Background: Overexpression of CCL2 (MCP-1) has been implicated in pathogenesis of metabolic conditions, such as obesity and T2D. A modular, open-source whole genome assembler by the AMOS Consortium. Primary monocytes (C) also show increased CCL2 secreted protein expression after co-stimulation with IFN- and LPS compared to stimulation with either IFN- or LPS. Introduction All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed on the journal to which the article will be submitted; gave final approval for the version to be published; and agreed to be accountable for all aspects of the work. 53 Overall, increasing evidence supports that IFN- primes cells and synergizes with LPS to activate inflammatory programs via diverse molecular mechanisms, such as chromatin remodeling and metabolic reprogramming. 28 , 48 Overall, the mechanisms underlying the IFN- priming effect appear to be complex, such as involving CD14 (LPS receptor) upregulation, 49 altered mRNA turnover, 50 inflammatory autocrine signaling, 28 and the role of CR3/CR4 complement receptors. including the software tools, is conditioned on your acceptance of the above terms, 1Immunology & Microbiology Department, Dasman Diabetes Institute, Kuwait City, Kuwait; 2Genetics & Bioinformatics, Dasman Diabetes Institute, Kuwait City, Kuwait; 3Animal and Imaging Core Facility, Dasman Diabetes Institute, Kuwait City, Kuwait; 4Institut Necker Enfants Malades (INEM), French Institute of Health and Medical Research (INSERM), Immunity & Metabolism of Diabetes (IMMEDIAB), Universit de Paris Cit, Paris, France However, inhibition of HDACs further enhanced the synergistic expression and production of CCL2 in response to IFN-/LPS. 46 , 47 Conclusion 
This is because macrophages are known transducers of IFNG signalling and are also first-responders to LPS via its target receptors TLR4/CD14. Blots were washed, incubated for 2h with HRP-conjugated secondary antibody (1:2500 dilution), and immunoreactive bands were developed and visualized using ChemiDoc MP Imaging Systems. Read our FAQs. Applied Figure 1 IFN- and LPS cooperatively induce CCL2 in monocytic cells/macrophages. Applied Biosystems develops and markets instrument-based systems, reagents, software, and contract services to the life science industry and research community. 28 Regarding the intracellular events, IFN- binds to a hetero-oligomeric (IFNGR1/IFNGR2), class II cytokine receptor and activates a Jak-Stat signaling pathway implicating the Janus family kinases (Jak1 and Jak2) and the transcription factor called signal transducer and activator of transcription 1 (STAT1). Software Repository at the European Bioinformatics Institute (EBI), The FTP site for Software Repository at The European Bioinformatics Institute (EBI), The world's largest open-source software development Web site. Human primary monocytes were isolated from peripheral blood samples collected from healthy individuals after their written informed consent, and the study was approved (Protocol #: RA-2010-003) by ethics committee of Dasman Diabetes Institute, Kuwait. Among chemokines, CCL2 and CXCL10 (IP-10) are known as IFN-regulated chemokines that function as potent attractors of monocytes, macrophages, T cells, natural killer (NK) cells, and antigen presenting dendritic cells (DCs) to the sites of inflammation, infection, or tissue damage. 
site you are consenting to these choices. Chromatin immunoprecipitation was done on cell lysates using H3K27ac antibody. Similar results were obtained using primary human monocytes ( Figure 1C ). However, the mechanisms leading to increased CCL2 expression in obesity are not fully understood. 55 , 56 We further show that the IFN-LPS synergy for CCL2 expression in monocytic cells was STAT1-dependent as the genetic knockdown of STAT1 led to a significant CCL2 suppression at the transcriptional and translational levels. The CB Insights tech market intelligence platform analyzes millions of data points on vendors, products, partnerships, and patents to help your team find their next technology solution. Future studies will be needed to identify the additional mechanisms of induction of inflammatory and repression of anti-inflammatory pathways by IFN-/LPS co-stimulation and to find out how they relate to monocyte/macrophage functions in the context of metabolic inflammation.
