Journal: JCI insight

Article Title: CETP inhibition enhances monocyte activation and bacterial clearance and reduces streptococcus pneumonia-associated mortality in mice.

doi: 10.1172/jci.insight.173205

Figure Lengend Snippet: Figure 4. CETPi increases macrophage infiltration into site of infection and macrophage activation while maintaining tissue homeostasis at different stages of sepsis. (A and B) Representative staining figures of M1 macrophages by marker CD86 in lung obtained 0 and 72 hours after infection by S. pneu- moniae from female APOA1.CETP mice treated with control or CETPi. Quantification was determined by random sampling spots on lungs of 3 different mice in each treatment and by measuring stained areas using ImageJ (H&E 0 hours, mean ± SD, 16.0 ± 2.6 [CETPi, n = 3] versus 2.7 ± 1.5 [Control, n = 3], unpaired t test, P = 0.002) (IHC 72 hours, mean ± SD, 6.0 ± 3.5 [CETPi, n = 3] versus 17.0 ± 7.9% [Control, n = 3], unpaired t test, P = 0.02). (C and D) Propor- tion of infiltrating inflammatory macrophages (CD11b+Ly6c+) and tissue repairing and inflammation resolving macrophages (CD11b+Ly6c–) in BAL samples obtained 24 and 72 hours after infection from female APOA1.CETP mice treated with control or CETPi (CD11b+Ly6c+ 72 hours, mean ± SD, 5.6 ± 1.8 [CETPi, n = 4] versus 14.3 ± 6.1% [Control, n = 3], unpaired t test with Bonferroni correction, P = 0.037) (CD11b+ Ly6c– 24 hours, mean ± SD, 62.1 ± 6.8 [CETPi, n = 4] ver- sus 45.6 ± 3.8 [Control, n = 3], unpaired t test with Bonferroni correction, P = 0.013; CD11b+ Ly6c– 72 hours, mean ± SD, 78.3 ± 9.1 [CETPi, n = 4] versus 34.7 ± 22.7 [Control, n = 3], unpaired t test with Bonferroni correction, P = 0.016). (E) Proportion of migrating monocytes (Ly6C++Ly6GDIM) in blood samples obtained 0 and 72 hours after infection from female APOA1.CETP mice treated with control or CETPi (mean ± SD, 12.0 ± 2.7 [CETPi, n = 5] versus 0.14 ± 0.06 [Control, n = 4], unpaired t test, P = 0.00005). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. ApoA1, apolipoprotein A-1; CETP, cholesteryl ester transfer protein; CD86, cluster of differentiation 86; CD11b, integrin α M; Ly6-C, lymphocyte antigen 6 locus C; Ly6-G, lymphocyte antigen 6 locus G.

Article Snippet: For IHC, tissue cross-sections were incubated with a primary macrophage membrane mark antibody kit (CD86 and CD206, 97624, CST) to identify specific cell types of macrophages followed by secondary antibodies using the alkaline phosphatase system to amplify signals.

Techniques: Infection, Activation Assay, Staining, Marker, Control, Sampling

Journal: JCI insight

Article Title: CETP inhibition enhances monocyte activation and bacterial clearance and reduces streptococcus pneumonia-associated mortality in mice.

