. We provide a mechanism by which the PTPN2 gene variants compromise intestinal epithelial barrier function and increase the risk of inflammatory disorders such as IBD.

Tumor-infiltrating leukocytes, in particular macrophages, play an important role in tumor behavior and clinical outcome. The spectrum of macrophage subtypes ranges from antitumor 'M1'-type to protumor 'M2'-type macrophages. Tumor-associated macrophages (TAMs) typically display phenotypic features of both M1 and M2, and the population distribution is thought to be dynamic and evolves as the tumor progresses. However, our understanding of how TAMs impact the tumor microenvironment remains limited by the lack of appropriate 3D in vitro models that can capture cell-cell dynamics at high spatial and temporal resolution. Using our recently developed microphysiological 'tumor-on-a-chip' (TOC) device, we present here our findings on the impact of defined macrophage subsets on tumor behavior. The TOC device design contains three adjacent and connected chambers in which both the upper and lower chambers are loaded with tumor cells, whereas the central chamber contains a dynamic, perfused, living microvascular network. Introduction of human pancreatic or colorectal cancer cells together with M1-polarized macrophages significantly inhibited tumor growth and tumor-induced angiogenesis. Protein analysis and antibody-based neutralization studies confirmed that these effects were mediated through production of C-X-C motif chemokines (CXCL9), CXCL10 and CXCL11. By contrast, M2-macrophages mediated increased tumor cell migration into the vascularized chamber and did not inhibit tumor growth or angiogenesis. In fact, single-cell RNA sequencing showed that M2 macrophages further segregated endothelial cells into two distinct subsets, corresponding to static cells in vessels versus active cells involved in angiogenesis. The impact of M2 macrophages was mediated mostly by production of matrix metalloproteinase 7 and angiopoietin 2. In summary, our data demonstrate the utility of the TOC device to mechanistically probe biological questions in a 3D in vitro microenvironment.

Therapeutic nucleic acids hold great promise for the treatment of genetic diseases, yet the delivery of this highly charged macromolecular drug remains a challenge in the field. Peptides are promising agents to mediate nucleic acid delivery because they can encode a biological function to overcome the trafficking barriers. Electrostatic nanocomplexes of nucleic acid and peptides can achieve effective delivery, but the balance between their stability and biological function must be finely tuned. In this work, we explore two peptide building blocks that have been studied in the literature: targeting ligands and intracellular trafficking peptides. We grafted these peptides on a polyethylene glycol (PEG) backbone with eight sites for substitution to create so-called "peptide spiders". These conjugates achieve stability via the well-known hydrophilic shielding effect of PEG. In addition, the coordination of peptide building blocks into multimers may create new biological properties, such as the well-known phenomena of increased binding avidity with multivalent ligands. In this work, we linked two trafficking peptides to the PEG backbone using either nonreducible or reducible chemistries and investigated the ability of these materials to carry silencing RNAs into mammalian cells. We then investigated these nanomaterials for their pharmacokinetic properties and silencing of undruggable targets in a mouse model of cancer. While reducible linkages were more potent at silencing in vitro, this effect was reversed when applied in the context of living animals. This work offers an insight into peptide-based delivery materials and investigates peptide-polymer linkages.

Ambient PM2.5 was collected during the winter season from Taiyuan, Shanxi, China; Jinan, Shandong, China; and Sacramento, California, USA, and used to create PMSX, PMSD, and PMCA extracts, respectively. Time-lag experiments were performed to explore the in vivo and in vitro toxicity of the PM extracts. In vivo inflammatory lung responses were assessed in BALB/c mice using a single oropharyngeal aspiration (OPA) of PM extract or vehicle (CTRL) on Day 0. Necropsies were performed on Days 1, 2, and 4 post-OPA, and pulmonary effects were determined using bronchoalveolar lavage (BAL) and histopathology. On Day 1, BAL neutrophils were significantly elevated in all PM- versus CTRL-exposed mice, with PMCA producing the strongest response. However, histopathological scoring showed greater alveolar and perivascular effects in PMSX-exposed mice compared to all three other groups. By Day 4, BAL neutrophilia and tissue inflammation were resolved, similar across all groups. In vitro effects were examined in human HepG2 hepatocytes, and U937 cells following 6, 24, or 48 h of exposure to PM extract or DMSO (control). Luciferase reporter and quantitative polymerase chain reaction assays were used to determine in vitro effects on aryl hydrocarbon receptor (AhR) activation and gene transcription, respectively. Though all three PM extracts activated AhR, PMSX produced the greatest increases in AhR activation, and mRNA levels of cyclooxygenase-2, cytochrome P450, interleukin (IL)-8, and interleukin (IL)-1β. These effects were assumed to result from a greater abundance of polycyclic aromatic hydrocarbons (PAHs) in PMSX compared to PMSD and PMCA.

Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.

There is strong evidence that exposure to fine particulate matter (PM2.5) and a high-fat diet (HFD) increase the risk of mortality from atherosclerotic cardiovascular diseases. Recent studies indicate that PM2.5 generated by combustion activates the Aryl Hydrocarbon Receptor (AHR) and inflammatory cytokines contributing to PM2.5-mediated atherogenesis. Here we investigate the effects of components of a HFD on PM-mediated activation of AHR in macrophages. Cells were treated with components of a HFD and AHR-activating PM and the expression of biomarkers of vascular inflammation was analyzed. The results show that glucose and triglyceride increase AHR-activity and PM2.5-mediated induction of cytochrome P450 (CYP)1A1 mRNA in macrophages. Cholesterol, fructose, and palmitic acid increased the PM- and AHR-mediated induction of proinflammatory cytokines in macrophages. Treatment with palmitic acid significantly increased the expression of inflammatory cytokines and markers of vascular injury in human aortic endothelial cells (HAEC) after treatment with PM2.5. The PM2.5-mediated activation of the atherogenic markers C-reactive protein (CRP) and S100A9, a damage-associated molecular pattern molecule, was found to be AHR-dependent and involved protein kinase A (PKA) and a CCAAT/enhancer-binding protein (C/EBP) binding element. This study identified nutritional factors interacting with AHR signaling and contributing to PM2.5-induced markers of atherogenesis and future cardiovascular risk.

BackgroundHIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency.ResultsTo quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts ("read-through," initiated, 5' elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival.ConclusionsHIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript.

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.

