Journal: Mediators of Inflammation

Article Title: Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1155/2014/898630

Figure Lengend Snippet: CMV blocks IDO activity and IDO-mediated antimicrobial effects in human MSC. (a) PBL, stimulated with OKT3, were cocultured with MSC in the presence or absence of CMV. As controls UV-inactivated CMV (uvCMV), the IDO-specific inhibitor 1-L-methyl-tryptophan (1-MT; 1.5 mM), or a neutralising anti-IFN- γ antibody ( α IFN- γ ; 10 ng/mL) was used. After three days IDO activity was determined and is presented as mean kynurenine content ± SEM of three independent experiments, each done in triplicate. (b) PBL (1 × 10 5 /well), stimulated with CD3-directed mAB OKT3, were cocultured with MSC (3 × 10 4 /well) in the absence or presence of CMV (MOI 5). After three days cultures were infected with S. aureus (10–100 cfu/well) and bacterial growth was determined photometrically. As a control, cultures were supplemented with L-tryptophan (Trp; 0.6 mM) at the time point of bacterial infection. Data are given as mean OD (620 nm) ± SEM of three experiments, each done in triplicate. Significant differences ( P < 0.05) as compared to the positive control are marked by asterisks.

Article Snippet: Recombinant human IFN- γ was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Activity Assay, Infection, Control, Positive Control

Journal: Mediators of Inflammation

Article Title: Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1155/2014/898630

Figure Lengend Snippet: CMV inhibits IDO induction by recombinant IFN- γ . (a) MSC (2 × 10 4 /well) which were infected with various amounts of CMV (MOI 0.1–10) were stimulated with IFN- γ (300 U/mL). After three days IDO activity was determined and is presented as mean kynurenine content ± SEM of three independent experiments, each done in triplicate. (b) MSC (1.5 × 10 6 /flask) were stimulated with IFN- γ (600 U/mL) in the absence or presence of CMV (MOI 5). Cells were harvested after 24 h and IDO protein was detected in Western blot analysis. β -Actin was utilized as a protein loading control, while the viral pp72 protein served as an infection control.

Article Snippet: Recombinant human IFN- γ was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Recombinant, Infection, Activity Assay, Western Blot, Control

Journal: Cells

Article Title: TRPA1 as a Key Regulator of Keratinocyte Homeostasis and Inflammation in Human Skin

doi: 10.3390/cells15020192

Figure Lengend Snippet: TRPA1 silencing modestly enhances keratinocyte expression of multiple chemokines. Keratinocytes obtained from three different donors were cultured for 18 h in the presence or absence of IFN-γ and TNF-α, in both control cells and silencing-treated cells, and then analyzed by real-time PCR, as described in the Methods section.

Article Snippet: Cytokine stimulations with recombinant human (rh) IFN-γ (200 U/mL), TNF-α (50 ng/mL), and IL-17A (50 ng/mL) (R&D Systems, Minneapolis, MN, USA), as well as treatments with TRPA1 agonists, were conducted on keratinocyte cultures kept in keratinocyte basal medium (KBM-GOLD, Clonetics, Lonza, Basel, Switzerland).

Techniques: Expressing, Cell Culture, Control, Real-time Polymerase Chain Reaction

Journal: Communications Biology

Article Title: Talaromyces marneffei suppresses macrophage inflammation by regulating host alternative splicing

