Human Monocytic Cell Line Research Articles

AACE2021-A-1057: The Effect of CLA and Il-13 Treatment on Differentiation Properties of Human Leukaemic Cells (THP-1)

The aim of this study was to investigate the effect of IL13 and CLA on differentiation properties of Macrophages to M2 type using an acute monocytic leukemia (THP1) and further measurement of CD206 (MR) and CD163 as a markers of differentiation using FACS analyzer. Also, to investigate whether M2 differentiation is up regulated through the activation of PPARγ, by the addition of its antagonist GW9662 to down regulate CD206 (MR) expression. The human monocytic cell line THP-1 cell was first derived from the peripheral blood of a 1 year old male with acute monocytic leukemia. Cell Culture Manipulation and sub-culture for the expression CD206 (MR) in CLA and IL-13-treated THP-1 cells. THEN CD206 (MR) expression in CLA and IL-13-treated THP-1 cells in the presence of GW9662. In this study the In-vitro differentiation of monocytes to M2 were enhanced by using Conjugated linoleic acid (CLA) isomers and IL-13, The result shows significant increase in MR (M2 marker) expression on the surface of differentiated cells as well there was significant decrease (P>.005) in the expression of CD 163 suggesting no up regulation occur. The expression of M2 markers (CD206) and PPARγ, a nuclear receptor controlling macrophage inflammation, correlate positively. This was clear when cells were treated with the PPARγ antagonist GW9662 (1μM), the amount of CD206 expression was reduced in both (IL13 and CLA). In conclusion the conducted study shows that CLA and IL-13 enhance the ability of Macrophages to differentiate from M1 to M2 thus promote the anti-inflammatory M2 macrophages phenotype also it demonstrated that PPARγ activation induces polarization of monocytes towards a beneficial M2 phenotype suggesting that CLA and IL-13 partly modulate their action through PPARγ activation.

Utilization of CRISPR interference to investigate the contribution of genes to pathogenesis in a macrophage model of Mycobacterium tuberculosis infection.

There is an urgent need for novel drugs that target unique cellular pathways to combat infections caused by Mycobacterium tuberculosis. CRISPR interference (CRISPRi)-mediated transcriptional repression has recently been developed for use in mycobacteria as a genetic tool for identifying and validating essential genes as novel drug targets. Whilst CRISPRi has been applied to extracellular bacteria, no studies to date have determined whether CRISPRi can be used in M. tuberculosis infection models. Using the human monocytic macrophage-like THP-1 cell line as a model for M. tuberculosis infection we investigated if CRISPRi can be activated within intracellular M. tuberculosis. The transcriptional repression of two candidate M. tuberculosis genes, i.e. mmpL3 and qcrB, leads to a reduction in viable M. tuberculosis within infected THP-1 cells. The reduction in viable colonies is dependent on both the level of CRISPRi-mediated repression and the duration of repression. These results highlight the utility of CRISPRi in exploring mycobacterial gene function and essentiality under a variety of conditions pertinent to host infection.

Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently.

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Soluble total antigen derived from Toxoplasma gondii RH strain prevents apoptosis, but induces anti-apoptosis in human monocyte cell line.

Apoptosis plays crucial role in the pathogenesis of toxoplasmosis, as it limits further development of the disease. The current study aimed to investigate the effects of different concentrations of soluble total antigen (STAg) of Toxoplasma gondii (Nicolle et Manceaux, 1908) on the apoptotic and anti-apoptotic pathways. PMA-activated THP-1 cell line was sensed by T. gondii STAg and the expression patterns of caspase-3, -7, -8, -9, Bax, Bcl-2, and Mcl-1 genes were evaluated. The results showed statistically significant concentration-dependent overexpression of both Bcl-2 (P-value < 0.0001) and Mcl-1 (P-value = 0.0147). The cas-7 showed overexpression in all concentrations (P-value < 0.0001). The cas-3 was suppressed in concentrations 100, 80, and 40 µg, but statistically significant downregulated in concentrations 10 and 20 µg. The Bax was suppressed in concentrations 100 to 20 µg, while it slightly downregulated 1.42 fold (P-value = 0.0029) in concentration 10 µg. The expression of cas-8 and -9 was suppressed in all concentrations. Our results indicated that T. gondii STAg downregulated and suppressed apoptotic and upregulated anti-apoptotic pathways. The upregulation of cas-7 in this study may indicate the role of T. gondii STAg in activation of inflammatory responses.