ABSTRACT The Zika virus (ZIKV) is teratogenic and considered a TORCH pathogen (toxoplasmosis [Toxoplasma gondii], rubella, cytomegalovirus, herpes simplex virus [HSV], and other microorganisms capable of crossing the blood-placenta barrier). In contrast, the related flavivirus dengue virus (DENV) and the attenuated yellow fever virus vaccine strain (YFV-17D) are not. Understanding the mechanisms used by ZIKV to cross the placenta is necessary. In this work, parallel infections with ZIKV of African and Asian lineages, DENV, and YFV-17D were compared for kinetics and growth efficiency, activation of mTOR pathways, and cytokine secretion profile using cytotrophoblast-derived HTR8 cells and monocytic U937 cells differentiated to M2 macrophages. In HTR8 cells, ZIKV replication, especially the African strain, was significantly more efficient and faster than DENV or YFV-17D. In macrophages, ZIKV replication was also more efficient, although differences between strains were reduced. Greater activation of the mTORC1 and mTORC2 pathways in HTR8 cells infected with ZIKV than with DENV or YFV-17D was observed. HTR8 cells treated with mTOR inhibitors showed a 20-fold reduction in ZIKV yield, versus 5- and 3.5-fold reductions for DENV and YFV-17D, respectively. Finally, infection with ZIKV, but not DENV or YFV-17D, efficiently inhibited the interferon (IFN) and chemoattractant responses in both cell lines. These results suggest a gating role for the cytotrophoblast cells in favoring entry of ZIKV, but not DENV and YFV-17D, into the placental stroma. IMPORTANCE Zika virus acquisition during pregnancy is associated with severe fetal damage. The Zika virus is related to dengue virus and yellow fever virus, yet fetal damage has not been related to dengue or inadvertent vaccination for yellow fever during pregnancy. Mechanisms used by the Zika virus to cross the placenta need to be deciphered. By comparing parallel infections of Zika virus strains belonging to the African and Asian lineages, dengue virus, and the yellow fever vaccine virus strain YFV-17D in placenta-derived cytotrophoblast cells and differentiated macrophages, evidence was found that Zika virus infections, especially by the African strains, were more efficient in cytotrophoblast cells than dengue virus or yellow fever vaccine virus strain infections. Meanwhile, no significant differences were observed in macrophages. Robust activation of the mTOR signaling pathways and inhibition of the IFN and chemoattractant response appear to be related to the better growth capacity of the Zika viruses in the cytotrophoblast-derived cells.

Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity.

Single cell analysis methods are increasingly being utilized to investigate how individual cells process information and respond to diverse stimuli. Soluble proteins play a critical role in controlling cell populations and tissues, but directly monitoring secretion is technically challenging. Microfabricated well arrays have been developed to assess secretion at the single cell level, but these systems are limited by low detection sensitivity. Semiconductor quantum dots (QD) exhibit remarkably bright and photostable luminescence signal, but to date they have not been evaluated in single cell secretion studies using microfabricated well arrays. Here, we used QDs in a sandwich immunoassay to detect secretion of the soluble cytokine tumor necrosis factor-α (TNF-α) from single cells. To enhance detection sensitivity, we employed two different strategies. First, we used a unique single QD imaging approach, which provided a detection threshold (180 attomolar) that was >100-fold lower than previously reported results using QDs. We also amplified QD binding to each captured TNF-α molecule using the bioorthogonal cycloaddition reaction between trans-cyclooctene and tetrazine, which further lowered detection threshold to 60 attomolar. This is 6 orders of magnitude more sensitive than organic fluorophores that have been used for single cell secretion studies, and far surpasses single molecule resolution within sub-picoliter microwells that are used to assess single cell secretion. Finally, single cell secretion studies were performed using phorbol 12-myristate 13-acetate (PMA) differentiated and lipopolysaccharide (LPS) activated U-937 cells. TNF-α secretion was detected from 3-fold more single cells using the QD-based method in comparison to rhodamine, which was accomplished by extending sensitivity into the range of ∼2 to 10 000 molecules captured per microwell. In future work, we will apply this technique to assess immune cell secretion dynamics under diverse stimuli and disease settings. We will also incorporate multiplexing capabilities to evaluate the secretome at the resolution of single molecules.

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

Background: MicroRNA is a form of non-coding RNAs that able to regulate gene expression. miR-424 is one of the members of the regulatory family, which plays an important role in the proliferation and differentiation of myeloid cells. Epigenetic changes can change the level of miR-424 under environmental factors. Therefore, the level of expression of miR-424 in U937 cells of the myeloid line was evaluated in this research under the influence of vitamin D3 (VitD3) and retinoic acid (RA). Methods: In this study, U937 cells were cultured in the presence of VitD3, and RA to evaluate cell proliferation, viability via the trypan blue exclusion test, and expression level of miR-424 by realtime PCR at specific times. Results: Cell proliferation has shown a significant decrease in the RA group versus other groups during incubation times (P < 0.05). In VitD3 group, there was a significant increase in cell proliferation after 24- and 48-hours incubation periods versus other groups. In the VitD3 and RA groups, the increase of cell proliferation caused the downregulation of miR-424. In addition, the upregulation of VitD3 group and downregulation of the RA group were significant versus the control group (P < 0.05). Conclusions: We concluded that the expression level of miR-424 was critically affected in the doseand time-dependent of RA and VitD3 treatment in the U937 cell line. Treatment with VitD3 decreased the expression of miR-424 and RA treatment increase miR-424 expression level in physiological doses. [ABSTRACT FROM AUTHOR]

