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: IFN‐λ4 confers a mixed phenotype on THP‐1‐derived macrophage‐like cells. ( A ) Histogram from one representative experiment showing that IFN‐λ4‐treated macrophage‐like cells had increased expression of CD80 but reduced expression of HLA‐DR (left) when activated with LPS, whereas IFN‐λ4 had no effect when activation was carried out with LPS and IFN‐γ. The graph on the right shows the normalized (for mock treatment) mean of positive events in the gate shown in the histogram and/or mean fluorescence intensity from two independent experiments with IFN‐λ4 treatment; error bars show sd . The significance was calculated using a one‐sample t ‐test (* P < 0.05). ( B and C , right) mRNA expression of different genes from M1‐activated (with LPS) ( B ) and M2‐activated (with macrophage CSF [M‐CSF], IL‐4, and IL‐10) ( C ) THP‐1‐derived macrophage‐like cells. The data show the mean from technical triplicates with error bars depicting sd , and are representative of at least two separate biological replicate experiments. * P < 0.05; ** P < 0.01, *** P < 0.001. ( C , left) Histogram of CD209‐, CD206‐, and CD163‐positive cells as determined by flow cytometry of THP1‐derived M2 macrophage‐like cells differentiated in the absence or presence of IFN‐λ4 (6 μg/ml). The data were obtained from a single experiment

Article Snippet: Human recombinant IFN‐λ4 (catalog #9165­IF; carrier‐free form) and IFN‐λ3 (catalog #5259‐IL/CF), containing <0.1 EU/μg of endotoxin, were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Expressing, Activation Assay, Fluorescence, Flow Cytometry

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: IFN‐λ4 influences M1‐monocyte‐derived macrophages (MDM) differentiation. ( A ) Schematic representation of the experimental design of M1‐MDM differentiation. Monocytes were obtained from a single donor. ( B ) The bar graph shows mRNA expression analysis by quantitative polymerase chain reaction (qPCR). The data show the mean and sd from six technical replicates derived from two biological replicate experiments ( n = 3 × 2). * P < 0.05; ** P < 0.01, *** P < 0.001

Article Snippet: Human recombinant IFN‐λ4 (catalog #9165­IF; carrier‐free form) and IFN‐λ3 (catalog #5259‐IL/CF), containing <0.1 EU/μg of endotoxin, were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

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: Important genes identified by RNA‐sequencing that were found to be differentially regulated after IFN‐λ4 treatment to differentiating monocyte‐derived macrophages (MDMs)

Article Snippet: Human recombinant IFN‐λ4 (catalog #9165­IF; carrier‐free form) and IFN‐λ3 (catalog #5259‐IL/CF), containing <0.1 EU/μg of endotoxin, were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding 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: Human recombinant IFN‐λ4 (catalog #9165­IF; carrier‐free form) and IFN‐λ3 (catalog #5259‐IL/CF), containing <0.1 EU/μg of endotoxin, were purchased from R&D Systems (Minneapolis, MN, USA).

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: Human recombinant IFN‐λ4 (catalog #9165­IF; carrier‐free form) and IFN‐λ3 (catalog #5259‐IL/CF), containing <0.1 EU/μg of endotoxin, were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation 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: Comparison of fold changes in cytokine secretion in cocultures of CD4 + cells and monocyte‐derived dendritic cells (MoDCs) differentiated in the presence of IFN‐λ3 or IFN‐λ4. MoDCs were differentiated from CD14 + cells obtained from four donors in the absence or presence of IFN‐λ3 or IFN‐λ4; coculture experiments were set up with CD4 + cells from 20 allogenic donors and cytokines were collected from supernatants and measured by ELISA. ( A ) Heatmap showing the fold changes of five cytokines collected from coculture supernatants involving MoDCs generated in the presence of IFN‐λ3 (left) or IFN‐λ4 (right) and allogenic CD4 + cells. The color key at the bottom shows row Z ‐score. The comparisons shown below the heatmap are between any two cytokines by paired t ‐tests. Only the comparisons that were statistically significant are shown. ( B ) Comparison of cytokine profiles between 20 allogenic CD4 + donors in coculture experiments involving MoDCs derived in presence of IFN‐λ3 or IFN‐λ4. The comparison is between IFN‐λ3 and IFN‐λ4 for a given cytokine. The bars depict mean values and error bars depict sd . A 2‐tailed t ‐test for dependent means was used for all statistical comparisons in ( A ) and ( B ); a 1‐tailed t ‐test did not show any new groups of cytokines that had significant differences in their means for IFN‐λ3; for IFN‐λ4, however, a 1‐tailed t ‐test showed significance in some new comparisons: IFN‐γ vs. IL‐4 ( P = 0.03) and IL‐4 vs. IL‐17 ( P = 0.03). For ( A ) and ( B ) * P < 0.05, ** P < 0.01; ns, not significant

Article Snippet: Human recombinant IFN‐λ4 (catalog #9165­IF; carrier‐free form) and IFN‐λ3 (catalog #5259‐IL/CF), containing <0.1 EU/μg of endotoxin, were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Comparison, Derivative Assay, Enzyme-linked Immunosorbent Assay, Generated

Journal: Journal of Virology

Article Title: A CRISPR Activation Screen Identifies Genes That Protect against Zika Virus Infection

doi: 10.1128/JVI.00211-19

Figure Lengend Snippet: Immunofluorescence of IFI6- and IFN-λ2-expressing ZIKV-infected cells. (A) Huh7 cell lines expressing IFI6 or nontargeting sgRNAs were stained with an IFI6-specific antibody Scale bar = 20 μm. (B) Medium from Huh7 cell lines expressing IFN-λ2 or NT sgRNAs was used to treat Huh7 cells. After 18 h, the cells were harvested and analyzed by qPCR using Mx1 primers. Recombinant IL-28b (IFN-λ3, R-IL-28B) was used as a positive control (10 ng/μl). ***, P < 0.01; ****, P < 0.001, one-way ANOVA Dunnett’s multiple-comparison test. (C and D) Huh7 cell lines expressing IFI6, IFN-λ2, or nontargeting sgRNAs were infected with ZIKV (MOI, 10) (C) or DENV (MOI, 1) (D) for 2 h. After 24 h (ZIKV) or 48 h (DENV), the cells were fixed and stained with flavivirus protein E-specific antibody and an antibody recognizing dsRNA. DAPI was used for nucleus staining. Scale bar = 100 μm. The relative mean fluorescence intensity of each image compared to the control was quantified using Fiji and is presented below each image.

Article Snippet: Interferon alpha B2 was purchased from PBL (NJ; catalog no. 11115-1), and recombinant human interleukin 28B (IL-28B)/IFN-λ 3 protein was from R&D Systems (catalog no. 5259-IL). . sgRNA library amplification and lentivirus production.

Techniques: Immunofluorescence, Expressing, Infection, Staining, Recombinant, Positive Control, Comparison, Fluorescence, Control