New azamacrocyclic binuclear Cu(II) aminocarboxylate complexes: Structural, magnetic, spectral and antiproliferative studies

New cationic complexes of Cu(II) with bridged aminocarboxylates: glycine and alanine, as well as octaazamacrocyclic ligand N,N',N'',N'''-tetrakis(2-pyridylmethyl)-1,4,8,11-tetraazacyclotetradecane (tpmc), with general formula [Cu2(L)tpmc](ClO4)4∙Y, (1): L=glycine, Y=2H2O and (2): L=alanine, Y=CH3CN were synthesized. Characterization of the complexes was performed by elemental analysis (C, H, N, Cu), UV/Vis, FTIR spectroscopy and magnetic measurements. The single crystal X-ray analyses results show that reported compounds were obtained as hydrates with formulae [Cu2(gly)tpmc](ClO4)4⋅6.5H2O (M1) and [Cu2(ala)tpmc](ClO4)4⋅5H2O (M2). They crystallize in the orthorhombic system, P212121 space group. The metal centers are bridged by the carboxylate group of the glycine/alanine and bound via nitrogen atoms of tmpc. In the complex M1/M2, geometry around Cu(II) coordination sphere is square pyramidal (M1: τ5 = 0.10/0.33; M2: τ5 = 0.04/0.30). In the crystal structure of M1 and M2 the amino acids exist as a zwitterion. The temperature dependence of magnetic susceptibility indicated very weak ferromagnetic interaction between two Cu(II) ions. The antiproliferative effect of the complexes, the free ligands, and cis-platin, as referent cytostatic drug, were tested against human leukemia monocytic cell line (THP-1), leukemic T cell lymphoblast (JURKAT) and Human Caucasian Burkitt′s lymphoma (RAMOS). The IC50 values for the complexes were from 37.9 ± 2.6 to 63.05 ± 0.72 µM.

Toxoplasma gondii GRA28 Is Required for Placenta-Specific Induction of the Regulatory Chemokine CCL22 in Human and Mouse.

ABSTRACTToxoplasma gondii is an intracellular protozoan pathogen of humans that can cross the placenta and result in adverse pregnancy outcomes and long-term birth defects. The mechanisms used by T. gondii to cross the placenta are unknown, but complex interactions with the host immune response are likely to play a role in dictating infection outcomes during pregnancy. Prior work showed that T. gondii infection dramatically and specifically increases the secretion of the immunomodulatory chemokine CCL22 in human placental cells during infection. Given the important role of this chemokine during pregnancy, we hypothesized that CCL22 induction was driven by a specific T. gondii-secreted effector. Using a combination of bioinformatics and molecular genetics, we have now identified T. gondii GRA28 as the gene product required for CCL22 induction. GRA28 is secreted into the host cell, where it localizes to the nucleus, and deletion of the GRA28 gene results in reduced CCL22 placental cells as well as a human monocyte cell line. The impact of GRA28 on CCL22 production is also conserved in mouse immune and placental cells both in vitro and in vivo. Moreover, parasites lacking GRA28 are impaired in their ability to disseminate throughout the animal, suggesting a link between CCL22 induction and the ability of the parasite to cause disease. Overall, these data demonstrate a clear function for GRA28 in altering the immunomodulatory landscape during infection of both placental and peripheral immune cells and show a clear impact of this immunomodulation on infection outcome.

Mitochondrial MicroRNAs Contribute to Macrophage Immune Functions Including Differentiation, Polarization, and Activation.