Hiroshi Saito,1 Yuya Nakamura,2,3 Masahiro Inagaki,2,4 Shiho Yamadera,5 Hideo Misawa,2,3 Naoki Sato,2,6 Tatsunori Oguchi,2 Takae Inagaki,7 Yuya Tsuji,2 Mayumi Tsuji,2 Isao Ohsawa,3 Hiromichi Gotoh,3 Yuji Kiuchi2 1JA Hiroshima Genneral Hospital, Hatsukaichi City, Hiroshima, Japan; 2Department of Pharmacology, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan; 3Saiyu Soka Hospital, Soka City, Saitama, Japan; 4Faculty of Arts and Sciences at Fujiyoshida, Showa University, Fujiyoshida City, Yamanashi, Japan; 5Department of Hospital Pharmaceutics, Showa University School of Pharmacy, Shinagawa-ku, Tokyo, Japan; 6Department of Research Center, Tanabe Pharmacy Inc., Chuo-ku, Tokyo, Japan; 7Graduate School of Nursing and Rehabilitation Science, Yokohama City, Kanagawa, JapanCorrespondence: Yuya Nakamura Email y.nakamura@med.showa-u.ac.jpPurpose: Lipopolysaccharides (LPS) induce inflammation by binding to the Toll-like receptor (TLR) 4 complex, including LPS-binding protein (LBP). The anti-inflammatory effects of linagliptin in LPS-induced inflammation in the TLR4-independent pathway have not been examined before. We examined the anti-inflammatory effects of linagliptin in the TLR4- and the LBP-independent pathway.Methods: U937 cells were cultured in the medium supplemented with 10% fetal bovine serum (FBS) and treated with 100 nM phorbol myristate acetate for 48 h. Cells were then left untreated or were treated with 10 μg/mL anti-TLR4 antibodies alone or in combination with linagliptin for 1 h in media supplemented with or without 10% FBS. The cells were divided into 5 groups: a) control cells (untreated) b) cells treated with LPS c) cells treated with 10 μg/mL anti-TLR4 antibodies d) cells treated with LPS and 10 μg/mL anti-TLR4 antibodies and e) cells treated with LPS, 10 μg/mL anti-TLR4 antibodies, and linagliptin. The LPS concentrations used were 50 pg/mL or 100 pg/mL for cells treated in the presence of 10% FBS and 100 pg/mL or 1 μg/mL for cells treated in the absence of FBS. Linagliptin concentrations of 1 nM, 10 nM, and 100 nM were used for treatment. The supernatants were analyzed for interleukin (IL)-6 production after 24 h of various treatments.Results: LPS increased IL-6 production compared to the untreated control cells, and anti-TLR4 antibody suppressed LPS-induced increased IL-6 levels. Linagliptin suppressed LPS-induced IL-6 production in a concentration-dependent manner in the presence of FBS. However, only 100 nM linagliptin could suppress LPS-induced IL-6 production in the absence of FBS.Conclusion: Concentration-dependent and -independent inflammatory suppression was observed following linagliptin treatment after LPS induction in an experimental model of TLR4 inhibition by anti-TLR4 antibodies. Our results showed that linagliptin may inhibit inflammation through multiple mechanisms centered around the TLR-4-mediated pathway.Keywords: lipopolysaccharide, U937 cells, fetal bovine serum, anti- toll-like receptor 4 antibody, interleukin-6

Understanding macrophage biology can improve comprehension of diverse biological processes and provide insights into novel therapeutic immunomodulatory strategies. Due to limited yield and technical difficulty in isolating primary macrophages, in vitro studies commonly use monocytes as precursor cells. Monocytic cell lines are a virtually unlimited source of macrophage precursors and two of the most frequently used cell lines are THP-1 and U937. Besides a great variability in macrophage differentiation protocols there is scarce information on possible differences in the biological responses of these cell lines. In this study, we used a standardized differentiation protocol using PMA and compared the response of macrophages derived from THP-1 and U937 cells to M1-and M2-polarizing conditions. THP-1-derived macrophages are more responsive to M1 stimuli and skewed towards M1 phenotype, whereas U937-derived macrophages were more responsive to M2 stimuli and skewed towards M2 phenotype. THP-1-derived macrophages also had greater production of ROS and phagocytic activity. Under M1-polarizing conditions, macrophages derived from both THP-1 and U937 reduced phagocytosis activity and the increased production of ROS. This information should be considered to make an informed choice on the cell line used as in vitro macrophage model, according to the experimental goals and biological context.

Fc gamma receptor (FcyR)-mediated antibody functions play a crucial role in preventing HIV infection. One such function, antibody-dependent phagocytosis (ADP), is thought to be involved in controlling other viral infections, but its role in HIV infection is unknown. We measured the ability of HIV-specific polyclonal and monoclonal antibodies (mAbs) to mediate the internalization of HIV-1 virions and HIV-1-decorated cells by phagocytes. To measure ADP of virions, we primarily used a green-fluorescent protein-expressing molecular clone of HIV-1JRFL, an R5, clinical isolate, in combination with polyclonal HIVIG or mAbs known to capture and/or neutralize HIV-1. THP-1 and U937 cells, as well as freshly isolated primary monocytes from healthy individuals, were used as phagocytic effector cells, and uptake of virions was measured by cytometry. We surprisingly found minimal or no ADP of virions with any of the antibodies. However, after coating virions with gp41 or with gp41-derived peptides, gp41- (but not gp120-) specific mAbs efficiently mediated phagocytosis. We estimated that a minimum of a few hundred gp41 molecules were needed for successful phagocytosis, which is similar to the number of envelope spikes on viruses that are readily phagocytosed (e.g. influenza virus). Furthermore, by employing fluorescence correlation spectroscopy, a well-established technique to measure particle sizes and aggregation phenomena, we found a clear association between virus aggregation and ADP. In contrast to virions themselves, virion-decorated cells were targets for ADP or trogocytosis in the presence of HIV-specific antibodies. Our findings indicate that ADP of virions may not play a role in preventing HIV infection, likely due to the paucity of trimers and the consequent inability of virion-bound antibody to cross-link FcyRs on phagocytes. However, ADP or trogocytosis could play a role in clearing HIV-infected cells and cells on the verge of infection.

Staphylococcus aureus (S. aureus) has been suggested to be an initiative pathogen in peri-implantitis because of the solid affinity to titanium. However, the detail pathogenesis for the peri-implantitis initiation by S. aureus is still lacking. This study aimed to in vitro examine the gelatinases' activities of monocytic U937 cell and human gingival fibroblast after challenges with S. aureus lipoteichoic acid (LTA) and peptidoglycan (PGN). Releases of gelatinases, including matrix metalloproteinase (MMP)-2 and −9, from cells were measured by zymography. The releases were further examined after being given the S. aureus LTA/PGN. Roles of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways on the enzyme releases were examined by administrating inhibitors. S. aureus LTA and PGN increased the activities of pro-MMP-9 from U937 cells and pro-MMP-2 and MMP-2 from gingival fibroblasts. By giving the NF-κB inhibitor, the enhanced gelatinase activities in both cells were attenuated. In U937 cells, the enhanced pro-MMP-9 could further be attenuated by MAPK inhibitors, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), P38 MAPK, and c-Jun N-terminal kinase (JNK) inhibitors; however, the attenuation by MAPK inhibitors could not be observed for MMP-2 in gingival fibroblasts. Nevertheless, in gingival fibroblasts, the pro-MMP-2 could be attenuated by JNK inhibitor. S. aureus could enhance gelatinase activities of gingival fibroblasts and U937 cells, via NF-κB. The MAPK pathway was also involved in MMP-9 activity of U937 cells; however, the involvement of MAPK in MMP-2 activity of gingival fibroblasts was questioned. [ABSTRACT FROM AUTHOR]

