stimulation with ifnγ  (R&D Systems)

Journal: Scientific Reports

Article Title: ELV-N32 and RvD6 isomer decrease pro-inflammatory cytokines, senescence programming, ACE2 and SARS-CoV-2-spike protein RBD binding in injured cornea

doi: 10.1038/s41598-021-92293-x

Figure Lengend Snippet: Lipid mediators attenuate IFNγ-induced ACE2 expression, senescence programming, and binding of Alexa 594-RBD in human corneal epithelial cells (HCEC). ( a ) Among several cytokines tested only IFNγ and α, induces ACE2 expression in HCEC (6 h after stimulation, analyzed by dd-PCR). ( b ) Effect of lipid mediators on gene expression of Ace2 , Cdkn2a , and Mmp1 of HCEC after adding IFNγ (100 ng/mL). ΔΔC T normalized fold change was used. p-values of statistical t test analysis in comparison to vehicle group are shown. Mean and SD are shown as the lines. ( c ) Alexa 594-RBD binding in HCEC. IFNγ (100 ng/mL) and lipid mediators (200 nM) were added to the HCEC for 12 h. Alexa 594-RBD (0.5 ng/well) was then added and images taken 24 h after. Fifteen images/condition analyzed. Representative images are shown (left side), and the Imaris based calculation was plotted (right-hand side). Data are presented as single image/each data point. The p-value of ANOVA-post hoc Dunnett's multiple comparisons test with vehicle as reference. Mean and SD are shown as the lines. ( d ) SASP Secretome (β-Gal staining) of HCEC 24 h after IFN-γ challenge and ± lipid mediators. Each point represents one image. The p-value of ANOVA-post hoc Dunnett's multiple comparisons test with vehicle as reference are shown. Mean and SD are shown as the lines. Representative images for each condition are in the right panel.

Article Snippet: IFNγ-stimulated Alexa 594-RBD binding was analyzed by Imaris (Fig. c and Supplementary Fig. ) correlates with increased ACE2 expression (Fig. b).

Techniques: Expressing, Binding Assay, Gene Expression, Comparison, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1007/s00018-024-05423-9

Figure Lengend Snippet: Lamin A/C modulation in myeloid compartment influences vaccinia virus susceptibility and protection across infection routes. ( A ) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna −/− mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as CD25 + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6- and 14-days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (* P < 0.05; ** P < 0.01; ns, not significant). ( B ) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna −/− mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (* P < 0.05; ** P < 0.01). ( C ) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna −/− mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6- and 14-days post-infection. ( D ) Viral load measurement in the tail skin was performed at 6- and 14-days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (* P < 0.05; ** P < 0.01; ns, not significant). This schematic was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution CC BY 4.0 License; https://smart.servier.com

Article Snippet: To assess IFNγ production intracellularly, CD4 T cells were stimulated with Cell Stimulation Cocktail (500X) (Tonbo) for 4 h at 37 oC to allow intracellular cytokine accumulation.

Techniques: Virus, Infection, Flow Cytometry, Derivative Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1007/s00018-024-05423-9

Figure Lengend Snippet: Lamin A/C deficiency in matured GM-CSF-BMDCs reduces CD4 T cell activation, proliferation and Th1 differentiation in vitro and in vivo. Flow cytometry was employed to assess the percentage of activated CD4 T cells, specifically gated on CD25 + or CD69 + cells; Th1-differentiated cells, specifically gated on IFNγ + cells; and proliferating CellTrace Violet + T cells. Maturation of GM-CSF-derived WT- and LysM-Lmna −/− -BMDCs was undertaken with ( A ) sonicated VACV extract, ( B ) Pam3CSK4, or ( C ) lipopolysaccharide (LPS), and, in all conditions, pulsed with OVA cognate OT-IIp. ( D ) WT- or Lmna −/− -BMDCs, matured with LPS and pulsed with the OVA 323–339 cognate OT-II peptide, were s.c. inoculated into CD45.1 OT-II recipient mice. Six days later, pLNs were extracted, and IFNγ-producing CD4 T cells were analyzed by flow cytometry. The data represent means ± SEM and are presented from a representative experiment out of 3 ( A and B , n = 3) or out of 4 ( C , n = 4) or out of 2 ( D , n = 4). Activation data were analyzed using One Way ANOVA and Bonferroni post-test, and proliferation and Th1 differentiation were assessed by unpaired Student’s t-test. Statistical significance was denoted by asterisks (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant). This schematic was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution CC BY 4.0 License; https://smart.servier.com