doi: 10.1172/jci.insight.173205

Figure Lengend Snippet: Figure 5. CETPi decreases caspase-1 and COX-2 protein expression in human and mouse cells. (A–C) COX-2 and Caspase-1 expression in PBMCs treated with increasing doses of CETPi. (D–E) COX-2 expression in THP1 cells treated with control or CETPi (1 μM) (mean ± SD, 1.00 ± 0.42 [Control, n = 3] versus 1.31 ± 0.38 [CETPi, n = 3], unpaired t test, P = 0.03). (F–G) Caspase-1 in THP1 cells treated with control or CETPi (1 μM) (mean ± SD, 1.00 ± 0.19 [Control, n = 3] versus 2.02 ± 0.11 [CETPi, n = 3], unpaired t test, P = 0.02). (H) Expression of ROS in THP1 cells treated with control or CETPi (2 μM), (mean ± SD, 2.45 ± 0.36 [CETPi, n = 6] versus 1.00 ± 0.19 [Control, n = 6], unpaired t test, P = 0.00005). (I–K) Expression of COX-2 and CETP in RAW264.7 cells after adenovirus transfection induced-CETP overexpression in increasing MOIs. (L–M) Expression of pro–caspase-1 and cleaved caspase-1 in RAW 264.7 cells after adeno- virus transfection–induced CETP overexpression at MOI = 1.6. Pro–caspase-1: mean ± SD, 1.00 ± 0.15 (Control, n = 3) versus 0.45 ± 0.22 (CETP overexpress, n = 3), unpaired t test, P = 0.02; cleaved caspase-1: mean ± SD, 1.00 ± 0.14 (Control, n = 3) versus 0.58 ± 0.16 (CETP overexpress, n = 3), unpaired t test, P = 0.03. Data displayed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Caspase-1, caspase-1/IL-1 converting enzyme; COX-2, prostaglandin-endoperoxide synthase 2; MOI, multiplicity of infection; PBMC, human peripheral blood mononuclear cell; RAW, murine macrophage cell; ROS, reactive oxygen species; THP1, human peripheral blood monocyte.

Article Snippet: For IHC, tissue cross-sections were incubated with a primary macrophage membrane mark antibody kit (CD86 and CD206, 97624, CST) to identify specific cell types of macrophages followed by secondary antibodies using the alkaline phosphatase system to amplify signals.

Techniques: Expressing, Control, Transfection, Over Expression, Virus, Infection

Journal: Cells

Article Title: Decidua Basalis Mesenchymal Stem Cells Favor Inflammatory M1 Macrophage Differentiation In Vitro

doi: 10.3390/cells8020173

Figure Lengend Snippet: M1 macrophage culture system. Untreated control experiment consisted of M1 macrophages cultured on a surface of 6-well culture plate in a medium containing GM-CSF, CMDBMSC experiment consisted of M1 macrophages cultured on a surface of 6-well culture plate in a medium containing GM-CSF and CMDBMSC (conditioned medium), SFDBMSC (soluble factor), and ICDBMSC (intercellular direct contact) experiments. In SFDBMSC and ICDBMSC experiment, cells (DBMSCs and monocytes) were separated by transwell chamber membrane culture system. For the experiments of SFDBMSC, DBMSCs were seeded on the upper compartments while monocytes were seeded in the lower compartment. For the experiments of ICDBMSC, DBMSCs were seeded on the reverse side of the membrane while monocytes were seeded on the upper side of the membrane. GM-CSF medium was added to SFDBMSC and ICDBMSC experiments. Effects of human DBMSCs on the morphology of human monocytes differentiated into macrophages by GM-CSF. ( A – F ) Represent phase-contrast microscopic images showing monocyte (round-shaped morphology) differentiation into M1-like macrophages (fried egg-shaped morphology) after six days of culture in a medium containing GM-CSF ( A ), in a medium containing GM-CSF and DBMSCs at a 20:1 monocyte: DBMSC ratio ( B ), at a 10:1 monocyte: DBMSC ratio ( C ), at a 1:1 monocyte: DBMSC ratio ( D ), in the presence of 10% CMDBMSC ( E ), or in the presence of 20% CMDBMSC ( F ). ( G – K ) Representative phase-contrast microscopic images showing monocyte-like cells after six days of culture in a medium containing GM-CSF and 30% CMDBMSC ( G ), 40% CMDBMSC ( H ), 50% CMDBMSC ( I ), 60% CMDBMSC ( J ), 80% CMDBMSC ( K ), or 100% CMDBMSC ( L ). Experiments were carried out in duplicate and repeated 30 times using 30 individual preparations of both monocyte-derived macrophages and DBMSCs. Scale bars represent 50 µm.