doi: 10.1038/s42003-023-05409-6

Figure Lengend Snippet: a JunB overexpression in NCOR2-013 overexpressing THP-1 macrophages was confirmed by RT-qPCR and WB ( n = 4 biological replicates, data are presented as mean values ± SD). b , c The JunB overexpressing THP-1 macrophages and control cells were infected with T. marneffei conidia (MOI = 10) for 24 h. The expression of TNF-α and IL-1β was detected by RT-qPCR ( b ) ( n = 3 biological replicates, data are presented as mean values ± SD). T. marneffei CFUs were detected in JunB overexpressing THP-1 macrophages and control cells ( c ). Four gradient serial dilutions (10 0 , 10 −1 , 10 −2 , 10 −3 ) were performed ( n = 9 biological replicates, data are presented as mean values ± SD). d , e The NCOR2-013 overexpressing THP-1 macrophages and control cells were stimulated with IFN-γ (100 ng/mL) for 24 h, and then infected with T. marneffei conidia (MOI = 10) for 24 h. The expression of TNF-α and IL-1β was detected by RT-qPCR ( d ) ( n = 3 biological replicates, data are presented as mean values ± SD). T. marneffei CFUs were detected in IFN-γ-stimulated NCOR2-013 overexpressing THP-1 macrophages and control cells ( e ). Four gradient serial dilutions (10 0 , 10 −1 , 10 −2 , 10 −3 ) were performed ( n = 8 biological replicates, data are presented as mean values ± SD). f , g The NCOR2-013 overexpressing THP-1 macrophages and control cells were stimulated with LPS (500 ng/mL) for 24 h, and then were infected with T. marneffei conidia (MOI = 10) for 24 h and 48 h. The expression of TNF-α and IL-1β was detected by RT-qPCR ( f ) ( n = 3 biological replicates, data are presented as mean values ± SD). T. marneffei CFUs were detected in LPS-stimulated NCOR2-013 overexpressing THP-1 macrophages and control cells ( g ). Four gradient serial dilutions (10 0 , 10 −1 , 10 −2 , 10 −3 ) were performed ( n = 9 biological replicates, data are presented as mean values ± SD). All data are shown as mean ± SD from three independent experiments. WB were shown were the representative blot. Two-tailed Student’s t test was used to determine significance, denoted by *( P < 0.05), **( P < 0.01), and ns (not significant). Supplementary material is available (Supplementary Figs. , ; Supplementary Data ). MOI multiplicity of infection, NC negative control, NCOR2-013 OE NCRO2-013 overexpression, JunB OE JunB overexpression, Tm T. marneffei .

Article Snippet: Human recombinant IFN-γ and GM-CSF antibodies were purchased from Solarbio Science & Technology (China), and the LPS (from E. coli ) was from Sigma-Aldrich (USA).

Techniques: Over Expression, Quantitative RT-PCR, Control, Infection, Expressing, Two Tailed Test, Negative Control

Journal: Nature Communications

Article Title: Ready-to-use iPSC-derived microglia progenitors for the treatment of CNS disease in mouse models of neuropathic mucopolysaccharidoses

doi: 10.1038/s41467-024-52400-8

Figure Lengend Snippet: A Schematic of the differentiation process for MG01. B Total MG01 cells produced in a T-75 flask for 3 hiPSC lines. C Viability of MG01 after thaw for each of the 3 hiPSC lines. D Representative flow-cytometry plots for CD45/CD14 and CX3CR1/CD11B in post-thaw MG01 and graphs depicting quantification of 3 biological replicates per line. E Panel of representative images depicting immunoreactivity of MG01 (Line 82) to CD45, CD68, P2RY12, TREM2, IBA1, TMEM119 and PU.1 after two days in culture. F Representative phase contrast images of polarized MG01 (Line 82) 12 hours after the addition of pHrodo-conjugated E. coli particles. Upper left insets depict the detected fluorescence of pHrodo particles after phagocytosis. G Pro-inflammatory cytokine (TNFα, CXCL10 and IL-6) expression levels after polarization of MG01 to M1-like state using LPS and IFNγ. H Quantification of representative phagocytosis assay (2DIV). Graph depicts pHrodo fluorescence after addition to MG01. Images were acquired every 30 m for a span of 24 h. Data points in B , and C represent biological replicates; in D represent a combination of biological and technical replicates, in G – H represent technical replicates from one representative experiment. All cell lines tested behaved similarly and data depicted in E – H is representative of all hiPSC-derived MG01. Graphs denote mean +/− SEM. Scale bars in panel E represent 50 µm. Panel A was in part created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: For M1-like microglia, an additional 50 μL of maturation medium supplemented with 200 ng/mL of LPS (Sigma-Aldrich, L4391) and 200 ng/mL of IFNγ (R&D Systems, 285-GMP) was added directly on top of the cells, unless otherwise stated in the figure legend.

Techniques: Produced, Flow Cytometry, Fluorescence, Expressing, Phagocytosis Assay, Derivative Assay

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Brief methods of bone marrow-derived macrophage induction.

Article Snippet: M1_IFNγ + LPS , M1 , 100 ng/mL IFNγ (Proteintech, Rosemount, IL, USA, HZ-1301), and 20 ng/mL LPS (sigma, USA, L4516).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Transcriptomic comparison of porcine macrophage phenotypes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) versus M2 (M1_IFNγ + LPS, and M1_GM-CSF). ( A ) PCA clustering M1_IFNγ + LPS ( n = 4), M1_GM-CSF ( n = 4), M2_IL4 + IL10 ( n = 3), M2_M-CSF ( n = 3); ( B ) Top enriched GO biological processes of 730 DEG genes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) and M2 (M2_IL4 + IL10, and M2_M-CSF); ( C ) Heatmap exhibiting 49 classical macrophage marker genes expression profile among M1_IFNγ + LPS, M1_GM-CSF, M2_IL4 + IL10, and M2_M-CSF; ( D ) The DEGs between M1 and M2 were studied by using the String online tool. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree.