A subset of microRNA (miRNA) has been shown to play an important role in mitochondrial (mt) functions and are named MitomiR. They are present within or associated with mitochondria. Most of the mitochondrial miRNAs originate from the nucleus, while a very limited number is encoded by mtDNA. Moreover, the miRNA machinery including the Dicer and Argonaute has also been detected within mitochondria. Recent, literature has established a close relationship between miRNAs and inflammation. Indeed, specific miRNA signatures are associated with macrophage differentiation, polarization and functions. Nevertheless, the regulation of macrophage inflammatory pathways governed specifically by MitomiR and their implication in immune-mediated inflammatory disorders remain poorly studied. Here, we propose a hypothesis in which MitomiR play a key role in triggering macrophage differentiation and modulating their downstream activation and immune functions. We sustain this proposition by bioinformatic data obtained from either the human monocytic THP1 cell line or the purified mitochondrial fraction of PMA-induced human macrophages. Interestingly, 22% of the 754 assayed miRNAs were detected in the mitochondrial fraction and are either exclusively or highly enriched cellular miRNA. Furthermore, the in silico analysis performed in this study, identified a specific MitomiR signature associated with macrophage differentiation that was correlated with gene targets within the mitochondria genome or with mitochondrial pathways. Overall, our hypothesis and data suggest a previously unrecognized link between MitomiR and macrophage function and fate. We also suggest that the MitomiR-dependent control could be further enhanced through the transfer of mitochondria from donor to target cells, as a new strategy for MitomiR delivery.

Cross-regulation of notch/AKT and serum/glucocorticoid regulated kinase 1 (SGK1) in IL-4-stimulated human macrophages

Notch signaling regulates the responses of macrophages to different stimuli in a context-dependent manner. The roles of Notch signaling in proinflammatory macrophages are well characterized, whereas its involvement, if any, in IL-4-stimulated macrophages (M(IL-4)) is still unclear. We observed that Notch signaling is functional in human M(IL-4). We performed transcriptome analysis of the Notch1 intracellular domain (NIC1)-overexpressing human monocytic cell line THP-1 with or without IL-4 stimulation to understand the global impact of Notch signaling in M(IL-4). The results revealed that NIC1-overexpressing THP-1 upregulated proinflammatory-associated genes and target genes of IL-4 signaling. We identified serum/glucocorticoid regulated kinase 1 (SGK1) as one of the genes increased by NIC1 overexpression in M(IL-4). To dissect the signaling pathway leading to SGK1 upregulation, we pretreated THP-1-derived macrophages with specific inhibitors of Notch (DAPT), AKT (LY294002) or ERK (U0126). Among these inhibitors, only LY294002 decreased the SGK1 mRNA levels in M(IL-4), indicating that the AKT pathway plays a key role in SGK1 transcription in M(IL-4). Furthermore, treatment of THP-1-derived macrophages with the SGK1 inhibitor (GSK650394) suppressed AKT phosphorylation, but not STAT6, in response to IL-4, indicating that SGK1 positively regulates AKT pathway in M(IL-4). Finally, GSK650394 treatment of human M(IL-4) increased the levels of PPARG mRNA and its protein, indicating a negative role of SGK1 in M(IL-4) function. Overall, we report that the Notch signaling and AKT pathways cooperatively regulate SGK1 expression in M(IL-4) where SGK1, in turn, plays an important role in suppressing IL-4-induced PPARγ expression.

Synthesis of 1-Aryloxymethyl-2,4,5-trifluorobenzene Analogs and Their Inhibitory Activity of CCL2 Expression

CCL2 overexpression is associated with macrophage recruitment during immune response and poor patient prognosis in various cancers, such as prostate, breast, pancreatic, and liver cancer. For developing CCL2 inhibitors, 1-aryloxymethyl-2,4,5-trifluorobenzene analogs were designed and synthesized by concise synthetic route including SN2 reaction, reduction, and oxime formation reaction. Furthermore, it was confirmed that they inhibited CCL2 production induced by 27-hydroxycholesterol through reverse transcription PCR in the human acute monocytic leukemia cell line (THP-1). Among them, N-((2-(2,4,5-trifluorobenzyloxy)naphthalene-1-yl)methyl)hydroxylamine (4) exhibited the highest CCL2 transcription inhibition activity.