This page is dedicated to featured products, resources, and valuable information pertaining to this rapidly developing area. Conclusion: Our findings suggest that IFN-, in combination with LPS, has the potential to augment inflammation via the H3K27ac-mediated induction of CCL2 in monocytic cells in the setting of obesity. Briefly, THP1 cells stimulated with IFN- (10 ng/mL) and/or LPS (10 ng/mL) were crosslinked with formaldehyde and digested with micrococcal Nuclease followed by sonication to yield fragments ranging from 200 to 800 bp. From pipettors and pipette tips, to CO2 incubators, fume hoods and cell imaging systems, you can shop all the essentials of the modern cell culture laboratory on LabX. third party. Shop new, used, and refurbished equipment on LabX and browse products for sale from hundreds of vendors. terms of use. These findings clearly highlight the importance of acetylation in this cooperativity. Proinflammatory M1-like polarization of macrophages requires a pro-oxidant Th1 microenvironment, marked by two initiation signals, such as IFN- and LPS or TNF-. Ccl2 mRNA and protein expression was quantified and statistically analyzed as described above. 61 As opposed to the stimulatory effect of IFN- priming on LPS-induced H3K27 acetylation and CCL2 expression that we observed, a suppressive effect of IFN- priming was observed on LPS-induced H3K27 acetylation and IL10 expression which, taken together, supports a broader regulatory role of IFN- priming in monocytes/macrophages. Your use of any resources provided herein, Protein concentration was measured using QuickStart Bradford Dye (Bio-Rad Laboratories). Briefly, 96-well microplates were prepared by coating overnight with diluted capture antibody (100 L/well). (D) Western blot showing phosphorylation of STAT1 after IFN- (10 ng/mL) treatment over time indicates the optimal STAT1 phosphorylation at 120 min.
Taken together, our work highlights the significance of IFN--LPS synergy, leading to CCL2 expression in monocytic cells via a mechanism involving H3K27 acetylation. Given that pharmacologic inhibitor of HATs, anacardic acid interrupts the histone acetylation, 38 monocytic cells were pre-treated with anacardic acid, and then co-stimulated with IFN- and LPS. 35 PBMCs were seeded in 6-well plates (Costar, Corning Incorporated) at a cell density of 3 106 cells per well and cultured in a starvation (serum-free) medium at 37C for 3 h. Non-adherent cells were removed by gentle repeated washing with a serum-free culture medium and then adherent monocytic cells were removed and incubated for 24 h in RPMI medium containing 2% FBS. CCL2 protein was determined by ELISA.
Chemokines are small chemoattractant proteins that impact the trafficking of immune cells to the sites of inflammation. CCL2 protein was measured in cell supernatants using commercial ELISA kit. Another study by Yan et al showed that HDAC9 deficiency led to reduced inflammation. All p-values < 0.05 were considered significant. Of note, H3K27 modification plays a key role in regulating gene expression, where acetylation (H3K27Ac) and methylation (H3K27me3) promote gene expression and repression/silencing, respectively. The blots were probed with rabbit anti-human antibody against H3K27, Histone H3, p-STAT1 or STAT1 in 1:1000 dilution at 4C overnight. 57 Like IFN-, LPS stimulation of macrophages also induces STAT1 phosphorylation which is required for promoting optimal expression of LPS-inducible genes. THP-1 monocytic cells were treated with anacardic acid (HATs inhibitor; 50 M) overnight or with TSA (HDACs inhibitor; 25 nM) for 6 h, followed by treatments with IFN- (10 ng/mL) and/or LPS (10 ng/mL) for 24h, and the treatment with vehicle alone served as control. Customers use these tools to analyze nucleic acids (DNA and RNA) and proteins in order to make scientific discoveries, develop new pharmaceuticals, and conduct standardized testing. Adherent cells were then washed and incubated in serum-free RPMI media for further three days before they were considered ready for treatment. LabX hosts an expansive portfolio of microscopy and imaging solutions. Notably, CCL2 gene and protein expression and was significantly diminished in cells treated with anacardic acid before IFN-/LPS co-stimulation, compared to likewise stimulated controls without pre-treatment with