Journal: Journal for immunotherapy of cancer

Article Title: Loss of ADAR1 in macrophages in combination with interferon gamma suppresses tumor growth by remodeling the tumor microenvironment.

doi: 10.1136/jitc-2023-007402

Figure Lengend Snippet: Figure 3 ADAR1 loss in macrophage with IFN-γ treatment affects the secretion of key cytokines through PKR/EIF2α signaling. (A, C) Human XL cytokine arrays for detecting differential factors between THP-1 cells with scrambled shRNA and shADAR1#1 under the treatment of IFN-γ (A) and between THP-1 cells with empty vector and WT ADAR, co-cultured with A549 (C). (B, D) Bar plots showing the expression levels of differential factors on ADAR1 knockdown (B) and ADAR1 overexpression (D). (E) Venn gram showing key cytokines identified by ADAR1 knockdown and overexpression experiments and their potential effects on the tumor microenvironment. (F) RT-qPCR-based mRNA expression levels of ADAR, CCL20, GDF15, IFN-G, IL-18, IL-18BP, and HAVCR2 in different THP-1 cells (transfected with scrambled shRNA, shADAR1#1 or shADAR1#2) with IFN-γ treatment. β-actin was used as an internal control. (G) RT-qPCR-based mRNA expression levels of Ccl20, Gdf15, Il-18, Il-18bp, and Havcr2 in BMDMs from C57BL/6 mice (Adarfl/fl and Adarfl/flLyz2Cre) treated with IFN-γ. Gapdh was used as an internal control. (H) Immunofluorescent staining for anti-dsRNA (J2) in THP-1 cells treated with IFN-γ. RNase III treatment was used as the negative control for the dsRNA signal. Scale bars, 10 µm. (I) Western blot showing the protein expression of p-PKRThr446/

Article Snippet: 2- AP (GlpBio, GC61906), Phorbol 12- myristate 13- acetate (PMA) (MedChemExpress, HY- 18739), Recombinant Human IFN-γ (Novoprotein, C014), Recombinant Mouse IL- 2 (Novoprotein, P04351), Recombinant M- CSF (Novoprotein, CB34), Recombinant Mouse IFN-γ (Novoprotein, C746), Recombinant Mouse TIM- 3 (Novoprotein, CM54), Recombinant Mouse CCL20 (PeproTech, 250–2), Recombinant Mouse GDF15 (R&D Systems, 8944- GD025), Recombinant Mouse IL- 18 Binding Protein Isoform d (Novoprotein, CM45), ShortCut RNase III (NEB, M0245S), poly (I:C) (GlpBio, GC14710).

Techniques: shRNA, Plasmid Preparation, Cell Culture, Expressing, Knockdown, Over Expression, Quantitative RT-PCR, Transfection, Control, Staining, Negative Control, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Loss of ADAR1 in macrophages in combination with interferon gamma suppresses tumor growth by remodeling the tumor microenvironment.

doi: 10.1136/jitc-2023-007402

Figure Lengend Snippet: Figure 5 The effect of ADAR1 loss and key cytokines on tube formation. (A) Schematic diagram of tube formation experiment of SVEC4-10 cells. (B) Tube formation of SVEC4-10 cells treated with conditioned media from BMDMs (Adarfl/fl vs Adarfl/flLyz2Cre) and CCL20, GDF15, or IFN-γ. Scale bar, 500 µm. (C) Bar plots showing relative vessel area, the total number of junctions, and the vessel length for SVEC4-10 tube formation. (D) Tube formation of SVEC4-10 cells treated with conditioned media from BMDMs (Adarfl/flLyz2Cre) and IFN-γ after pretreatment with or without 2-AP (5 mM). Scale bar, 500 µm. (E) Bar plots showing relative vessel area, the total number of junctions, and vessel length for SVEC4-10 tube formation. (F) Tube formation of HUVEC cells treated with conditioned media from THP-1 cells (transfected with shADAR1#1) and IFN-γ after pretreatment with or without 2-AP (5 mM). Scale bar, 200 µm. (G) Bar plots showing relative vessel area, the total number of junctions, and the vessel length for HUVEC tube formation. (C, E, and G) P values are based on unpaired Student’s t-test. Data are presented as mean±SD. ADAR, adenosine deaminases acting on RNA; BMDMs, bone marrow-derived macrophages; IFN, interferon; 2-AP, 2-aminopurine.

