title: Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli.
abstract: Macrophages are versatile cells of the immune system which react to various external stimuli through different polarization patterns which adjust the cells to the required function whether it is removal of pathogens or necrotic cells, tissue repair or propagation of inflammation. As much of macrophage behavior is determined by their polarization, appropriate models to study macrophage polarization are required. Previously we have published a protocol for transfection of THP-1 macrophages, which in brief pre-differentiates THP-1 monocytes for 48h using 100ng/ml PMA, followed by detachment of the cells and eletroporation using Lonza nucleofector technology and finally includes further 48h of differentiation with 100ng/ml PMA. When we applied this protocol to study interleukin (IL) 10 dependent polarization, the cells were inert to the IL10 stimulus, as indicated by a failure to induce IL10 target genes such as SOCS3. Further investigation revealed that the cells were classically activated by the differentiation agent phorbol 12-myristate 13-acetate (PMA) as shown by induction of chemokine receptor CCR7. Subsequent reduction of PMA concentration during THP-1 macrophage differentiation significantly improved their response to IL10 as SOCS3 increased more than 40-fold. This increased responsiveness of the THP-1 macrophages was also confirmed for polarization with LPS and IFNγ. Up-regulation of classical activation markers CCL3, CCR7 and TNFα was enhanced from 18-, 21- and 70-fold, respectively, to 48-, 222- and 951-fold, respectively. Reduction of PMA concentration did not negatively affect macrophage differentiation or transfection efficiency. Expression of macrophage differentiation markers CD11b and CD68 as well as cell morphology remained unchanged. In addition transfection efficiency and rates of apoptosis and necrosis remained unaffected. Thus our revised protocol combines high transfection efficiency and cell vitality with a strong response to polarizing stimuli and therefore constitutes a suitable model for studying macrophage polarization in vitro.
authors.affiliation: Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany. Electronic address: marten.maess@uni-jena.de.
authors.forename: Marten B
authors.initials: MB
authors.lastname: Maeß
authors.affiliation: Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany. Electronic address: berith.wittig@uni-jena.de.
authors.forename: Berith
authors.initials: B
authors.lastname: Wittig
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padua, Largo Meneghetti 2, 35131 Padua, Italy. Electronic address: andrea.cignarella@unipd.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cignarella
authors.affiliation: Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany. Electronic address: stefan.lorkowski@uni-jena.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Lorkowski
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D039481:CD11b Antigen / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D054405:Chemokine CCL3 / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D016753:Interleukin-10 / Q000494:pharmacology; D053710:Interleukin-10 Receptor beta Subunit / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D009336:Necrosis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D054400:Receptors, CCR7 / Q000235:genetics / Q000378:metabolism; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D039481:CD11b Antigen; D054405:Chemokine CCL3; D007371:Interferon-gamma; D016753:Interleukin-10; D053710:Interleukin-10 Receptor beta Subunit; D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; D054400:Receptors, CCR7; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D013755:Tetradecanoylphorbol Acetate; D014409:Tumor Necrosis Factor-alpha; C515429:CCL3 protein, human; C515370:CCR7 protein, human; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C081846:IL10RB protein, human; C509295:ITGAM protein, human; C106644:SOCS3 protein, human
keywords: 7-AAD; 7-aminoactinomycin; Activation; CCL3; CCR7; FCS; IFNγ; IL; IL10; IL10RB; LPS; PE; PKC; PMA; Polarization; RPL37A; RT-qPCR; SOCS3; TGFβ1; THP-1 macrophages; TNFα; Transfection; chemokine (C–C motif) ligand 3; chemokine (C–C motif) receptor 7; fetal calf serum; interferon γ; interleukin; interleukin 10 receptor β chain; lipopolysaccharide; phorbol 12-myristate 13-acetate; phycoerythrin; protein kinase C; reverse transcriptase quantitative polymerase chain reaction; ribosomal protein L37A; siRNA; suppressor of cytokine signaling 3; tumor growth factor β1; tumor necrosis factor α.