anacardic acid ( Figure 4A and B ). Herein, we present the data, suggesting that IFN-/LPS co-stimulation has the potential to augment inflammation via the increased CCL2 production by monocytic cells in metabolic condition, such as obesity. The field of Bioprocessing technology is expanding, with advances in upstream and downstream process development and innovations in scalable equipment. Relative mRNA expression was expressed as fold expression over average of control gene expression. Next, we investigated the priming effect of one treatment on the stimulatory response to the other. The blots were then washed three times with TBS and incubated for 2 h with HRP-conjugated secondary antibody (Promega, Madison, WI, USA) in 1:2500 dilution. Shop now. Immunoreactive bands were developed using an Amer sham ECL Plus Western Blotting Detection System (GE Health Care, Buckinghamshire, UK) and visualized by ChemiDoc MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA). The extensive life sciences portfolio includes: With a wide selection of options within each category, youll find a product to fit your unique needs and budget. The data show significant suppression of (B) CCL2 mRNA (***p< 0.001) and (C) CCL2 secreted protein (**p< 0.01) in cells co-stimulated with IFN- and LPS as compared to those stimulated with IFN- or LPS alone. Get answers. Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2CT method. The CB Insights tech market intelligence platform analyzes millions of data points on venture capital, startups, patents , partnerships and news mentions to help you see tomorrow's opportunities, today. Relative changes in Ccl2 gene expression were calculated using 2CT method and expressed as fold change over its expression in control (vehicle treatment). In addition to monocytic cells and primary monocytes, THP-1-derived macrophages co-stimulated with IFN- and LPS also display elevated expression of CCL2 transcripts (D) and secreted protein (E). IFN- and TNF- are known to induce expression of several proinflammatory genes in macrophages. Fisher Scientific SAS - Boulevard Sbastien Brant - F67403 Illkirch Cedex - FranceS.A.S. Cells were harvested for RNA isolation, and conditioned media were collected to measure CCL2 production in supernatants. For example, phagocytic and microbicidal activities, as well as expression of cytokines, chemokines, and antigen presenting molecules. Similar results were obtained using primary monocytes and macrophages. Combined exposure to IFN-/LPS resulted in a further increase in CCL2 gene expression and protein release ( Figure 1A and B ). Acetylation level was detected by qPCR using primers specific to the closest regions of transcription start site of CCL2 promotor ( Figure 3C ). After 40 hrs, transfected cells were treated with IFN- (10 ng/mL) and LPS (10 ng/mL) for 24 hrs. Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) All data were expressed as mean SEM values (n = 3) and group means between two data sets were compared using Students t-test. Results Funding 62 Furthermore, we found that inhibition of acetyltransferases (HATs) by anacardic acid, which promote acetylation, resulted in suppression of the additive effect of IFN- and LPS on CCL2 production. The 3 most popular patent topics include: Molecular biology, Fluid dynamics, Biotechnology, DNA sequencing, Genetics, Nadeem Akhter,1 Shihab Kochumon,1 Amal Hasan,1 Ajit Wilson,1 Rasheeba Nizam,2 Ashraf Al Madhoun,2,3 Fatema Al-Rashed,1 Hossein Arefanian,1 Fawaz Alzaid,2,4 Sardar Sindhu,1,3 Fahd Al-Mulla,1 Rasheed Ahmad1 CBI websites generally use certain cookies to enable better interactions with. To further identify acetylation levels on the CCL2 promoter regions, monocytic cells were treated with IFN-, LPS, or IFN-+LPS for 24 hrs and cell lysates were prepared. was read using a microplate reader at 450 nm, with wavelength correction set to 540 nm or 570 nm. THP-1 cells were differentiated into macrophages as previously described. CCL2 is a key chemokine involved in monocytic migration to the site of inflammation. CBI websites generally use certain cookies to enable better interactions with our sites and services. Applied Biosystems's headquarters is located at 850 Lincoln Centre Drive, Foster City. Disclosure. Relative STAT1 mRNA expression was expressed as fold expression over average of