Normal human monocytic cell line CRL9855 can be transformed into macrophages with very low concentrations of phorbol 12-myristate 13-acetate

"Monocytes are leukocytes that can differentiate into tissue macrophages and act in immune surveillance and tissue reconstruction. They are frequently used in vitro to study inflammatory response, lymphocyte activation, and phagocytosis. A widely used model is the in vitro differentiation of various monocytic cell lines including THP-1 and U937 using phorbol 12- myristate 13-acetate (PMA). These cell lines have various limitations because of their malignant background. Data available on utilizing normal human monocytes cell lines are scarce. Our study aims to identify the optimum concentration of PMA needed to maximize the number of adherent macrophages using the normal monocytic cell line CRL-9855SC (ATCC CRL-9855). Using impedance readings and time-lapse microscopy, we determined that the optimum PMA concentration is 25ng/mL for 48 hourshrs. Using these optimized conditions, we were able to induce the formation of adherent and proliferating macrophages, which can be further used for morphology and functional studies."

Oxalate Alters Cellular Bioenergetics, Redox Homeostasis, Antibacterial Response, and Immune Response in Macrophages.

Individuals with calcium oxalate (CaOx) kidney stones can have secondarily infected calculi which may play a role in the development of recurrent urinary tract infection (UTI). Uropathogenic Escherichia coli (UPEC) is the most common causative pathogen of UTIs. Macrophages play a critical role in host immune defense against bacterial infections. Our previous study demonstrated that oxalate, an important component of the most common type of kidney stone, impairs monocyte cellular bioenergetics and redox homeostasis. The objective of this study was to investigate whether oxalate compromises macrophage metabolism, redox status, anti-bacterial response, and immune response. Monocytes (THP-1, a human monocytic cell line) were exposed to sodium oxalate (soluble oxalate; 50 µM) for 48 hours prior to being differentiated into macrophages. Macrophages were subsequently exposed to calcium oxalate crystals (50 µM) for 48 hours followed by UPEC (MOI 1:2 or 1:5) for 2 hours. Peritoneal macrophages and bone marrow-derived macrophages (BMDM) from C57BL/6 mice were also exposed to oxalate. THP-1 macrophages treated with oxalate had decreased cellular bioenergetics, mitochondrial complex I and IV activity, and ATP levels compared to control cells. In addition, these cells had a significant increase in mitochondrial and total reactive oxygen species levels, mitochondrial gene expression, and pro-inflammatory cytokine (i.e. Interleukin-1β, IL-1β and Interleukin-6, IL-6) mRNA levels and secretion. In contrast, oxalate significantly decreased the mRNA levels and secretion of the anti-inflammatory cytokine, Interleukin-10 (IL-10). Further, oxalate increased the bacterial burden of primary macrophages. Our findings demonstrate that oxalate compromises macrophage metabolism, redox homeostasis, and cytokine signaling leading to a reduction in anti-bacterial response and increased infection. These data highlight a novel role of oxalate on macrophage function.