The toxicity of heated tobacco products (HTP) on the immune cells remains unclear. Here, U937-differentiated macrophages were exposed to a single and short-term exposure (30 minutes) of HTP vapor or cigarette smoke (CS) in an air-liquid interface (ALI) system to evaluate the effects on macrophages' early activation and polarization. In our system, HTP released lower amounts of polycyclic aromatic hydrocarbons (PAHs), but higher nicotine levels than CS into the cell culture supernatant. Both tobacco products triggered the expression of the α-7 nicotinic receptor (α7 nAChR) and reactive oxygen species (ROS) production. When challenged with a bacterial product, lipopolysaccharide (LPS), cells exposed to HTP or CS failed to respond properly and enhance ROS production upon LPS stimuli. Furthermore, both tobacco products also impaired bacterial phagocytosis and the exposures triggered higher IL-1β secretion. The α7 nAChR antagonist treatment rescued the effects caused only by HTP exposure. The CS-exposed group switched macrophage to the pro-inflammatory M1, while HTP polarized to the suppressive M2 profile. Associated, data highlight that HTP and CS exposures similarly activate macrophages; nonetheless, the α7 nAChR pathway is only involved in HTP actions, and the distinct subsequent polarization caused by HTP or CS may influence the outcome of host defense., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Acute myeloid leukemia (AML) is the most prevalent type of hematopoietic malignancy. Despite recent therapeutic advancements, the high relapse rate associated with extramedullary involvement remains a challenging issue. Moreover, therapeutic targets that regulate the extramedullary infiltration of AML cells are still not fully elucidated. The Aryl Hydrocarbon Receptor (AHR) is known to influence the progression and migration of solid tumors; however, its role in AML is largely unknown. This study explored the roles of AHR in the invasion and migration of AML cells. We found that suppressed expression of AHR target genes correlated with an elevated relapse rate in AML. Treatment with an AHR agonist on patient-derived AML cells significantly decreased genes associated with leukocyte trans-endothelial migration, cell adhesion, and regulation of the actin cytoskeleton. These results were further confirmed in THP-1 and U937 AML cell lines using AHR agonists (TCDD and FICZ) and inhibitors (SR1 and CH-223191). Treatment with AHR agonists significantly reduced Matrigel invasion, while inhibitors enhanced it, regardless of the Matrigel's stiffness. AHR agonists significantly reduced the migration rate and chemokinesis of both cell lines, but AHR inhibitors enhanced them. Finally, we found that the activity of AHR and the expression of NMIIA are negatively correlated. These findings suggest that AHR activity regulates the invasiveness and motility of AML cells, making AHR a potential therapeutic target for preventing extramedullary infiltration in AML.

Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action.

Background: Syringin, a phenylpropanoid glycoside, has exhibited numerous biological properties including inhibitory activities against various immune and inflammatory disorders. In this study, syringin isolated from Tinospora crispa was evaluated for its ability to down-regulate activated nuclear factor-kappa B (NF-κB), phosphoinositide-3-kinase-Akt (PI3K-Akt) and mitogen-activated protein kinases (MAPKs) signal transducing networks in U937 macrophages activated by lipopolysaccharide., Methods: The attenuating effects of syringin on the productions of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), and the expressions of signaling molecules of the signaling pathways were investigated by using ELISA, Western blot, and qRT-PCR., Results: Syringin downregulated the NF-κB, MAPKs, and PI3K-Akt signal networks by significantly reducing PGE 2 production in the macrophages via suppression of COX-2 gene and protein expression levels. It also reduced TNF-α and IL-1β secretion and their mRNA expression, suppressed phosphorylation of NF-κB (p65), IKKα/β, and IκBα, and restored ability of IκBα to degrade. Syringin dose-dependently attenuated Akt, p38 MAPKs, JNK, and ERK phosphorylation. Also, the expression of corresponding upstream signaling molecules toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) were down-regulated in response to syringin treatment., Conclusion: The suppressive effect of syringin on the inflammatory signaling molecules in MyD88-dependent pathways suggested it's potential as a drug candidate for development into an agent for treatment of various immune-mediated inflammatory disorders., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Although traditional chemotherapy kills a fraction of tumor cells, it also activates the stroma and can promote the growth and survival of residual cancer cells to foster tumor recurrence and metastasis. Accordingly, overcoming the host response induced by chemotherapy could substantially improve therapeutic outcome and patient survival. In this study, resistance to treatment and metastasis has been attributed to expansion of stem-like tumor-initiating cells (TICs). Molecular analysis of the tumor stroma in neoadjuvant chemotherapy-treated human desmoplastic cancers and orthotopic tumor xenografts revealed that traditional maximum-tolerated dose chemotherapy, regardless of the agents used, induces persistent STAT-1 and NF-κB activity in carcinoma-associated fibroblasts. This induction results in the expression and secretion of ELR motif-positive (ELR+) chemokines, which signal through CXCR-2 on carcinoma cells to trigger their phenotypic conversion into TICs and promote their invasive behaviors, leading to paradoxical tumor aggression after therapy. In contrast, the same overall dose administered as a low-dose metronomic chemotherapy regimen largely prevented therapy-induced stromal ELR+ chemokine paracrine signaling, thus enhancing treatment response and extending survival of mice carrying desmoplastic cancers. These experiments illustrate the importance of stroma in cancer therapy and how its impact on treatment resistance could be tempered by altering the dosing schedule of systemic chemotherapy.

Background: Intercellular cross-talking was suggested in matrix metalloproteinase (MMP)-9 expression with unknown mechanisms. Studies showed cyclophilin A (CypA) playing an important role in regulating MMP-9 expression in varied diseases. The aim of the study was to examine the CyPA on the MMP-9 augmentation in monocytic U937 cells after Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) treatment and human gingival fibroblast (hGF) co-culture.Methods: In independent culture or co-culture of hGF and U937 cell, quantitative real-time polymerase chain reaction (qPCR) and zymography were selected to examine the mRNA and protein activity of MMP-9, respectively. The CyPA expression was determined by qPCR.Results: LPS could enhance MMP-9 mRNA expression and enzyme activity in U937 cell. However, the enhancements were not observed in hGF. Similarly, LPS enhanced CyPA mRNA in U937, but not in hGF. After co-cultured with hGF, however, MMP-9 and CyPA in U937 increased regardless of the presence/absence of LPS. In U937 cells, the extra-supplied CyPA increased MMP-9 mRNA and enzyme activity, whereas the CyPA inhibitor, cyclosporine A, suppressed the LPS- and co-culture-enhanced MMP-9. Moreover, the inhibitors for MAP kinase, including PD98059 (ERK) and SP600125 (JNK), suppressed the CyPA-enhanced MMP-9 in U937.Conclusion: Through the CyPA pathway, the LPS and the hGF could augment the MMP-9 expression in the U937 cells. [ABSTRACT FROM AUTHOR]

mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orally-administered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2. Using time-of-flight mass cytometry, we demonstrated that MLN0128 selectively targeted and functionally inhibited AML stem/progenitor cells with high AKT/mTOR signaling activity. Using the reverse-phase protein array technique, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples and identified several pro-survival pathways that antagonize MLN0128-induced cellular stress. A combined blockade of AKT/mTOR signaling and these pro-survival pathways facilitated AML cell killing. Our findings provide a rationale for the clinical use of MLN0128 to target AML and AML stem/progenitor cells, and support the use of combinatorial multi-targeted approaches in AML therapy.

Whether tumor suppressor WWOX (WW domain-containing oxidoreductase) stimulates immune cell maturation is largely unknown. Here, we determined that Tyr-33-phosphorylated WWOX physically binds non-phosphorylated ERK and IκBα in immature acute lymphoblastic leukemia MOLT-4 T cells and in the naïve mouse spleen. The IκBα·ERK·WWOX complex was shown to localize, in part, in the mitochondria. WWOX prevents IκBα from proteasomal degradation. Upon stimulating MOLT-4 with ionophore A23187/phorbol myristate acetate, endogenous IκBα and ERK undergo rapid phosphorylation in

Nitroglycerin (NTG) markedly enhances nitric oxide (NO) bioavailability. However, its ability to mimic the anti-inflammatory properties of NO remains unknown. Here, we examined whether NTG can suppress endothelial cell (EC) activation during inflammation and developed NTG nanoformulation to simultaneously amplify its anti-inflammatory effects and ameliorate adverse effects associated with high-dose NTG administration. Our findings reveal that NTG significantly inhibits human U937 cell adhesion to NO-deficient human microvascular ECs in vitro through an increase in endothelial NO and decrease in endothelial ICAM-1 clustering, as determined by NO analyzer, microfluorimetry, and immunofluorescence staining. Nanoliposomal NTG (NTG-NL) was formulated by encapsulating NTG within unilamellar lipid vesicles (DPhPC, POPC, Cholesterol, DHPE-Texas Red at molar ratio of 6:2:2:0.2) that were ~155 nm in diameter and readily uptaken by ECs, as determined by dynamic light scattering and quantitative fluorescence microscopy, respectively. More importantly, NTG-NL produced a 70-fold increase in NTG therapeutic efficacy when compared with free NTG while preventing excessive mitochondrial superoxide production associated with high NTG doses. Thus, these findings, which are the first to reveal the superior therapeutic effects of an NTG nanoformulation, provide the rationale for their detailed investigation for potentially superior vascular normalization therapies.

Background: Patients with diabetes are particularly susceptible to Legionella pneumophila (LP) infection, but the exact pathogenesis of LP infection in diabetic patients is still not fully understood. Herein, we investigated the effect of diabetes on immune function during LP infection in vitro and in vivo., Methods: The time course of LP infection in macrophages under normal and high-glucose (HG) conditions was examined in vitro. Western blot was used to determine nucleotide-binding oligomerization domain 1 (NOD1), kinase 1/2 (ERK1/2), mitogen-activated protein kinase p38 (MAPK p38), and c-Jun N-terminal kinases (JNK). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell Counting Kit-8 (CCK8) assay assessed U937 cell viability after treating cells with different concentrations of high sugar medium and ML130 (NOD1 inhibitor). For the in vivo study, normal and streptozocin-induced diabetic guinea pigs were infected with LP for 6, 24, and 72 h, after which NOD1, MAPK-related signals, TNF-α, and IL-6 expression in lung tissues were assessed using immunohistochemistry, western blot, and RT-PCR., Results: HG attenuated the upregulation of NOD1 expression and reduced TNF-α and IL-6 secretion caused by LP compared with LP-infected cells exposed to normal glucose levels (all p < 0.05). In diabetic guinea pigs, HG inhibited the upregulation of NOD1 expression in lung tissues and the activation of p38, ERK1/2, and cJNK caused by LP infection compared to control pigs (all p < 0.05)., Conclusion: HG attenuates the response of macrophages to LP infection by inhibiting NOD1 upregulation and the activation of MAPK signaling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To investigate the effects of the serine/threonine kinase family member 1 ( PIM1 ) gene on the proliferation and apoptosis of acute myeloid leukemia (AML) U937 cells, and the regulation effect on Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway., Methods: Bone marrow mononuclear cells from newly diagnosed adult AML patients and patients with iron deficiency anemia were collected and PIM1 mRNA expression was detected by RT-qPCR. AML cell line U937 cells were divided into U937 group (U937 cells were cultured normally), Si-PIM1 group (U937 cells were transfected with low expression adenovirus vector containing PIM1 mRNA), Si-NC group (U937 cells were transfected with low expression adenovirus vector without PIM1 mRNA), coumermycin A1 (CoA1) group (JAK2 activator CoA1 was added to U937 cells at a concentration of 20 μmol/L), and Si-PIM1+CoA1 group (U937 cells were transfected with adenoviral vector containing low expression of PIM1 mRNA and added with CoA1 at a concentration of 20 μmol/L). After culture for 24 h, the expressions of PIM1 mRNA and protein, JAK2/STAT3 pathway, cell cycle and apoptosis-related proteins in U937 cells were detected by RT-qPCR and Western blot, the cell proliferation activity was detected by MTT assay, and flow cytometry was used to detect cell cycle changes and apoptosis rate., Results: The PIM1 mRNA expression level in bone marrow mononuclear cells in AML patients was higher than that in patients with iron deficiency anemia ( P < 0.05). Compared with U937 group, PIM1 mRNA and protein, phosphorylated JAK2 (p-JAK2)/JAK2, phosphorylated STAT3 (p-STAT3)/STAT3, Cyclin D1, cyclin-dependent kinase 2 (CDK2) protein, cell proliferation activity, S phase and G 2 /M phase proportions were decreased in Si-PIM1 group (all P < 0.05), while p27, Caspase-3 protein, G 0 /G 1 phase proportion and apoptosis rate were increased (all P < 0.05). However, the changes of above indicators in CoA1 group were just opposite to those in Si-PIM1 group, indicating that CoA1 could reverse the effect of Si-PIM1 on U937 cells. There were no significant differences in above indexes of U937 cells between U937 group, Si-PIM1+CoA1 group and Si-NC group ( P >0.05)., Conclusion: Knockdown of PIM1 gene expression can inhibit U937 cell proliferation and promote apoptosis, in order to alleviate ALM process, which may be related to the inhibition of JAK2/STAT3 pathway activation.