Article Snippet: To assess IFNγ production intracellularly, CD4 T cells were stimulated with Cell Stimulation Cocktail (500X) (Tonbo) for 4 h at 37 oC to allow intracellular cytokine accumulation.

Techniques: Activation Assay, In Vitro, In Vivo, Flow Cytometry, Derivative Assay, Sonication

Journal: PNAS Nexus

Article Title: Vascular endothelial cell-specific disruption of the profilin1 gene leads to severe multiorgan pathology and inflammation causing mortality

doi: 10.1093/pnasnexus/pgad305

Figure Lengend Snippet: Effect of endothelial Pfn1 depletion on immunomodulatory cytokine/chemokine secretion in vitro and in vivo. A and B) Relative abundance of indicated cytokines/chemokines circulating in the serum of Pfn1 WT vs Pfn1 EC-KO mice collected either 8–10 days (mid-stage, panel A) or 15–18 days (late-stage, panel B) after the last TMX administration. Data representative of at least three separate litters with “ n ” representing the total number of mice in each group pooled from different litters. C and D) Real-time quantitative RT-PCR ( panel C ; n = 3 experiments) and immunoblot ( panel D ) validations of Pfn1 depletion in Ad-Cre–infected EC with Ad-GFP–infected cells serving as control (18S RT-PCR and GAPDH blots serve as the housekeeping gene control and loading control for RT-PCR and immunoblot experiments, respectively). E) Relative abundance of indicated cytokines/chemokines in the CM of Ad-Cre–infected EC relative to Ad-GFP–infected control cells (data represent the average fold change based on three independent experiments). F) Relative abundance of IL6 mRNA expression in circulating primary F4/80+ cells isolated from mice with the indicated genotypes ( n = 6 mice/group pooled from two separate litters). G) Relative abundance of IL6 mRNA expression in macrophages differentiated from primary BMDM and exposed to the CM of Ad-Cre– vs Ad-GFP–infected EC, either in the absence or in the presence of IFNγ stimulation (data summarized from five independent experiments). * P < 0.05 and ** P < 0.01.

Article Snippet: After 7 days, macrophages were replated and exposed to the CM derived from EC with or without additional stimulation of 100 ng/ml IFNγ (PeproTech, Cranbury NJ) for 24 h.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Western Blot, Infection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation

Journal: Biomaterials

Article Title: Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings

doi: 10.1016/j.biomaterials.2018.07.012

Figure Lengend Snippet: Gene expression of murine bone marrow derived macrophages over time when cultured on bCaP with 250 ng IFNγ on the exterior and 10 μg SIMV below the bCaP barrier layer as compared to culture on the bCaP layer only. (A-C) M1 markers. (D and E) M2 markers. * P < 0.05.

Article Snippet: After bCaP application, 0, 250 or 500 ng of the M1 stimulating molecule IFNγ (Cat#300–02, Peprotech Inc, NJ) in 0.5 ml PBS was adsorbed for one-hour on the same side of the bCaP coated disks with SIMV and then rinsed with PBS.

Techniques: Gene Expression, Derivative Assay, Cell Culture

Journal: Biomaterials

Article Title: Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings

doi: 10.1016/j.biomaterials.2018.07.012

Figure Lengend Snippet: Simvastatin dose response study. Gene expression of human THP-1 macrophages cultured on bCaP with either 2 or 10 μg SIMV beneath the bCaP with LPS and IFNγ in the media for the first three days. (A-C) M1 macrophage markers and (D and E) M2 macrophage markers. (F) Schematic representation of the experimental configuration. qRT-PCR data presented as fold change over the housekeeping GAPDH and normalized to M0 macrophages cultured on bCaP without drug. * P < 0.05.