Article Snippet: Monocytes were seeded in 6-well plates in M1 macrophage differentiation medium (RPMI-1640 medium containing 50 ng/mL GM-CSF (R and D Systems), 10% FBS, 2 mM L-glutamine, and antibiotics indicated above), and then cultured at 37°C as described above for six days ( ).

Techniques: Control, Cell Culture, Membrane, Derivative Assay

Journal: Cells

Article Title: Decidua Basalis Mesenchymal Stem Cells Favor Inflammatory M1 Macrophage Differentiation In Vitro

doi: 10.3390/cells8020173

Figure Lengend Snippet: Reversibility effects of DBMSCs on macrophage differentiation. Human monocytes differentiated into macrophages by GM-CSF in the presence of DBMSCs. DBMSCs were removed from the monocyte cultures on Day 3, and monocyte-derived macrophages were then washed and cultured again in fresh M1-like macrophage differentiation medium without DBMSCs for a further three days. Macrophage differentiation was analyzed by morphological analysis using microscopic examination (Panels A , B , and C ) and flow cytometric analysis of cell surface molecules (CD14, CD163, CD204, CD206, and B7-H4). ( A – C ) Representative phase-contrast microscopic images showing monocyte (round-shaped morphology) differentiation into M1-like macrophages (fried egg-shaped morphology) after six days of culture ( A ), in a medium containing GM-CSF and DBMSCs at a 20:1 monocyte: DBMSC ratio ( B ), in the presence of 20% CMDBMSC ( C ) . After six days in culture, compared to untreated macrophages, CMDBMSCs significantly increased the expression of CD14 ( D ) and CD163 ( E ) on macrophages, but had no significant effect on the expression of CD204 ( F ), CD206 ( G ), and B7-H4 ( H ) on macrophages. Compared to untreated macrophages, SFDBMSC significantly increased the expression of CD14 ( D ), CD163 ( E ), and CD206 ( G ) on macrophages, but had no significant effect on the expression of CD204 ( F ), and B7-H4 ( H ) on macrophages. In addition, ICDBMSCs significantly decreased the expression of CD163 ( E ), CD204 ( F ), CD206 ( G ) on macrophages, but had no significant effect on the expression of CD14 ( D ) and B7-H7 ( H ) compared with the effect on untreated macrophages. Levels of expression are presented as median fluorescent intensity (MFI) as determined by flow cytometry. Experiments were carried out in duplicate and repeated 10 times using 10 individual preparations of both monocyte-derived macrophages and DBMSCs. * p < 0.05. Bars represent standard errors.

Article Snippet: Monocytes were seeded in 6-well plates in M1 macrophage differentiation medium (RPMI-1640 medium containing 50 ng/mL GM-CSF (R and D Systems), 10% FBS, 2 mM L-glutamine, and antibiotics indicated above), and then cultured at 37°C as described above for six days ( ).

Techniques: Derivative Assay, Cell Culture, Expressing, Flow Cytometry

Journal: Cells

Article Title: Decidua Basalis Mesenchymal Stem Cells Favor Inflammatory M1 Macrophage Differentiation In Vitro

doi: 10.3390/cells8020173

Figure Lengend Snippet: T cell proliferation by M1-like macrophages (M1) generated in the presence of DBMSCs. After culturing M1 alone or with CMDBMSC (CMM1), SFDBMSC (SFM1), and ICDBMSC (ICM1), M1 were harvested and added to allogeneic CD4 + T cells at 5:1, 10:1, and 20:1 T cells:M1 (M1, CMM1, SFM1, and ICM1) ratios. M1 pre-treated with DBMSCs (CMM1, SFM1, and ICM1) or without (M1) significantly increased T cell proliferation at all indicated ratios. Similarly, ICM1 significantly increased T cell proliferation at all indicated ratios compared to that with M1, while SFM1 significantly increased T cell proliferation at the 5:1 T cells: SFM1 ratio compared to that with M1. By contrast, CMM1 significantly decreased T cell proliferation at the 10:1 and 20: 1 T cells: CMM1 ratios compared to that with M1 ( A ). In addition, the secretion of IL-10 by T cells stimulated with M1, CMM1, SFM1, and ICM1 was significantly reduced ( B ), while the secretion of IL-12 and IFN-γ was significantly increased, p < 0.05 ( C and D ). Experiments were carried out in triplicate and repeated 10 times using 10 individual preparations of T cells and macrophages harvested from 10 individual experiments of macrophages cultured with DBMSCs (CMDBMSC, SFDBMSC, and ICDBMSC). * p < 0.05, Bars represent standard error.