SALBUTAMOL AMELIORATES THE PHENOTYPE OF THE SKIN INFLAMMATORY DISEASE PSORIASIS ACCORDING TO SKIN SPHEROID MODELS

Psoriasis is a multifactorial chronic inflammatory disorder resulting by the interplay of genetics, the immune system and the environment. It is characterized by the hyperproliferation of epithelial cells, generating red, itchy psoriatic plaques which have no cure but have great negative impact in patients’ life. Although corticosteroids or vitamin D analogs might help recovery to some extent, there is yet no total cure for the disease. In this study, we sought to generate three-dimensional (3D) stress-related psoriatic skin spheroids with the screening of the potential efficacy of a β2-adrenergic receptor agonist, salbutamol. 3D Culture spheroids with human dermal fibroblasts (HDF), human epithelial keratinocytes (HEK) and human monocytic cell line (THP-1) were generated as a representative model of skin and the protocol of stress-related modelling was conducted. The efficacy of the drug salbutamol was evaluated by the changes in mRNA and protein expression levels of selected genes, as well as by several metabolic assays. We developed a method for culturing spherical organoid models of psoriasis in vitro. We tested the potential theurapetic effects of salbutamol on psoriasis spheroids. Spheroids treated with salbutamol indicated the effictiveness of the treatment. 3D spheroid system was found partially efficient for mimicking the physiological features of psoriasis in vitro. This present work may be a starting point for future investigation as it is the first to generate a stress-related psoriatic model and first to try a β2 agonist as a potential treatment option. Considering the effects and suitability of topical application of salbutamol, its efficacy should not be underestimated and should be investigated further for translating this knowledge into clinics.

Binding of gamma-glutamyl transferase to TLR-4 allows the activation of tissue factor expression in human monocytes

Abstract Background Gamma-glutamyl transferase (GGT) plays a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the initiator of the clotting cascade. GGT may modulate TF expression, an assumption verified by previous studies carried out in human Peripheral Blood Mononuclear Cells (PBMCs). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic activity since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. Purpose To assess whether GGT-induced TF stimulation is a consequence of binding to Toll-Like Receptor (TLR)-4 and activation of NF-κB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from a leukemia patient, were incubated with hrGGT (0.5 ng/μl for PBMCs and 1ng/μl for THP-1). LPS-Rs (0.5 ng/μl for PBMCs and 1 ng/μl for THP-1), CLI-095 (3x10–6 M) and BAY-11-7082 (10–5 M) were used to block TLR-4 receptors, TLR4 signaling and NF-κB respectively. TF pro-coagulant activity (PCA) was assessed using of StartMax coagulometer and results were expressed in pg/ml after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110±70 to 510±43, n=7, p&amp;lt;0.01) and THP-1 cells (PCA from 170±64 to 460±80, n=15, p&amp;lt;0.001).In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72±17% n=4, p&amp;lt;0.01) a TLR-4 antagonist, CLI-095 (PCA:-74±34%, n=7, p&amp;lt;0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71±32%, n=7, p&amp;lt;0.001), a NF-κB inhibitor. Similar results were obtained in THP-1 cells [LPS-Rs: −76±15%, n=6, p&amp;lt;0.01; CLI-095: −100±6,6%, n=6, p&amp;lt;0.01; BAY-11-7082: −100±2,1%, n=6, p&amp;lt;0.01]. hrGGT activates NF-κB in hTLR4-positive HEK cell lines while doesn't induces effect in TLR4-negative HEK cells. Conclusions Besides confirming the cytokine-Like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells, these data identify for the first time the possible role of TLR-4 as the receptor of GGT and NfkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque. Funding Acknowledgement Type of funding sources: None.

In Vivo PET Imaging of Monocytes Labeled with [89Zr]Zr-PLGA-NH2 Nanoparticles in Tumor and Staphylococcus aureus Infection Models.