control (scrambled siRNA) gene expression. All data are expressed as mean SEM (n 3). In this complex, Jak1/Jak2 transactivates each other and phosphorylate IFNGR1, forming a paired set of STAT1 docking sites on the ligand-activated IFN- receptor. Discussion The lysates were clarified by centrifugation at 14,000 g for 10 min and supernatants were collected. The data show increased H3K27 acetylation (H3K27 ac) in the cells stimulated with IFN- or IFN-+LPS compared to cells stimulated with LPS only (***p< 0.001). In partial agreement with this, a previous study showed that IFN- stimulation induced CCL2 expression in human astrocytoma cells. Applied Biosystems has filed 576 patents. To measure CCL2 concentrations, averages of duplicate readings for each standard, control, and sample were calculated, and the average zero standard O.D. Wells for single or combined treatments were also incubated for 24 hrs simultaneously, with untreated well serving as control. Use of these cookies, which may be stored on your device, permits us to improve and customize your experience. Now you can shop LabX to find antibodies, reagents, kits, and related tools for your research and clinical needs. 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Peripheral blood mononuclear cells (PBMCs) were isolated using HistoPaque density gradient method as described. 58 Whether sequentially or simultaneously treated with IFN- and LPS, STAT1 appears to be a convergence point for proinflammatory immune responses in monocytes or macrophages, 59 which is in agreement with the synergy between these two proinflammatory stimuli for CCL2 expression. Inhibition or Activation of Acetylation by Pharmacological Agents Affects the Synergy Between IFN- and LPS IFN- is known to prime cells and potentiate the effects of LPS stimulation. Chronic upregulation of CCL2 is often mediated by epigenetic mechanisms consisting of histone modification, and nucleosome positioning. # DY 279, R&D Systems Inc.). In this regard, we found that priming effect of IFN- followed by LPS stimulation was significantly more robust, than other way around, in driving CCL2 gene and protein expression in monocytic cells ( Figure 1F and G ) Materials and Methods The use of all the resources provided herein and any associated materials is at user's Cell lysates were used for determination of H3K27 acetylation by Western blotting. 29 , 30 All p-values < 0.05 were considered significant (*p< 0.05 and **p< 0.01). The profile is currenly unclaimed by the seller. Herein, we show that IFN- and LPS, the two inflammatory initiators of inflammation, may interact synergistically to enhance CCL2 expression in monocytes/macrophages. Alternatively, monocytic cells were primed by incubation with IFN- (10 ng/mL), washed and incubated with LPS (10 ng/mL) for 24 hrs. Chromatin immunoprecipitation was done on cell lysate as described in methodology using antibodies specific to H3K27, Histone H3, and normal rabbit IgG for overnight at 4C. ELISA Total RNA was purified from cells lysed in RLT buffer, using RNeasy kit and following the manufacturers instructions (Qiagen, Valencia, CA; USA). Copyright 2022 CB Information Services, Inc. All rights reserved. Biosystems and we neither support nor make any warranty for such resources. 40 Our data show that stimulation of monocytes/macrophages with IFN- and/or LPS induces CCL2 expression. Find products and equipment for pharma and biopharma research, development, and processing all in one place. To confirm the role of histone acetylation, we examined whether the inhibition of histone acetyl transferases (HATs) impacted the IFN-/LPS synergy for CCL2 gene expression and protein secretion. Applied Biosystems's latest funding round is Merger. H3K27 acetylation levels induced by treatments compared to control (vehicle) were detected by qPCR using primers specific to the closest regions of transcription start site at the CCL2 promotor.
Statistical analysis was performed using GraphPad Prism software (version 6.07, La Jolla, CA, USA). The data show that only the IFN- priming followed by LPS stimulation led to elevated CCL2 mRNA (F) and secreted protein (G) expression in monocytic cells compared to controls stimulated with IFN- or LPS alone. (DI) THP1 cells were stimulated with IFN- (10 ng/mL) and/or LPS (10 ng/mL) by incubation for 24h at 37C.