Article Snippet: Monocytes were seeded in 6-well plates in M1 macrophage differentiation medium (RPMI-1640 medium containing 50 ng/mL GM-CSF (R and D Systems), 10% FBS, 2 mM L-glutamine, and antibiotics indicated above), and then cultured at 37°C as described above for six days ( ).

Techniques: Generated, Cell Culture

Journal: Signal Transduction and Targeted Therapy

Article Title: Exploration and functionalization of M1-macrophage extracellular vesicles for effective accumulation in glioblastoma and strong synergistic therapeutic effects

doi: 10.1038/s41392-022-00894-3

Figure Lengend Snippet: a . Schematic illustration of TAMs phenotype analysis from tumor samples of glioma patients. b . M1 macrophage (iNOS), M2 macrophage (CD163), and proliferation (Ki67) immunostaining of histological sections of tumor-adjacent tissues as control and in both low-grade gliomas (LGG: diffuse astrocytoma, n = 22) and high-grade gliomas (HGG: anaplastic astrocytoma, n = 20; glioblastoma multiforme, n = 22) resected from glioma patients. Quantitative analysis of the corresponding M2/M1 ratios was shown on the right side. The proliferation-related Ki67 marker index was positively correlated with the M2/M1 ratio. All images have the same scale of 50 μm. c . M2/M1 ratio analysis of 167 HGG and 522 LGG cases acquired from The Cancer Genome Atlas (TCGA) database. Each dot represented a single individual. d . Survival curves of glioma patients from TCGA database. The OncoLnc tool was used to explore the survival correlations for M2/M1 ratio data. e . Immunostaining of histological sections (left) and quantitative analysis (right) of noncolocalization percentage of microglia (TMEM119, green) and M1 macrophage (iNOS, red) of human glioma tissue. All images have the same scale of 50 μm. Nuclei: DAPI, blue ( n = 6). f . Immunostaining of histological sections (left) and quantitative analysis (right) of noncolocalization percentage of microglia (TMEM119, green) and M2 macrophage (CD163, red) of human glioma tissue. All images have the same scale of 50 μm. Nuclei: DAPI, blue ( n = 6). g . Schematic illustration of TAM phenotype analysis from tumor samples of U87MG (human glioblastoma cells) /G422 (mouse glioblastoma cells) /GL261 (mouse glioma cells)-cell-derived xenograft tumor-bearing mice. h . M1 macrophage (iNOS), M2 macrophage (CD163), and proliferation (Ki67) immunostaining of histological sections of normal tissue, U87MG, G422, and GL261-bearing tissue in mice. All images have the same scale of 50 μm. i . Microglia (TMEM119, green) and M1 macrophage (iNOS, red) immunostaining of histological sections of U87MG, G422, and GL261-bearing tissue (Top). Microglia and M2 macrophage (CD163, red) immunostaining of histological sections of U87MG, G422, and GL261-bearing tissue (bottom). All images have the same scale of 50 μm. Nuclei: DAPI, blue. Data in b , e , and f are presented as the mean ± S.D. Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test ( b ) or unpaired two-tailed Student’s t -test ( c ) and survival analysis was calculated by two-sided Log-rank Mantel-Cox tests ( d )

Article Snippet: The expression of CD9/CD81/ALIX/TSG101 (EV marker), iNOS (M1 marker) and F4/80 (macrophages marker) in M1 macrophage and M1EVs were analyzed by ProteinSimple® Wes TM capillary western blot analyzer (PS-MK15; ProteinSimple, USA).