Simple SummaryImmune cells are increasingly used for therapy in cancer and other diseases. To better understand immune-cell kinetics, cell-tracking with highly sensitive imaging modalities is required. The aim of this study was to develop a new strategy for the in vivo tracking of a small number of cells, using positron emission tomography (PET). We labeled poly(lactic-co-glycolic acid) nanoparticles containing a primary endcap (PLGA-NH2) with the radionuclide zirconium-89. The nanoparticles were characterized for size, polydispersity index, zetapotential and radiolabel retention. Subsequently, they were used for the ex vivo radiolabeling of a monocyte cell line (THP-1). We demonstrated that these radiolabeled monocyte cells can be traced in vivo in mouse tumor and infection models.The exponential growth of research on cell-based therapy is in major need of reliable and sensitive tracking of a small number of therapeutic cells to improve our understanding of the in vivo cell-targeting properties. 111In-labeled poly(lactic-co-glycolic acid) with a primary amine endcap nanoparticles ([111In]In-PLGA-NH2 NPs) were previously used for cell labeling and in vivo tracking, using SPECT/CT imaging. However, to detect a low number of cells, a higher sensitivity of PET is preferred. Therefore, we developed 89Zr-labeled NPs for ex vivo cell labeling and in vivo cell tracking, using PET/MRI. We intrinsically and efficiently labeled PLGA-NH2 NPs with [89Zr]ZrCl4. In vitro, [89Zr]Zr-PLGA-NH2 NPs retained the radionuclide over a period of 2 weeks in PBS and human serum. THP-1 (human monocyte cell line) cells could be labeled with the NPs and retained the radionuclide over a period of 2 days, with no negative effect on cell viability (specific activity 279 ± 10 kBq/106 cells). PET/MRI imaging could detect low numbers of [89Zr]Zr-THP-1 cells (10,000 and 100,000 cells) injected subcutaneously in Matrigel. Last, in vivo tracking of the [89Zr]Zr-THP-1 cells upon intravenous injection showed specific accumulation in local intramuscular Staphylococcus aureus infection and infiltration into MDA-MB-231 tumors. In conclusion, we showed that [89Zr]Zr-PLGA-NH2 NPs can be used for immune-cell labeling and subsequent in vivo tracking of a small number of cells in different disease models.

C/EBPβ is a key transcription factor of ox-LDL inducing THP-1 cells to release multiple pro-inflammatory cytokines.

CCAAT/enhancer binding protein β (C/EBPβ) plays an important role during atherogenesis. However, how C/EBPβ functions remains unclear. In this study, we explore the relationship between C/EBPβ and oxidized LDL-induced multiple pro-inflammatory cytokines released in monocytes. THP-1 cells (human monocyte cell line) were stimulated by ox-LDL, ChIP was used to detect the binding function of C/EBPβ to target genes, small interfering RNA was used to knock down the expression of C/EBPβ, Western Blot was used to detect protein expression, and ChIP-seq was used to detect different groups of C/EBPβ bound gene fragments. The integrative genomics viewer (IGV), model-based analysis of ChIP-seq (MACS) were used to visualize the results of ChIP-seq. GO (gene ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome data bases enrichment analysis were performed by the ClusterProfiler software. Ingenuity pathway analysis (IPA) was used to analyze the results of ChIP-seq and to summarize the data within the database. We identified C/EBPβ as a key protein that regulated IL-1β, IL-6 through database. Then our results confirmed that C/EBPβ could bind directly to the gene of IL-18 and C/EBPβ plays a role in the increased expression and secretion of IL-18 protein after ox-LDL stimulation of THP-1. Using ChIP-seq, we found that the enhanced transcriptional function of C/EBPβ after ox-LDL treatment triggered changes in C/EBPβ-regulated downstream pathways. In the ChIP-seq results, we extracted inflammatory cytokines with significant expression differences, and by comparing them with the database of inflammatory cytokines that C/EBPβ directly regulated, we screened five inflammatory cytokines, CXCL8, IL17B, TNFSF11, CSF3, and CCL2, and the results showed that knockdown of C/EBPβ expression inhibited ox-LDL-induced secretion of CXCL8, TNFSF11, CSF3, and CCL2 by THP-1. Our results suggest that ox-LDL stimulation enhances C/EBPβ-regulated transcription in THP-1 and C/EBPβ upregulate the release of multiple pro-inflammatory cytokines including IL-18, IL-1β, and IL-6 through direct binding to genes.