Article Snippet: After bCaP application, 0, 250 or 500 ng of the M1 stimulating molecule IFNγ (Cat#300–02, Peprotech Inc, NJ) in 0.5 ml PBS was adsorbed for one-hour on the same side of the bCaP coated disks with SIMV and then rinsed with PBS.

Techniques: Gene Expression, Cell Culture, Quantitative RT-PCR

Journal: Biomaterials

Article Title: Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings

doi: 10.1016/j.biomaterials.2018.07.012

Figure Lengend Snippet: IFNγ dose response study in combination with 10 μg SIMV. Gene expression of human THP-1 macrophages cultured on bCaP with IFNγ on the exterior and SIMV below the bCaP barrier layer. (A-C) M1 macrophage markers. (D and E) M2 macrophage markers. (F) Schematic representation of the sequential delivery system. Data presented as fold change over the housekeeping gene GAPDH. * P < 0.05.

Article Snippet: After bCaP application, 0, 250 or 500 ng of the M1 stimulating molecule IFNγ (Cat#300–02, Peprotech Inc, NJ) in 0.5 ml PBS was adsorbed for one-hour on the same side of the bCaP coated disks with SIMV and then rinsed with PBS.

Techniques: Gene Expression, Cell Culture

Journal: Biomaterials

Article Title: Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings

doi: 10.1016/j.biomaterials.2018.07.012

Figure Lengend Snippet: Gene expression for THP-1 cultured on bCaP surface with or without 250 ng IFNγ or 10 μg SIMV or both over 6 days. (A-C) M1 macrophage markers and (D and E) M2 macrophage markers. * P < 0.05.

Article Snippet: After bCaP application, 0, 250 or 500 ng of the M1 stimulating molecule IFNγ (Cat#300–02, Peprotech Inc, NJ) in 0.5 ml PBS was adsorbed for one-hour on the same side of the bCaP coated disks with SIMV and then rinsed with PBS.

Techniques: Gene Expression, Cell Culture

Journal: Biomaterials

Article Title: Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings

doi: 10.1016/j.biomaterials.2018.07.012

Figure Lengend Snippet: Cytokine protein expression of THP-1 human peripheral blood macrophages over time when cultured on bCaP with 250 ng IFNγ on the exterior and/or 10 μg SIMV below the bCaP barrier layer as compared to culture on the bCaP layer only. (A-C) Cytokines associated with an M1 phenotype. (D) Cytokine associated with M2. * P < 0.05.

Article Snippet: After bCaP application, 0, 250 or 500 ng of the M1 stimulating molecule IFNγ (Cat#300–02, Peprotech Inc, NJ) in 0.5 ml PBS was adsorbed for one-hour on the same side of the bCaP coated disks with SIMV and then rinsed with PBS.

Techniques: Expressing, Cell Culture

Journal: Biomaterials

Article Title: Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings

doi: 10.1016/j.biomaterials.2018.07.012

Figure Lengend Snippet: A comparison of the gene expression of primary bone marrow macrophages from young adult and old mice during culture on bCaP with 10 μg SIMV below the bCaP barrier layer. The M1 stimuli LPS and IFNγ were in the culture medium for the first 3 days. (A-C) M1 macrophage markers. (D, E) M2 macrophage markers. Data normalized to GAPDH and then expressed as fold change over M0 macrophages cultured on bCaP. * P < 0.05.

Article Snippet: After bCaP application, 0, 250 or 500 ng of the M1 stimulating molecule IFNγ (Cat#300–02, Peprotech Inc, NJ) in 0.5 ml PBS was adsorbed for one-hour on the same side of the bCaP coated disks with SIMV and then rinsed with PBS.

Techniques: Comparison, Gene Expression, Cell Culture