Techniques: Immunostaining, Control, Marker, Derivative Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Exploration and functionalization of M1-macrophage extracellular vesicles for effective accumulation in glioblastoma and strong synergistic therapeutic effects

doi: 10.1038/s41392-022-00894-3

Figure Lengend Snippet: a . Schematic illustration of the fabrication process for M1EVs (derived from M1 macrophages), M0EVs (derived from M0 macrophages), EMVs (derived from erythrocytes), and PEG NPs (derived from PEG-PLGA materials). b . Representative fluorescence images of U87MG-bearing mice after intravenous ( i.v .) injection with M1EVs, M0EVs, EMVs, and PEG NPs (all labeled with DiR) at different time points. c . Ex vivo images of the major organs dissected from mice in different groups at 48 h after i.v . injection. d . Quantitative analysis of corresponding fluorescence signals in panel ( c ). e . In vivo time-lapse two-photon images of the diffusion of M1EVs, M0EVs, EMVs, and PEG NPs acrossed the brain microvascular endothelial cells at 48 h after i.v . injection (left). Tetramethylrhodamine isothiocyanate-Dextran was used to label blood vessels (red). M1EVs, M0EVs, and EMVs labeled with DiO (green); PEG NPs labeled with FITC (green), and corresponding formulation distributions in tumor tissue (right). All images have the same scale of 50 μm. f . Immunofluorescence images of histological sections of M2 and M1 macrophages (left), and quantitative analysis of M2/M1 ratios (right) at 48 h after i.v . injection. All images have the same scale of 50 μm. iNOS (red, M1 marker), CD163 (green, M2 marker) ( n = 3). g . Ex vivo images of the major organs dissected from G422 bearing mice in different groups (mouse source) at 48 h after i.v . injection (left), and corresponding analysis of ex vivo fluorescence signals of the major organs dissected from mice in different groups (right). h . Immunofluorescence images of histological sections (G422-bearing mice) of M2 and M1 macrophages (right). Scale bar: 50 μm, iNOS (red, M1 marker), CD163 (green, M2 marker) ( n = 3). i . Ex vivo images of the major organs dissected from GL261 bearing-mice in different groups at 48 h after i.v . injection (left), and corresponding analysis of ex vivo fluorescence signals of the major organs dissected from mice in different groups (right). j . Immunofluorescence images of histological sections (GL261-bearing mice) of M2 and M1 macrophages (right). Scale bar: 50 μm, iNOS (red, M1 marker), CD163 (green, M2 marker) ( n = 3). Data in d , f , g , and i are presented as the mean ± S.D. Statistical significance was calculated, compared with the M1EVs group, by one-way ANOVA with a Kruskal-Wallis test ( d , g , i ). ns, not significant

Article Snippet: The expression of CD9/CD81/ALIX/TSG101 (EV marker), iNOS (M1 marker) and F4/80 (macrophages marker) in M1 macrophage and M1EVs were analyzed by ProteinSimple® Wes TM capillary western blot analyzer (PS-MK15; ProteinSimple, USA).

Techniques: Derivative Assay, Fluorescence, Injection, Labeling, Ex Vivo, In Vivo, Diffusion-based Assay, Formulation, Immunofluorescence, Marker

Journal: Signal Transduction and Targeted Therapy

Article Title: Exploration and functionalization of M1-macrophage extracellular vesicles for effective accumulation in glioblastoma and strong synergistic therapeutic effects