Understanding Inflammation-associated Ophthalmic Pathologies: A Novel 3D Co-culture Model of Monocyte-myofibroblast Immunomodulation

ABSTRACT Purpose Inflammation is associated with, and may be causal of, a variety of ophthalmic pathologies. These pathologies are currently difficult to model in vitro because they involve complex interactions between the innate immune system, stromal cells, and other cells that normally maintain ocular tissue homeostasis. Using transscleral drainage channel fibrosis after glaucoma surgery as an example of inflammation-associated ocular fibrosis, we have assessed a simple but novel 3D cell culture system designed to reveal the immunomodulatory impacts of ocular connective tissue cells on monocytes, a major cellular component of the circulating immune system. Methods Primary human Tenon’s capsule fibroblasts derived from five unrelated patients were activated into myofibroblasts in 3D collagen matrices under isometric tension, with and without exposure to an inflammatory cytokine-enhanced milieu, and co-cultured with an immortalized human monocyte cell line (THP-1 cells). Quantitative PCR analyses were performed on 8 candidate genes to assess the impacts of inflammatory cytokines on the myofibroblasts and the monocytes in mono-cultures and compared to cells in co-culture to clearly distinguish any co-culture-induced impacts on gene expression. Results Our data indicate that both Tenon’s capsule myofibroblasts in 3D mono-culture and THP-1 monocytes in suspension mono-culture were responsive to inflammatory cytokine stimuli. Co-culture with Tenon’s capsule myofibroblasts significantly modulated the gene expression responses of THP-1 monocytes to inflammatory cytokine stimulation, indicative of an immunomodulatory “feedback” system between these cell types. Conclusion Our findings provide proof of principle for the use of simple 3D co-culture systems as a means to enhance our understanding of ocular stromal cell interactions with cells of the innate immune system and to provide more informative in vitro models of inflammation-associated ophthalmic pathologies.

DISULFIRAM EXERTS DOSE-DEPENDENT ACTIVATING AND INHIBITORY EFFECTS ON THE HUMAN INFLAMMASOME

TOPIC: Critical Care TYPE: Original Investigations PURPOSE: Gasdermin-D (GSDMD) is a pore-forming molecule that serves as the final effector of pyroptosis (a form of inflammatory cell death) following activation of the cellular inflammasome in monocytes. Disulfiram (DSM), a drug used in the treatment of alcohol abuse, has recently been shown to inhibit GSDMD via disruption of pore formation after its cleavage by the inflammatory caspases (1, 4, and 5). The aim of this study was to elucidate the inhibitory effects of DSM on GSDMD-mediated pyroptosis and explore its role as a potential therapeutic agent in sepsis. METHODS: A series of experiments was conducted using THP1 cells (a human monocytic cell line) or purified human monocytes from the blood of healthy donors. Samples were pretreated with DSM in various concentrations 30 minutes prior to inflammasome activation with bacterial lipopolysaccharide (LPS). Cells were then assessed under light microscopy for swelling and fluorescent microscopy for the formation of ASC specks (activated oligomers of the adaptor protein required for caspase activation). Cell death was assessed using a lactate dehydrogenase (LDH) cytotoxicity assay. Interleukin-1-beta (IL1s) release was measured by ELISA. GSDMD cleavage, pro-IL1s synthesis, and caspase-1 cleavage were assessed using immunoblots. RESULTS: Unexpectedly, low dose DSM accentuated inflammasome activation. Dramatic inflammasome induction was observed at a concentration of 10uM, a dose associated with near peak inhibition in the prior study. However, higher doses of 50 to 100 uM suppressed pyroptosis as evidenced by loss of cell swelling and ASC speck formation, as well as LDH and IL-1s release. The DSM effect occurred downstream of caspase-1 activation as evidenced by preserved intracellular pro-IL1s synthesis and caspase-1 cleavage in the presence of DSM. Contrary to the prior report, GSDMD cleavage appeared to be inhibited at these higher doses. CONCLUSIONS: DSM unexpectedly exhibits upstream activation of the inflammasome at low doses but downstream inhibition at high doses. CLINICAL IMPLICATIONS: Although DSM's inhibitory effects on inflammasome function make it a promising agent for the future treatment of acute inflammatory conditions such as sepsis and covid-19, our results indicate that further study is needed to better characterize its possible activating effects at lower concentrations. DISCLOSURES: Collaboration via BARDA grant funding relationship with Beckman Coulter, Inc. Please note: 9/19/2019-8/31/2014 Added 04/27/2021 by Elliott Crouser, source=Web Response, value=Grant/Research Support No relevant relationships by Gregory Eisinger, source=Web Response No relevant relationships by Mikhail Gavrilin, source=Web Response No relevant relationships by Wissam Osman, source=Web Response No relevant relationships by Evan Prather, source=Web Response No relevant relationships by Mark Wewers, source=Web Response