Article Snippet: 2- AP (GlpBio, GC61906), Phorbol 12- myristate 13- acetate (PMA) (MedChemExpress, HY- 18739), Recombinant Human IFN-γ (Novoprotein, C014), Recombinant Mouse IL- 2 (Novoprotein, P04351), Recombinant M- CSF (Novoprotein, CB34), Recombinant Mouse IFN-γ (Novoprotein, C746), Recombinant Mouse TIM- 3 (Novoprotein, CM54), Recombinant Mouse CCL20 (PeproTech, 250–2), Recombinant Mouse GDF15 (R&D Systems, 8944- GD025), Recombinant Mouse IL- 18 Binding Protein Isoform d (Novoprotein, CM45), ShortCut RNase III (NEB, M0245S), poly (I:C) (GlpBio, GC14710).

Techniques: Transfection, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Loss of ADAR1 in macrophages in combination with interferon gamma suppresses tumor growth by remodeling the tumor microenvironment.

doi: 10.1136/jitc-2023-007402

Figure Lengend Snippet: Figure 8 Schematic summary of this study. ADAR1-deficient macrophages combined with IFN-γ treatment reprogram the tumor microenvironment by two mechanisms: (1) inhibit angiogenesis by decreased secretion of GDF15 and CCL20 and increased secretion of IFN-γ, and (2) activate CD8+ T cells by decreased secretion of TIM-3 and IL-18BP and increased secretion of IL-18. These effects collectively convert a “cold tumor” into a “hot tumor.” Combined treatment with ADAR1- deficient macrophages and IFN-γ may represent an effective therapeutic approach. dsRNA, double-stranded RNA; IFN, interferon; IL, interleukin; uORF, upstream Open Reading Frame.

Article Snippet: 2- AP (GlpBio, GC61906), Phorbol 12- myristate 13- acetate (PMA) (MedChemExpress, HY- 18739), Recombinant Human IFN-γ (Novoprotein, C014), Recombinant Mouse IL- 2 (Novoprotein, P04351), Recombinant M- CSF (Novoprotein, CB34), Recombinant Mouse IFN-γ (Novoprotein, C746), Recombinant Mouse TIM- 3 (Novoprotein, CM54), Recombinant Mouse CCL20 (PeproTech, 250–2), Recombinant Mouse GDF15 (R&D Systems, 8944- GD025), Recombinant Mouse IL- 18 Binding Protein Isoform d (Novoprotein, CM45), ShortCut RNase III (NEB, M0245S), poly (I:C) (GlpBio, GC14710).

Techniques:

Journal: bioRxiv

Article Title: Identification of a distinct cluster of GDF15 high macrophages exhibiting anti-inflammatory activities

doi: 10.1101/2024.01.11.575315

Figure Lengend Snippet: Immunofluorescence double labeling showing the existence of GDF15 high macrophages in lung tissues. (A) Results obtained in healthy human lung tissues from 3 independent subjects. Two representative microscopic fields were shown. Arrowheads indicated the CD68 + GDF15 high macrophages. (B) Results obtained in human lung tissues with COPD from 4 independent subjects. Arrowheads indicated the CD68 + GDF15 high macrophages. (C) Results obtained in rat lung tissues without and with experimental PAH. The nuclei were counterstained with DAPI (blue). White arrowheads indicated CD68 + GDF15 high macrophages. The red box highlighted the presence of CD68 + GDF15 high macrophages (white arrowheads); the green box highlighted the presence of CD68 + GDF15 low macrophages (red arrowheads). The bar graphs showed the absolute and relative abundances of CD68 + GDF15 high macrophages in normal and PAH lungs. Data were expressed as mean ± SEM. * P < 0.05, unpaired t -test, n = 6.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Identification of a distinct cluster of GDF15 high macrophages exhibiting anti-inflammatory activities