Four new diterpenoids, including three secolathyrane diterpenoids (1-3) and one lathyrane diterpenoid (4), together with seven known diterpenoids, were obtained in the shelled seeds of Euphorbia lathyris. In particular, 1-3 possess a rare split ring structure, and currently only one compound with the same skeleton has been identified in E. lathyris. Compound 4 furnishes an unprecedented oxygen bridge structure. The structures were identified using various spectral techniques, including NMR, HR-ESI-MS, single-crystal X-ray diffraction and calculated electronic circular dichroism (ECD). The biosynthetic pathway of 1-4 was inferred. Furthermore, the cytotoxic activities of all compounds (1-11) were measured on three human tumor cells. New compounds 2 and 3 showed moderate cytotoxic activities against U937 cells with IC 50 values of 22.18 and 25.41 μM, respectively., Competing Interests: Declaration of competing interest No competing interests were declared by the authors., (Copyright © 2024. Published by Elsevier B.V.)

Although the anticancer activity of ONC212 has been reported, the precise mechanism underlying its apoptotic effects remains unclear. In this study, we investigated the apoptotic mechanism of ONC212 in acute myeloid leukemia (AML) cells. ONC212 induces apoptosis, MCL1 downregulation, and mitochondrial depolarization in AML U937 cells. Ectopic MCL1 expression alleviates mitochondria-mediated apoptosis in ONC212-treated U937 cells. ONC212 triggers AKT phosphorylation, inducing NOX4-dependent ROS production and promoting HuR transcription. HuR-mediated ATF4 mRNA stabilization stimulates NOXA and SLC35F2 expression; ONC212-induced upregulation of NOXA leads to MCL1 degradation. The synergistic effect of ONC212 on YM155 cytotoxicity was dependent on increased SLC35F2 expression. In addition, YM155 feedback facilitated the activation of the ONC212-induced signaling pathway. A similar mechanism explains ONC212- and ONC212/YM155-induced AML HL-60 cell death. The continuous treatment of U937 cells with the benzene metabolite hydroquinone (HQ) generated U937/HQ cells, exhibiting enhanced responsiveness to the cytotoxic effects of ONC212. In U937/HQ cells, ONC212 triggered apoptosis through NOXA-mediated MCL1 downregulation, enhancing YM155 cytotoxicity. Collectively, our data suggested that ONC212 upregulated SLC35F2 expression and triggered NOXA-mediated MCL1 degradation in U937, U937/HQ, and HL-60 cells by activating the AKT/NOX4/HuR/ATF4 pathway. The ONC212-induced signaling pathway showed anti-AML activity and enhanced YM155 cytotoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

C-type lectins play a crucial role as pathogen-recognition receptors for the dengue virus, which is responsible for causing both dengue fever (DF) and dengue hemorrhagic fever (DHF). DHF is a serious illness caused by the dengue virus, which exists in four different serotypes: DEN-1, DEN-2, DEN-3, and DEN-4. We conducted a genetic association study, during a significant DEN-2 outbreak in southern Taiwan, to explore how variations in the neck-region length of L-SIGN (also known as CD209L, CD299, or CLEC4M) impact the severity of dengue infection. PCR genotyping was utilized to identify polymorphisms in variable-number tandem repeats. We constructed L-SIGN variants containing either 7- or 9-tandem repeats and transfected these constructs into K562 and U937 cells, and cytokine and chemokine levels were evaluated using enzyme-linked immunosorbent assays (ELISAs) following DEN-2 virus infection. The L-SIGN allele 9 was observed to correlate with a heightened risk of developing DHF. Subsequent results revealed that the 9-tandem repeat was linked to elevated viral load alongside predominant T-helper 2 (Th2) cell responses (IL-4 and IL-10) in K562 and U937 cells. Transfecting K562 cells in vitro with L-SIGN variants containing 7- and 9-tandem repeats confirmed that the 9-tandem repeat transfectants facilitated a higher dengue viral load accompanied by increased cytokine production (MCP-1, IL-6, and IL-8). Considering the higher prevalence of DHF and an increased frequency of the L-SIGN neck's 9-tandem repeat in the Taiwanese population, individuals with the 9-tandem repeat may necessitate more stringent protection against mosquito bites during dengue outbreaks in Taiwan., Competing Interests: The authors declare no conflicts of interest.

Ilama leaves are an important source of secondary metabolites with promising anticancer properties. Cancer is a disease that affects a great number of people worldwide. This work aimed to investigate the in vivo, in vitro and in silico anticancer properties of three acyclic terpenoids (geranylgeraniol, phytol and farnesyl acetate) isolated from petroleum ether extract of ilama leaves. Their cytotoxic activity against U-937 cells was assessed using flow cytometry to determine the type of cell death and production of reactive oxygen species (ROS). Also, a morphological analysis of the lymph nodes and a molecular docking study using three proteins related with cancer as targets, namely, Bcl-2, Mcl-1 and VEGFR-2, were performed. The flow cytometry and histomorphological analysis revealed that geranylgeraniol, phytol and farnesyl acetate induced the death of U-937 cells by late apoptosis and necrosis. Geranylgeraniol and phytol induced a significant increase in ROS production. The molecular docking studies showed that geranylgeraniol had more affinity for Bcl-2 and VEGFR-2. In the case of farnesyl acetate, it showed the best affinity for Mcl-1. This study provides information that supports the anticancer potential of geranylgeraniol, phytol and farnesyl acetate as compounds for the treatment of cancer, particularly with the potential to treat non-Hodgkin's lymphoma., Competing Interests: The authors declare no conflicts of interest.