Article Snippet: M1_IFNγ + LPS , M1 , 100 ng/mL IFNγ (Proteintech, Rosemount, IL, USA, HZ-1301), and 20 ng/mL LPS (sigma, USA, L4516).

Techniques: Comparison, Marker, Expressing

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Comparison of transcriptomics in different phenotypes of porcine macrophages. ( A ) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method, M1_IFNγ + LPS, and M1_GM-CSF; ( B ) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method M2_IL4 + IL10 and M2_M-CSF; ( C ) GO pathway enrichment analysis of two polarized macrophages, M1_IFNγ + LPS and M1_GM-CSF; ( D ) GO pathway enrichment analysis of two polarized macrophages, M2_IL4 + IL10 and M2_M-CSF; ( E ) The DEGs were studied by using the String online tool, M1_IFNγ + LPS, and M1_GM-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 1; ( F ) The DEGs are studied by using the String online tool, M2_IL4 + IL10, and M2_M-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 2.

Article Snippet: M1_IFNγ + LPS , M1 , 100 ng/mL IFNγ (Proteintech, Rosemount, IL, USA, HZ-1301), and 20 ng/mL LPS (sigma, USA, L4516).

Techniques: Comparison

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Validation of M1_IFNγ + LPS, M1_GMCSF, M2_M-CSF, and M2_IL4 + IL10 signatures. ( A ) SS2-infected macrophages were enriched in M1_IFNγ + LPS based on GSEA (Data source: Microarray analysis); ( B ) PRRSV-infected macrophages were enriched in M1_GM-CSF based on GSEA (Data source: RNA-seq); ( C ). T. gondii -infected macrophages were enriched in M2_IL4 + IL10 based on GSEA (Data source: RNA-seq); ( D ) T. gondii Me49 -infected macrophages were enriched in M2_M-CSF based on GSEA (Data source: RNA-seq).

Article Snippet: M1_IFNγ + LPS , M1 , 100 ng/mL IFNγ (Proteintech, Rosemount, IL, USA, HZ-1301), and 20 ng/mL LPS (sigma, USA, L4516).

Techniques: Biomarker Discovery, Infection, Microarray, RNA Sequencing

Journal: Pancreas

Article Title: Antimicrobial Peptide Human Neutrophil Peptide 1 as a Potential Link Between Chronic Inflammation and Ductal Adenocarcinoma of the Pancreas

doi: 10.1097/mpa.0000000000001054

Figure Lengend Snippet: FIGURE 5. In vitro stimulation of pancreatic cancer cell lines (A–B: Colo 357; C–D: PANC-1; E–F: T3M4) with proinflammatory cytokines. Stimulation of all cancer cell lines with IL-1β (A, C, E) and IFN-γ (B, D, F) significantly increased the amount of HNP-1 in the supernatants (*P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: In Vitro Stimulation of Cancer Cells With Proinflammatory Cytokines The effects of proinflammatory cytokines on tumor cellspecific expression of HNP-1 were studied by adding recombinant bioactive human tumor necrosis factor α (TNF-α), recombinant bioactive human interleukin (IL) 1β, and recombinant bioactive human interferon (IFN) γ (all R&D Systems, Minneapolis, Minn) to the cell cultures.

Techniques: In Vitro

Journal: iScience

Article Title: Chimeric antigen receptor NK-92 cell function is modulated by HLA class I expression of target cells

doi: 10.1016/j.isci.2025.112523

Figure Lengend Snippet: HLA-I surface expression of target cells modulates secretion of TNFα and IFNγ by CD276-specific CAR NK-92 cells (A) Secretion of TNFα (left y axis, black) and IFNγ (right y axis, blue) after co-incubation of CD276-directed CAR NK-92 cells with the indicated target cell lines as measured by ELISA (E:T ratio 1:1; given are individual data points plus superimposed means ± SE; n = 4). (B–E) HLA-I-directed siRNA treatment increases TNFα and IFNγ secretion for target cells expressing high levels of HLA-I, whereas no changes are observed for siRNA-treated cell lines with low levels of HLA-I. Values (means from n = 2–3 experiments per cell line) were normalized to those obtained for cells treated with non-targeting siRNA control. Absolute cytokine levels are given in . Numbers indicate p values as calculated by paired t-test.