doi: 10.1101/2024.01.11.575315

Figure Lengend Snippet: GDF15 high macrophages could be derived by in vitro differentiation of mononuclear cells. (A and B) Immunofluorescence staining and flow cytometry results confirming that in vitro differentiation of human peripheral blood mononuclear cells (PBMNCs) with GM-CSF for 7 days yielded CD68 + macrophages. (C and D) Flow cytometry and immunofluorescence double labeling results showing that the PBMNC-derived macrophages contained a minor population of GDF15 high cells (arrowheads in D) (example from 3 independent experiments). (E and F) Fluorescence microscopy and flow cytometry data showing that GM-CSF differentiation of rat bone marrow mononuclear cells (BMMNCs) in vitro yielded macrophages (GFP expressing) of a high purity (∼90%). CD68pro-GFP rats had a GFP transgene under the control of CD68 promoter. Cells from normal rats showing no GFP fluorescence served as a negative control (left panel in E). (G and H) Flow cytometry and immunofluorescence double labeling data (from 3 independent experiments) showing that the BMMNC-derived macrophages (from CD68pro-GFP rats) contained a minor population of GDF15 high cells (arrowheads in H). The flow cytometry data in panels C and G were from cells gated for GFP + . The nuclei were counterstained with DAPI (blue).

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, In Vitro, Immunofluorescence, Staining, Flow Cytometry, Labeling, Fluorescence, Microscopy, Expressing, Control, Negative Control

Journal: bioRxiv

Article Title: Identification of a distinct cluster of GDF15 high macrophages exhibiting anti-inflammatory activities

doi: 10.1101/2024.01.11.575315

Figure Lengend Snippet: Flow cytometry results showing that GDF15 high macrophages did not exhibit a typical M1 or M2 phenotype. Experiments were performed in human PBMNC-derived macrophages, using CD86, CD80 and IL-1β as the M1 markers, and CD206, CD163 and IL-4 as the M2 markers. Data were from a single test using pooled samples from 4 healthy volunteers.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Derivative Assay

Journal: bioRxiv

Article Title: Identification of a distinct cluster of GDF15 high macrophages exhibiting anti-inflammatory activities

doi: 10.1101/2024.01.11.575315

Figure Lengend Snippet: Molecular characterization of human PBMNC-derived GDF15 high macrophages with scRNA-seq. (A) Graphical outline of the experimental procedure. (B) UMAP plots showing the identified cell clusters (C1 to C7) based on the scRNA-seq data from total 73,768 cells combined from samples from 3 healthy volunteers, 3 PAH patients harboring mutations in BMPR2 gene, and 3 PAH patients without BMPR2 mutations. The inset showed that macrophages from healthy controls and PAH patients had virtually identical clustering profiles when analyzed separately. The putative nomenclatures for C1 to C7 were given below the graph. (C) Violin plots showing the expression patterns of identified marker genes for C1 to C7. (D) UMAP plots showing expression patterns of the top 10 genes that were overexpressed in GDF15 high macrophages (C5) as compared to GDF15 low cells. (E) UMAP plots showing expression patterns of the top 9 genes encoding secreted proteins which were overexpressed in GDF15 high macrophages as compared to GDF15 low cells. (F) Cell-cell communication network map created using CellChat showing the possible effector cells of the GDF15 high macrophage. (G and H) Predicted ligand-receptor pairs potentially involved in the signaling of reciprocal communications between GDF15 high macrophage and other cell types as listed in panel F.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Expressing, Marker

Journal: bioRxiv

Article Title: Identification of a distinct cluster of GDF15 high macrophages exhibiting anti-inflammatory activities

doi: 10.1101/2024.01.11.575315

Figure Lengend Snippet: GDF15 high macrophages exhibited reduced inflammatory activation in vitro. (A) Expression patterns of potential substitute cell surface markers for GDF15 based on the scRNA-seq data. (B) Flow cytometry results showing that rat BMMNC-derived macrophages contained a minor fraction of TNFSF9 high cells, whose expression level was correlated with that of GDF15 (from 3 independent experiments). (C) Flow cytometry verification of the correlation between TNFSF9 and GDF15 expressions in human PBMNC-derived macrophages (from 2 independent experiments). (D) Real-time PCR results showing that GDF15 high macrophages (H) exhibited reduced expressions of TNF-α, IL-1β and IL-6 in response to LPS stimulation (1 μg/mL for 6 hr), as compared to GDF15 low cells (L). Rat BMMNC-derived macrophages were FACS purified using TNFSF9 as a substitute marker for GDF15, and primed with IFN-γ (10 ng/mL for 12 hr). (E) Boyden chamber cell migration assay showing that GDF15 high macrophages (H) exhibited reduced migratory activity as compared to GDF15 low cells (L) in the absence and presence of LPS stimulation. (F) Representative fluorescent microscopic images and quantitative data showing that GDF15 high macrophages exhibited reduced phagocytic activity in the presence of LPS stimulation as compared to GDF15 low cells. Phagocytosis was assessed by internalization of fluorochrome-labeled latex beads (orange color). The macrophages were from CD68pro-GFP rats. Data were mean ± SEM. * P < 0.05, one-way ANOVA, n = 3 in D; 6 in E; 6 in F. NS, no significance.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, In Vitro, Expressing, Flow Cytometry, Derivative Assay, Real-time Polymerase Chain Reaction, Purification, Marker, Cell Migration Assay, Activity Assay, Labeling