Applied Biosystems GeneAmp PCR System 2700, Bio-Rad Laboratories, Inc. MyCycler Personal Thermal Cycler, Esco Technologies Swift Maxi Thermal Cyclers, Life Technologies QuantStudio 3 Real-Time PCR System, Life Technologies QuantStudio 5 Real-Time PCR System, Applied Biosystems ABI ViiA 7 Real-Time 384 & 96 &. 45 Another contributory factor in this synergy may be the strong stimulatory signaling co-induced by LPS and IFN- via the NF-B pathway. var d = new Date(); document.getElementById("footerYear").innerHTML = d.getFullYear(); Thermo Fisher Scientific Inc. Restez inform des toutes dernires nouveauts, Pinces et dcapsuleuses pour vials dchantillonnage automatique, Vials dchantillonnage automatique, capsules et bouchons, Ractifs pour chromatographie et spectromtrie de masse, Colonnes et cartouches de chromatographie, Produits pour chromatographie sur couche mince, Produits analytiques et de chromatographie, Balances, poids dlalonnage et accessoires de pese, Agitateurs, agitateurs basculants et agitateurs rotatifs, Agitateurs magntiques et barreaux dagitation, Mobilier, matriel de stockage, chariots & hottes, Plaques chauffantes et agitateurs magntiques chauffants, quipement de stockage dans lazote liquide, Accessoires pour rfrigrateurs et conglateurs, Kits et bandelettes de test du chlore et du pH, Appareils portables de mesure de loxygne dissous, Contenants pour objets pointus et tranchants, quipements de scurit, gants, lunettes & nettoyage, Lunettes, lunettes de scurit et quipements de protection du visage, Stockage et mise au rebut des matires dangereuses, Blouses, tabliers et vtements de laboratoire, Verrerie et articles en plastique pour travaux pratiques, quipements pour laboratoire de sciences dans lenseignement, STEM (Sciences, technologie, ingnierie et mathmatiques) et orientation professionnelle, Supports de filtres et matriel de filtration, Analyse des boissons et denres alimentaires, Analyse de leau et analyse environnementale, Portoirs pour tubes et matriel de stockage, Racks pour flacons et matriel de stockage, Invitrogen Lipofectamine 3000 Transfection Reagent, Invitrogen Essential 8 Flex - weekend free PSC culture, Invitrogen Countess II FL Automated Cell Counter, Invitrogen Attune NxT Acoustic Focusing Cytometer, Invitrogen Novex WedgeWell Tris-Glycine Gels, Thermo Scientific Pierce Power System semi-dry blotting and staining device, Applied Biosystems SimpliAmp Thermal Cycler, Applied Biosystems Anza Restriction Enzyme Cloning System, Invitrogen SuperScript IV Reverse Transcriptase, Thermo Scientific Phusion High-Fidelity DNA Polymerase, Applied Biosystems PowerUp SYBR Universal Master Mix, Applied Biosystems TaqMan Fast Advanced Master Mix, Applied Biosystems StepOne and StepOnePlus Real-Time PCR Systems, Conditions gnrales dutilisation du site Web, Centre dinformation sur la confidentialit, cell, protein and molecular biology technologies, cell culture media, supplements, cells and reagents. You can read more about your cookie choices at our privacy policyhere. 18 Following this monocytic extravasation into the adipose tissue and differentiation into resident M1-like inflammatory macrophages, CCL2 production further increases in both plasma and adipose tissue of obese mice and humans. We incubated monocytic cells with vehicle, IFN-, LPS, or IFN-+LPS for 4 hrs. LabX has a large variety of resources for the expanding world of materials science. They are not validated products and are provided "as is" and without warranty. Cells were stimulated with IFN- (10 ng/mL) and/or LPS (10 ng/mL), for 24 h at 37C.
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