Article Snippet: For IFNγ treatment, around 600,000-900,000 target cells (depending on cell line) were incubated with Recombinant Human IFNγ (R&D Systems, #10067-IF-025) for 48 or 72 hours using an IFNγ concentration of 100 ng/ml and respective volumes of PBS as negative control.

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: iScience

Article Title: Chimeric antigen receptor NK-92 cell function is modulated by HLA class I expression of target cells

doi: 10.1016/j.isci.2025.112523

Figure Lengend Snippet: Breast cancer and non-transformed, normal cells express HER2 on their surface and are lysed by HER2-specific CAR NK-92 cells (A) Representative histograms of HER2-associated fluorescence intensity and respective IgG isotype controls of the lowest (U-87MG, blue and dark gray) and highest (MDA-MB-453, red and light gray) expressing tumor cell lines. (B and C) HER2 surface expression (B) and HER2-specific CAR NK-92-mediated (E:T = 2.5:1, black, and 0.5:1, blue) lysis of MDA-MB-231, MDA-MB-453, and MCF7 breast cancer and U-87MG glioblastoma cells (C) as measured by flow cytometry and calcein release assay, respectively. (D and E) HER2 surface expression (D) and HER2-specific CAR NK-92-mediated (E:T = 2.5:1) lysis of non-transformed SVGA, hCMEC/D3 and HFF cells (E) as analyzed in (B and C). (F) Secretion of TNFα (left y axis, black) and IFNγ (right y axis, blue) after co-incubation of HER2-directed CAR NK-92 cells and the indicated target cell lines as measured by ELISA (E:T ratio 1:1. Data in (b-f) represent individual data points with superimposed means ± SE (n = 4–6). (G) Representative histograms of HER2-associated fluorescence intensity and respective IgG isotype controls of MDA-MB-453 cells after HER2-directed (red and light gray) or non-targeting control (blue and dark gray) siRNA treatment. (H) Mean HER2 surface expression after HER2 siRNA treatment normalized to the respective non-targeting siRNA controls (n = 2–3 experiments per cell line). (I) Lysis rates by HER2-directed CAR NK-92 cells (E:T ratio 2.5:1) of MDA-MB-231, MDA-MB-453, MCF7 and SVGA cells treated with non-targeting or HER2-directed siRNA ( n = 3). Numbers indicate p values as calculated by paired t-test.

Article Snippet: For IFNγ treatment, around 600,000-900,000 target cells (depending on cell line) were incubated with Recombinant Human IFNγ (R&D Systems, #10067-IF-025) for 48 or 72 hours using an IFNγ concentration of 100 ng/ml and respective volumes of PBS as negative control.

Techniques: Transformation Assay, Fluorescence, Expressing, Lysis, Flow Cytometry, Release Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: iScience

Article Title: Chimeric antigen receptor NK-92 cell function is modulated by HLA class I expression of target cells

doi: 10.1016/j.isci.2025.112523

Figure Lengend Snippet: IFNγ upregulates HLA-I expression on target cell lines, but, nevertheless, increases cytotoxicity of HER2-specific CAR NK-92 cells (A) Representative histograms of HLA-I-associated fluorescence intensity and respective IgG isotype controls of MDA-MB-453 cells incubated with IFNγ (blue and dark gray) or without IFNγ (red and light gray). (B and C) IFNγ incubation upregulates mean (n = 2–3 per cell line) HLA-I surface expression normalized to vehicle control (B) and increases specific lysis (C) by HER2-directed CAR NK-92 cells (E:T ratio 2.5:1) for MDA-MB-453, MDA-MB-231, MCF7, and SVGA cells. (D) Representative histograms of HLA-I-associated fluorescence intensity and respective IgG isotype controls of MDA-MB-453 target cells after IFNγ incubation (blue and dark gray), or IFNγ incubation with additional HLA-I directed siRNA treatment (red and light gray). (E–G) HLA-I downregulation by siRNA during IFNγ incubation reduced mean (n = 2–3 per cell line) HLA-I surface expression (E) and increased relative (F) and absolute (G) specific lysis by HER2-directed CAR NK-92 cells (E:T ratio 2.5:1) for MDA-MB-453, MCF7 and SVGA, but not MDA-MB-231 cells. Numbers indicate p values as calculated by paired t-test. Absolute values for each experiment are shown in .

Article Snippet: For IFNγ treatment, around 600,000-900,000 target cells (depending on cell line) were incubated with Recombinant Human IFNγ (R&D Systems, #10067-IF-025) for 48 or 72 hours using an IFNγ concentration of 100 ng/ml and respective volumes of PBS as negative control.

Techniques: Expressing, Fluorescence, Incubation, Control, Lysis