The hypomethylation agent decitabine (DAC), in combination with other apoptosis inducers, is considered a potential modality for cancer treatment. We investigated the mechanism underlying the combined cytotoxicity of DAC and YM155 in acute myeloid leukemia (AML) cells because of increasing evidence that YM155 induces apoptosis in cancer cells. Co-administration of DAC and YM155 resulted in synergistic cytotoxicity in AML U937 cells, which was characterized by the induction of apoptosis, NOXA-dependent degradation of MCL1 and survivin, and depolarization of mitochondria. Restoration of MCL1 or survivin expression attenuated DAC/YM155-induced U937 cell death. DAC initiated AKT and p38 MAPK phosphorylation in a Ca 2+ /ROS-dependent manner, thereby promoting autophagy-mediated degradation of β-TrCP mRNA, leading to increased Sp1 expression. DAC-induced Sp1 expression associated with Ten-eleven-translocation (TET) dioxygenases and p300 was used to upregulate the expression of SLC35F2. Simultaneously, the activation of p38 MAPK induced by DAC, promoted CREB-mediated NOXA expression, resulting in survivin and MCL1 degradation. The synergistic cytotoxicity of DAC and YM155 in U937 cells was dependent on elevated SLC35F2 expression. Additionally, YM155 facilitated DAC-induced degradation of MCL1 and survivin. A similar mechanism explained DAC/YM155-mediated cytotoxicity in AML HL-60 cells. Our data demonstrated that the synergistic cytotoxicity of DAC and YM155 in AML cell lines U937 and HL-60 is dependent on AKT- and p38 MAPK-mediated upregulation of SLC35F2 and p38 MAPK-mediated degradation of survivin and MCL1. This indicates that a treatment regimen that amalgamates YM155 and DAC may be beneficial for AML., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The hematopoietic U937 cells are able to differentiate into monocytes, macrophages, or osteoclasts when stimulated, respectively, with vitamin D3 (VD3), phorbol 12-myristate 13-acetate (PMA) or PMA plus VD3. We have previously demonstrated that magnesium (Mg) strongly potentiates the osteoclastic differentiation of U937 cells. In this study, we investigated whether such an effect may be ascribed to a capacity of Mg to modulate the monocyte differentiation of U937 cells and/or to an ability of Mg and VD3 to act directly and independently on the early phases of the osteoclastic differentiation. To address this issue, we subjected U937 cells to an individual and combined treatment with Mg and VD3 and then we analyzed, by flow cytometry and quantitative real-time polymerase chain reaction, the expression of a number of genes related to the early phases of the differentiation pathways under consideration. The results obtained indicated that Mg favors the monocyte differentiation of U937 cells induced by VD3 and at the same time, Mg contrasts the inhibitory effect that VD3 exerts on the osteoclastic differentiation in the absence of PMA. The crucial and articulated role played by Mg in diverse pathways of the osteoclastic differentiation of U973 cells is emphasized. [ABSTRACT FROM AUTHOR]

Quaking (QKI) is a tumor-suppressor gene encoding a conserved RNA-binding protein, whose expression is downregulated in several solid tumors. Here we report that QKI plays an important role in the immune response and suppression of leukemogenesis. We show that the expression of Qki is reduced in lipopolysaccharide (LPS)-challenged macrophages, suggesting that Qki is a key regulator of LPS signaling pathway. Furthermore, LPS-induced downregulation of Qki expression is miR-155-dependent. Qki overexpression impairs LPS-induced phosphorylation of JNK and particularly p38 MAPKs, in addition to increasing the production of anti-inflammatory cytokine IL-10. In contrast, Qki ablation decreases Fas expression and the rate of Caspase3/7 activity, while increasing the levels of IL-1α, IL-1β and IL-6, and p38 phosphorylation. Similarly, the p38 pathway is also a target of QKI activity in chronic lymphocytic leukemia (CLL)-derived MEC2 cells. Finally, B-CLL patients show lower levels of QKI expression compared with B cells from healthy donor, and Qki is similarily downregulated with the progression of leukemia in Eµ-miR-155 transgenic mice. Altogether, these data implicate QKI in the pathophysiology of inflammation and oncogenesis where miR-155 is involved.

Mesoderm Inducer in Xenopus Like1 (MIXL1), a paired-type homeobox transcription factor induced by TGF-β family of ligands is required for early embryonic specification of mesoderm and endoderm. Retrovirally transduced Mixl1 is reported to induce acute myelogenous leukemia (AML) with a high penetrance. But the mechanistic underpinnings of MIXL1 mediated leukemogenesis are unknown. Here, we establish the protooncogene c-REL to be a transcriptional target of MIXL1 by genome wide chromatin immune precipitation. Accordingly, expression of c-REL and its downstream targets BCL2L1 and BCL2A2 are elevated in MIXL1 expressing cells. Notably, MIXL1 regulates c-REL through a zinc finger binding motif, potentially by a MIXL1-Zinc finger protein transcriptional complex. Furthermore, MIXL1 expression is detected in the cancer genome atlas (TCGA) AML samples in a pattern mutually exclusive from that of HOXA9, CDX2 and HLX suggesting the existence of a core, yet distinct HOX transcriptional program. Finally, we demonstrate MIXL1 to be induced by BMP4 and not TGF-β in primary human hematopoietic stem and progenitor cells. Consequently, MIXL1 expressing AML cells are preferentially sensitive to the BMPR1 kinase inhibitor LDN-193189. These findings support the existence of a novel MIXL1-c REL mediated survival axis in AML that can be targeted by BMPR1 inhibitors. (MIXL1- human gene, Mixl1- mouse ortholog, MIXL1- protein).

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.

Acute myeloid leukemia (AML) is a malignant cancer of the hematopoietic system. Although the effectiveness of arsenic compounds has been recognized and applied clinically, some patients are still found resistant to this chemotherapy. In this study, we investigated that a synthetic thyroid hormone analog (TA), 2-iodo-4-nitro-1-(o-tolyloxy) benzene, had a strong apoptosis effect on U937 cells. U937 cells were treated with TA, and examinted the generation of reactive oxygen species (ROS), dysfunction of mitochondria, expression of pro-apoptosis and anti-apoptosis, and cleavage of caspase-3 and Poly (ADP-ribose) polymerase (PARP). Further, it is also evaluated that insight molecular mechanism and signaling pathways involved in the study. It is found that TA significantly induced apoptosis in U937 cells through production of ROS, dysfunction of mitochondria, and activation of caspase cascade. It was also observed that MAPK signaling pathway including ERK, JNK, and P38 signals are involved in the induction of apoptosis. Moreover, marked activation of autophagy and ER stress markers such as LC3, P62, Beclin1 and GRP78, CHOP were observed, respectively. Pretreatment with ER stress inhibitor tauroursodeoxycholic acid (TUDCA) and autophagy inhibitor 3-Methyladenine (3-MA) have successfully attenuated and aggravated TA-induced apoptosis, respectively. We further confirmed the active involvement of ER stress and autophagy signals. In conclusion, TA induced apoptosis through ER stress and activation of autophagy, and the latter is not conducive to TA-induced cell death. Our results may provide a new insight into the strategic development of novel therapy for the treatment of AML. [ABSTRACT FROM AUTHOR]

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways.