doi: 10.1038/s41392-022-00894-3

Figure Lengend Snippet: a . TEM image of M1EVs. Scale bar: 100 nm. b . ProteinSimple ® capillary immunoassay (Wes) analysis of CD9, CD81, ALIX, TSG101, iNOS, F4/80, and GAPDH in M1 macrophages and M1EVs. c . Confocal laser scanning microscopy (CLSM) images of AQ4N-M1EVs (Top, green: M1EVs; red: AQ4N) and Ce6-M1EVs (bottom, green: M1EVs; red: Ce6). All images have the same scale of 1 μm. d . Representative flow cytometry analysis images of M1EVs (top) and TA-M1EVs (M1EVs containing AQ4N and TRMRA in place of Ce6 due to the overlayed spectrum with AQ4N) (bottom). e . Production of ROS with Ce6, CPPO/Ce6, CC-M1EVs, and CCA-M1EVs in buffers with different H 2 O 2 concentrations, where A 0 and A were the absorbance of ABDA at 399 nm before and after H 2 O 2 addition ( n = 3). f . Cumulative AQ4N release profiles of CCA-M1EVs before and after H 2 O 2 treatment in PBS buffer ( n = 3). g . Consumption of oxygen with different formulations after H 2 O 2 treatment in PBS buffer ( n = 3). h . Quantification of the AQ4/AQ4N ratio after different treatments based on high-performance liquid chromatography (HPLC) analysis. i . Illustration of in vitro BBB and TME model. The Transwell TM co-culture system containing bEnd.3 cells in the upper chamber and a combination of U87MG glioma cells and macrophages in the bottom chamber under hypoxic condition. j . CLSM images of bEnd.3 cells with different treatments. Scale bar: 5 μm. (green: ZO-1, red: EVs). k . Accumulative penetration efficiency of M1EVs, CC-M1EVs, A-M1EVs, and CCA-M1EVs labeled with DiD through a monolayer bEnd.3 layer at different time points ( n = 3). l . Flow cytometry analysis of the M2/M1 ratio in the lower chamber after incubation with different EV designs ( n = 3). m . Production of H 2 O 2 with different treatments in the lower chamber (Amplex Red Hydrogen Peroxide Assay Kit) ( n = 3). n . Assessment of intracellular ROS (labeled by DCFH-DA) of U87MG cells in the lower chamber ( n = 3). o . Flow cytometry analysis of the cell-death-inducing effect of different formulations on U87MG cells in the lower chamber (Annexin V and PI in the dead cell apoptosis kit) ( n = 3). Statistical significance was calculated via one-way ANOVA with a Kruskal-Wallis test ( e , g , l , m , n , and o ) or unpaired two-tailed Student’s t -test ( f ). ns, not significant

Article Snippet: The expression of CD9/CD81/ALIX/TSG101 (EV marker), iNOS (M1 marker) and F4/80 (macrophages marker) in M1 macrophage and M1EVs were analyzed by ProteinSimple® Wes TM capillary western blot analyzer (PS-MK15; ProteinSimple, USA).

Techniques: Confocal Laser Scanning Microscopy, Flow Cytometry, High Performance Liquid Chromatography, In Vitro, Co-Culture Assay, Labeling, Incubation, Amplex Red Hydrogen Peroxide Assay, Two Tailed Test

Journal: Journal of Leukocyte Biology

Article Title: Monocytes differentiated into macrophages and dendritic cells in the presence of human IFN‐λ3 or IFN‐λ4 show distinct phenotypes

doi: 10.1002/JLB.3A0120-001RRR

Figure Lengend Snippet: RNA‐sequencing (RNA‐seq) analysis shows that IFN‐λ4 confers a modified M1 phenotype on monocyte‐derived macrophages (MDMs). MDMs differentiated from CD14+ monocytes (as shown in Fig. ) from a single donor were subjected to paired‐end RNA‐seq analysis. Duplicate samples that had mock or IFN‐λ4 treatment during differentiation with GM‐CSF were used. ( A ) Volcano plot showing the differentially expressed genes (DEGs) in IFN‐λ4‐treated MDMs compared with untreated cells; a cut‐off of 1.5‐fold change and P = 0.05 were used. ( B ) Heatmap of the top 100 up‐ and down‐regulated DEGs are shown, with duplicate IFN‐λ4‐treated and mock‐treated samples in different colors. Some of the important genes are shown on the right (also included in Table ). ( C ) Reactome pathway enrichment analysis bubble plot of the DEGs (IFN‐λ4 vs. mock) showing the top four most affected pathways

Article Snippet: CD14 + cells from four more donors (PromoCell) were differentiated into M1‐MDMs in the presence or absence of IFN‐λ4 and further stimulated with LPS, and the expression of some important cytokines were assessed by ELISA (Fig. ).