Metformin alleviates intestinal epithelial barrier damage by inhibiting endoplasmic reticulum stress-induced cell apoptosis in colitis cell model.

To investigate the effect and mechanism of metformin on intestinal epithelial barrier injury in ulcerative colitis. A cell model of colitis was established by co-culture of human colon cancer cell line Caco-2 and human monocyte cell line THP-1. The colitis model cells were treated with metformin at concentration of for Flow cytometry was used to detect Caco-2 cell apoptosis, and Western blotting was used to detect the protein expression of tight junction proteins and endoplasmic reticulum stress-related proteins. After metformin treatment, the apoptosis rate of Caco-2 cells was decreased from (14.22±2.34)% to 0.61)% (=3.119, <0.05), and the expression levels of tight junction protein-1 and claudin-1 increased (=5.172 and 3.546, both <0.05). In addition, the expression levels of endoplasmic reticulum-related proteins glucose regulated protein (GRP) 78, C/EBP homologous protein (CHOP) and caspase-12, as well as the phosphorylation level of PRKR-like endoplasmic reticulum kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) decreased (all <0.05). Metformin may alleviate the intestinal epithelial barrier damage in colitis by reducing intestinal epithelial cell apoptosis and increasing the expression of tight junction proteins, which may be associated with the inhibition of endoplasmic reticulum stress-induced apoptotic pathway.

Immunomodulatory effects exerted by extracellular vesicles from Staphylococcus epidermidis and Staphylococcus aureus isolated from bone-anchored prostheses

Staphylococcus aureus and Staphylococcus epidermidis are the bacteria that most frequently cause osteomyelitis. This study aimed to determine whether staphylococci isolated from osteomyelitis associated with septic loosening of orthopedic prostheses release extracellular vesicles (EVs) and, if so, to determine tentative immunomodulatory effects on the human monocytic cell line THP-1. EVs were isolated from bacterial cultures using filtration and ultracentrifugation and characterized by scanning electron microscopy, nanoparticle tracking analysis and Western Blot. The cytotoxic effect of EVs was analyzed by NucleoCounter and lactate dehydrogenase (LDH) analyses. Confocal laser scanning microscopy was employed to visualize the uptake of EVs by THP-1 cells. Activation of the transcription factor nuclear factor-κB (NF-κB) was determined in THP1-Blue™ NF-κB cells, and the gene expression and secretion of cytokines were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. All investigated strains, irrespective of their biofilm formation ability, were able to secrete EVs in vitro. The S. aureus strains produced significantly more EVs than the S. epidermidis strains. Both S. aureus-derived EVs and S. epidermidis-derived EVs were internalized by THP-1 cells, upregulated Toll-like receptor 3 (TLR3) gene expression, activated NF-κB, and promoted the gene expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, matrix metallopeptidase (MMP)-9 and IL-10. Whereas EVs from both staphylococcal species upregulated the proapoptotic DNA damage-inducible transcript 4 (DDIT4) gene and downregulated the antiapoptotic B-cell lymphoma 2 (Bcl-2) gene, cytolysis was preferentially induced in S. aureus EV-stimulated cells, possibly related to the expression of cytolytic proteins predominantly in S. aureus EVs. In conclusion, staphylococcal EVs possess potent cytolytic and immunomodulatory properties.

Pseudogene-Mediated Gene Conversion After CRISPR-Cas9 Editing Demonstrated by Partial CD33 Conversion with SIGLEC22P.

Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocyte cell line. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines, which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.