Journal: bioRxiv

Article Title: Identification of a distinct cluster of GDF15 high macrophages exhibiting anti-inflammatory activities

doi: 10.1101/2024.01.11.575315

Figure Lengend Snippet: GDF15 high macrophages exerted anti-inflammatory effects via paracrine mechanisms. (A to C) RAW264.7 cells co-cultured with rat BMMNC-derived GDF15 high (H) or GDF15 low (L) macrophages were left untreated or stimulated with LPS for 4 hr. Results for the expression of pro-inflammatory cytokines (A, real-time PCR), cell migratory activity (B), and phagocytic activity (C) were shown. (D to F) Unsorted rat BMMNC-derived macrophages co-cultured with rat GDF15 high (H) or GDF15 low (L) macrophages were left untreated or stimulated with LPS for 4 hr. Results for the expression of pro-inflammatory cytokines (D, real-time PCR), cell migratory activity (E), and phagocytic activity (F) were shown. Data were mean ± SEM. * P < 0.05, one-way ANOVA, n = 4-5 in A; 6 in B; 3 in C; 3-4 in D; 6 in E; 6 in F. NS, no significance.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

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

Journal: bioRxiv

Article Title: Identification of a distinct cluster of GDF15 high macrophages exhibiting anti-inflammatory activities

doi: 10.1101/2024.01.11.575315

Figure Lengend Snippet: GDF15 might be a macrophage-derived anti-inflammatory factor. (A) Real-time PCR results showing that treatment with exogenous GDF15 (20 ng/mL) inhibited LPS- induced expression of pro-inflammatory cytokines in RAW264.7 cells. (B) Flow cytometry results showing that GDF15 treatment had no effects on phagocytosis in RAW264.7 cells without or with LPS stimulation. (C) Representative images and quantitative data of Boyden chamber assay showing that exogenous GDF15 inhibited migration of LPS-challenged RAW264.7 cells. Cells on the membrane were stained with Giemsa. (D) Effects of conditioned medium from GDF15 high macrophages (H), as compared to the medium from GDF15 low cells (L), on the expression of pro-inflammatory cytokines in RAW264.7 cells. All experiments were performed in the presence of LPS stimulation. α-GDF15, GDF15neutralizing antibody; IgG, non-specific immunoglobulin control. (E) The same experiments as those in D carried out in rat BMMNC-derived macrophages. Data were mean ± SEM. * P < 0.05, one-way ANOVA; † P < 0.05, unpaired t -test, n = 3-5 in A; 3 in B; 6 in C; 3 in D; 3 in E. NS, no significance.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Boyden Chamber Assay, Migration, Membrane, Staining, Control

Journal: bioRxiv

Article Title: Identification of a distinct cluster of GDF15 high macrophages exhibiting anti-inflammatory activities

doi: 10.1101/2024.01.11.575315

Figure Lengend Snippet: Detection of GDF15 high macrophages in various human tissues. GDF15 high macrophages (arrowheads) were identified using immunofluorescence double labeling with anti-CD68 (green color) and anti-GDF15 (red color) antibodies in (A) colon tissues from both healthy subjects and patients with ulcerative colitis, (B) kidneys (the normal peri-tumor tissue) (tested in one sample only) and (C) atherosclerotic plaques in the carotid artery (representative data from 6 independent samples showing similar results). The nuclei were counterstained with DAPI (blue). Data were mean ± SEM. NS, no significance, unpaired t -test, n = 3 for Healthy; 4 for Colitis.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Labeling