Techniques: RNA Sequencing Assay, Modification, Derivative Assay

Journal: Journal of Leukocyte Biology

Article Title: Monocytes differentiated into macrophages and dendritic cells in the presence of human IFN‐λ3 or IFN‐λ4 show distinct phenotypes

doi: 10.1002/JLB.3A0120-001RRR

Figure Lengend Snippet: Recombinant IFN‐λ3 shows superior specific activity compared to recombinant IFN‐λ4 in IFN‐stimulated genes (ISGs) stimulation in various cell types. ( A ) A range of concentrations were tested for the two cytokines in A549 (left; procedure followed same as in Fig. ) and PMA‐differentiated THP‐1 cells (right; THP‐1 cells were treated with PMA for 48 h and then treated with IFN‐λ3 or IFN‐λ4 for 24 h) and quantitative polymerase chain reaction (qPCR) was carried out to measure the ISG expression. (b) Western blot showing the expression of pSTAT1 in M2‐monocyte‐derived macrophage (MDM) cells generated from human PBMC‐derived CD14 + cells (obtained by negative selection) as described in Section 2 (“Materials and Methods”). ( C ) ISG stimulation activity of IFN‐λ3 and IFN‐λ4 was tested at the given concentrations in in vitro generated monocyte‐derived dendritic cells (MoDCs) and M1‐MDMs from a single donor as described in Section 2 (CD14 + monocytes were isolated from PBMCs of a healthy volunteer by positive selection for experiments shown in ( C ) as described in Section 2. For A and C : The data show mean from technical triplicates from one experiment with error bars depicting sd

Article Snippet: CD14 + cells from four more donors (PromoCell) were differentiated into M1‐MDMs in the presence or absence of IFN‐λ4 and further stimulated with LPS, and the expression of some important cytokines were assessed by ELISA (Fig. ).

Techniques: Recombinant, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Derivative Assay, Generated, Selection, In Vitro, Isolation

Journal: Journal of Leukocyte Biology

Article Title: Monocytes differentiated into macrophages and dendritic cells in the presence of human IFN‐λ3 or IFN‐λ4 show distinct phenotypes

doi: 10.1002/JLB.3A0120-001RRR

Figure Lengend Snippet: Monocyte‐derived macrophages (MDMs) differentiated in the presence of IFN‐λ3 or IFN‐λ4 show altered cytokine secretion. ( A ) Activated M1‐MDMs differentiated in the presence of IFN‐λ4 show lower IL‐1β and higher IL‐10 secretion. CD14 + cells obtained from five independent donors were differentiated into M1‐MDMs and stimulated with LPS (as per scheme in Fig. ), and cytokine secretion was measured by ELISA. The data show mean values from five independent donors with error bars representing sd . Filled circles and filled triangles represent MDMs derived from each of the five donors (in different colors) differentiated without and with IFN‐λ4 respectively, joined by a trend line. A 1‐tailed t ‐test for two dependent means was carried out to calculate statistical significance. * P < 0.05; ns, not significant. ( B ) Cytokine profiles of activated M1‐ and M2‐MDMs differentiated in the presence of IFN‐λ3 or IFN‐λ4. The data show mean values from four independent donors with error bars representing sd . CD14 + cells from each donor were split into three aliquots and differentiated into M1‐ or M2‐MDMs in the absence or presence of IFN‐λ3 or IFN‐λ4; after activation with LPS, cytokines were collected from supernatants and measured by ELISA. A 2‐tailed t ‐test for two independent means was used to calculate statistical significance; only significant comparisons are shown; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: CD14 + cells from four more donors (PromoCell) were differentiated into M1‐MDMs in the presence or absence of IFN‐λ4 and further stimulated with LPS, and the expression of some important cytokines were assessed by ELISA (Fig. ).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay