Journal: Journal for Immunotherapy of Cancer

Article Title: Wrinkle in the plan: miR-34a-5p impacts chemokine signaling by modulating CXCL10/CXCL11/CXCR3-axis in CD4 + , CD8 + T cells, and M1 macrophages

doi: 10.1136/jitc-2020-001617

Figure Lengend Snippet: Gene expression analysis of the effects of miR-34a-5p over-expression in M1 macrophages. (A) Experimental workflow. (B-C) Over-representation analysis of 296 mRNAs, which were at least 1.5-fold downregulated on miR-34a-5p over-expression by GeneTrail3. (B) Representative significant enriched pathways in Gene Ontology (GO) category “biological process.” (C) Representative significant enriched pathways in the GO category “molecular function.” (D) Protein–protein interaction networks of the 20 most downregulated mRNAs using the STRING database V.11 ( https://string-db.org/ ). (E) Protein–protein interaction networks of the 20 most upregulated mRNAs using the STRING database V.11.

Article Snippet: M1 macrophages were differentiated from freshly obtained PBMC using M1 macrophage Generation Medium DXF according to the manufacturer’s protocol (PromoCell GmbH, Heidelberg, Germany).

Techniques: Expressing, Over Expression

Journal: Journal for Immunotherapy of Cancer

Article Title: Wrinkle in the plan: miR-34a-5p impacts chemokine signaling by modulating CXCL10/CXCL11/CXCR3-axis in CD4 + , CD8 + T cells, and M1 macrophages

doi: 10.1136/jitc-2020-001617

Figure Lengend Snippet: 20 most upregulated and downregulated mRNAs in miR-34a-5p transfected M1 macrophages. Upper panel displays the 20 most downregulated mRNAs and the lower panel the 20 most upregulated mRNAs

Article Snippet: M1 macrophages were differentiated from freshly obtained PBMC using M1 macrophage Generation Medium DXF according to the manufacturer’s protocol (PromoCell GmbH, Heidelberg, Germany).

Techniques: Transfection

Journal: Journal for Immunotherapy of Cancer

Article Title: Wrinkle in the plan: miR-34a-5p impacts chemokine signaling by modulating CXCL10/CXCL11/CXCR3-axis in CD4 + , CD8 + T cells, and M1 macrophages

doi: 10.1136/jitc-2020-001617

Figure Lengend Snippet: (A–C) Effects of miR-34a-5p over-expression on the endogenous levels and secretion of CXCL10 and CXCL11 in M1 macrophages. Monocytes were differentiated in M1 macrophages, transfected with ANC, or miR-34a-5p mimic, and activated with IFN-γ and LPS. Subsequently, the CXCL10 and CXCL11 endogenous levels were measured by western blots (A, one representative western blot is displayed) and quantified from three independent experiments from two different donors (B) Three asterisks correspond to p value ≤0.001 and one asterisk to a p value 0.01

Article Snippet: M1 macrophages were differentiated from freshly obtained PBMC using M1 macrophage Generation Medium DXF according to the manufacturer’s protocol (PromoCell GmbH, Heidelberg, Germany).

Techniques: Over Expression, Transfection, Western Blot, Enzyme-linked Immunosorbent 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: 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