The 8;21 translocation is the most common chromosomal aberration occurring in acute myeloid leukemia (AML). This translocation causes expression of the RUNX1-ETO (AML1-ETO) fusion protein, which cooperates with additional mutations in leukemia development. We report here that interferons (IFNs) and IFN-stimulated genes are a group of genes consistently up-regulated by RUNX1-ETO in both human and murine models. RUNX1-ETO-induced up-regulation of IFN-stimulated genes occurs primarily via type I IFN signaling with a requirement for the IFNAR complex. Addition of exogenous IFN in vitro significantly reduces the increase in self-renewal potential induced by both RUNX1-ETO and its leukemogenic splicing isoform RUNX1-ETO9a. Finally, loss of type I IFN signaling via knockout of Ifnar1 significantly accelerates leukemogenesis in a t(8;21) murine model. This demonstrates the role of increased IFN signaling as an important factor inhibiting t(8;21) fusion protein function and leukemia development and supports the use of type I IFNs in the treatment of AML.

Bacterial and viral mRNAs are often polycistronic. Akin to alternative splicing, alternative translation of polycistronic messages is a mechanism to generate protein diversity and regulate gene function. Although a few examples exist, the use of polycistronic messages in mammalian cells is not widely appreciated. Here we report an example of alternative translation as a means of regulating innate immune signaling. MAVS, a regulator of antiviral innate immunity, is expressed from a bicistronic mRNA encoding a second protein, miniMAVS. This truncated variant interferes with interferon production induced by full-length MAVS, whereas both proteins positively regulate cell death. To identify other polycistronic messages, we carried out genome-wide ribosomal profiling and identified a class of antiviral truncated variants. This study therefore reveals the existence of a functionally important bicistronic antiviral mRNA and suggests a widespread role for polycistronic mRNAs in the innate immune system.

The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of αvβ3 and that the resulting ternary complex formation (integrin-FKN-CX3CR1) is critical for CX3CR1 signaling and FKN-induced integrin activation. However, only certain cell types express CX3CR1. Here we studied if FKN-CD can activate integrins in the absence of CX3CR1. We describe that WT FKN-CD activated recombinant soluble αvβ3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate αvβ3 in the absence of CX3CR1 through the direct binding of FKN-CD to αvβ3. WT FKN-CD activated αvβ3 on CX3CR1-negative cells (K562 and CHO) but K36E/R37E did not, suggesting that FKN-CD can activate integrin at the cellular levels in a manner similar to that in cell-free conditions. We hypothesized that FKN-CD enhances ligand binding to the classical RGD-binding site (site 1) through binding to a second binding site (site 2) that is distinct from site 1 in αvβ3. To identify the possible second FKN-CD binding site we performed docking simulation of αvβ3-FKN-CD interaction using αvβ3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive αvβ3 (site 2), which is located at a crevice between αv and β3 on the opposite side of site 1 in the αvβ3 headpiece. We studied if FKN-CD really binds to site 2 using a peptide that is predicted to interact with FKN-CD in site 2. Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD. This suggests that FKN-CD actually binds to site 2, and this leads to integrin activation. We obtained very similar results in α4β1 and α5β1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.

The prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP exists as the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. mCRP is highly pro-inflammatory, but pCRP is not. The mechanism of pro-inflammatory action of mCRP is unclear. We studied the role of integrins in pro-inflammatory action of mCRP. Docking simulation of interaction between mCRP and integrin αvβ3 predicted that mCRP binds to αvβ3 well. We found that mCRP actually bound to integrins αvβ3 and α4β1 well. Antagonists to αvβ3 or α4β1 effectively suppressed the interaction, suggesting that the interaction is specific. Using an integrin β1 mutant (β1-3-1) that has a small fragment from the ligand binding site of β3, we showed that mCRP bound to the classical RGD-binding site in αvβ3. We studied the role of integrins in CRP signaling in monocytic U937 cells. Integrins αvβ3 and α4β1 specifically mediated binding of mCRP to U937 cells. mCRP induced AKT phosphorylation, but not ERK1/2 phosphorylation, in U937 cells. Notably, mCRP induced robust chemotaxis in U937 cells, and antagonists to integrins αvβ3 and α4β1 and an inhibitor to phosphatidylinositide 3-kinase, but not an MEK inhibitor, effectively suppressed mCRP-induced chemotaxis in U937 cells. These results suggest that the integrin and AKT/phosphatidylinositide 3-kinase pathways play a role in pro-inflammatory action of mCRP in U937 cells. In contrast, pCRP is predicted to have a limited access to αvβ3 due to steric hindrance in the simulation. Consistent with the prediction, pCRP was much less effective in integrin binding, chemotaxis, or AKT phosphorylation. These findings suggest that the ability of CRP isoforms to bind to the integrins is related to their pro-inflammatory action.

Background: The conservative character of the cell cycle outlined that any dysregulation in the regulatory components of this process in normal cells opens a gate toward neoplastic transformation. Objectives: Given the critical role of cyclin-dependent kinases (CDKs) in cancer pathogenesis and based on their frequent aberrancy in human leukemia, the present study aimed at evaluating the suppressive effect of a multi-CDK inhibitor AT7519 on acute myeloid leukemia-derived U937 cells. Methods: To assess the anti-leukemic effects of the inhibitor on acute myeloid leukemia (AML) cells, we used MTT and trypan blue assays. Flow cytometric analysis and q-RT-PCR were also applied to evaluate the impact of AT7519 on cell cycle and apoptosis. Results: The results suggested that suppression of CDK in U937 cells hampered the proliferation of leukemic cells through a G2/M arrest mediated by p21 gene. Additionally, the anti-survival impact of AT7519 on these cells was shown to be along with the apoptosis initiation not only through the increment of pro-apoptotic gene expression but also through diminishing the mRNA levels of both Pin1 and Survivin. Notably, the potent anti-leukemic property of this agent has become more prominent when we found that the blockage of CDKs in AML cells could synergize with the cytotoxic effect of vincristine (VCR). To the best of our knowledge, little is known about the molecular mechanisms of resistance to AT7519 and we proposed that the effectiveness of this agent was partially attenuated through either c-Myc or autophagy activation in U937 cells. Conclusions: This study suggests that the pharmacological targeting of CDKscould probablyunwindthe complexity of therapeutic obstacles on the way of acute leukemia, either in the context of mono- or combined-modal strategy. [ABSTRACT FROM AUTHOR]