Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: ZBP1 is mainly expressed in macrophages and alters the ratio of M1 and M2 macrophages in sepsis‐induced myocardial dysfunction. A single‐nucleus mRNA sequencing (snRNA‐seq) was performed in hearts from LPS‐treated WT or Zbp1 −/− mice (for each group, single‐cell suspensions from three hearts were pooled as one sample). (A) UMAP plot of cell clusters in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. CM, cardiomyocyte; EC, endothelial cell; FB, fibroblast; Mac, macrophage; NP, neutrophil. (B,C) UMAP plot and dot plot of Zbp1 expression in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. (D,E) UMAP plot of macrophage in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. M1, M1 macrophage; M2, M2 macrophage. (F–H) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images (F) and quantification of positive cells (G,H) are shown [scale bar = 50 µm, n = 5]. Mean ± SEM; * P < 0.05, *** P < 0.001, ns, no significance. WT, wild‐type; ZBP1, Z‐DNA binding protein 1; LPS, lipopolysaccharide; snRNA‐seq, single‐nucleus mRNA sequencing.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Sequencing, Expressing, Marker, Staining, Binding Assay

Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: Myeloid‐specific Zbp1 deficiency protects against sepsis‐induced myocardial dysfunction. (A) Schematic showing experimental design to investigate the impact of myeloid‐specific Zbp1 deficiency on septic myocardial dysfunction (LPS: 10 mg/kg; 6 h). (B) Zbp1 gene knockout efficiency in hearts and bone marrow cells was detected by Western blot. (C–E) Representative images of transthoracic M‐mode echocardiographic trance, hematoxylin and eosin (H&E) staining, immunohistochemistry staining of CD11b, Ly6C and Ly6G at 6 h after intraperitoneal injection of LPS in Zbp1 fl/fl or Zbp1 cko mice. M‐mode analysis of EF (D) and FS (E) before (pre) and 6 h after (post) LPS treatment. [scale bar = 20 µm, n = 7 in each group]. (F–H) Quantitative analysis of CD11b + , Ly6C + area and Ly6G + cells in myocardial tissues ( n = 7 in each group). (I,J) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images and quantification of positive cells are shown [scale bar = 50 µm, n = 5 in each group]. Mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. LPS, lipopolysaccharide; EF, ejection fraction; FS, fractional shortening; DAPI, 4′,6‐diamidino‐2‐phenylindole.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Gene Knockout, Western Blot, Staining, Immunohistochemistry, Injection, Marker

Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: Blocking STAT1 prevents cardiac dysfunction caused by sepsis. (A) Schematic showing experimental design to investigate the impact of STAT1 inhibition on septic myocardial dysfunction (Flu, fludarabine, STAT1 inhibitor, 50 mg/kg/day; Sol, solvent). (B–D) Western blotting detection and quantitative analysis of STAT1 and ZBP1 in myocardial tissues at 6 h after intraperitoneal injection of NS or LPS (10 mg/kg) in solvent or fludarabine‐treated mice ( n = 5 in each group). (E–G) Representative images of transthoracic M‐mode echocardiographic trance, hematoxylin and eosin (H&E) staining, immunohistochemistry staining of CD11b, Ly6C and Ly6G at 6 h after intraperitoneal injection of LPS in solvent or fludarabine‐treated mice. M‐mode analysis of EF (F) and FS (G) before (pre) and 6 h after (post) LPS treatment. [Scale bar = 20 µm, n = 8–9 in each group]. (H–J) Quantitative analysis of CD11b + , Ly6C + area and Ly6G + cells in myocardial tissues ( n = 5 in each group). (k–l) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images and quantification of positive cells are shown [scale bar = 50 µm, n = 5 in each group]. Mean ± SEM; * P < 0.05, *** P < 0.001. ZBP1, Z‐DNA binding protein 1; LPS, lipopolysaccharide; STAT1, signal transducer and activator of transcription 1; EF, ejection fraction; FS, fractional shortening; DAPI, 4′,6‐diamidino‐2‐phenylindole.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Blocking Assay, Inhibition, Solvent, Western Blot, Injection, Staining, Immunohistochemistry, Marker, Binding Assay

Journal: Nature Communications

Article Title: Synthesis of bioactive (1→6)-β-glucose branched poly-amido-saccharides that stimulate and induce M1 polarization in macrophages

doi: 10.1038/s41467-022-32346-5

Figure Lengend Snippet: Fold change of median fluorescence intensity of a CD80, b CD206, and c CD163 compared to an unstained control. Two-tailed unpaired t test were performed for a LPS vs P5 ( p = 0.0002), LPS vs Lentinan ( p = 0.0002), LPS vs Laminarin ( p = 0.0132). Percentage of positive cells of d CD80, e CD206, and f CD163 compared to an unstained control. Error bars represent standard deviation. Two-tailed unpaired t test were performed for d LPS vs P5 ( p < 0.0001), LPS vs Lentinan ( p = 0.0003), LPS vs Laminarin ( p = 0.0296). Unless otherwise stated, data are means ± SD of three separate experiments ( N = 3). ns p > 0.05, * p < 0.05, **** p ≤ 0.0001 compared using two-tailed unpaired t test. Abbreviations used: Ctrl = Control, Dex = Dextran, Lent = Lentinan, Lam = Laminarin.

Article Snippet: After 24 h of incubation, cells were lifted from the plate with cold PBS and Accutase cell detachment solution and stained for CD68 (StemCell 60105FI) and either CD80 (Biolegend 333611), CD163 (Biolegend 305221), or CD206 (R&D Systems FAB25342P).

Techniques: Fluorescence, Control, Two Tailed Test, Standard Deviation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combinatorial macrophage induced innate immunotherapy against Ewing sarcoma: Turning "Two Keys" simultaneously.

doi: 10.1186/s13046-024-03093-w

Figure Lengend Snippet: Fig. 5 Characterization of markers for ES-phagocytic macrophages. A Gating strategy for evaluating cell surface protein expression on phagocytic versus non-phagocytic macrophages. In-vitro phagocytosis assay was carried out incubating A673 cells (Non- or DOX- or MAG- or D&M-treated) labeled with CMFDA (detected by FITC channel) and M-CSF Mφ. Cells were harvested and stained with APC-CD11b and PE-CD38 antibodies followed by flow cytometry analysis. The viable cells were plotted in FITC (CMFDA) x APC view to visualize cell populations in the in-vitro phagocytosis assay. A gate was drawn to include cells that were FITC positive and APC positive as the phagocytic macrophage population, and another gate was drawn to include cells that were FITC- APC + as the non-phagocytic macrophage population. The FITC + APC + and FITC- APC + cells were then plotted separately in a PE x APC view to visualize expression of CD38 in both cell populations. A quat gate was drawn to visualize APC + PE + as the CD38 + and APC + PE- as the CD38- macrophages. B Comparison of percent of CD14, CD38, CD163, CD206 or CD209 positive and negative cells in the phagocytic and non-phagocytic macrophages incubated with non- or DOX- or MAG- or D&M-treated A673 cells. Macrophages alone (no A673) served as a control. *p < 0.05, **p < 0.01, ***p < 0.001 (Fisher’s Exact test). Shown are results in a representative experiment. The same trend was seen in three independent biological repeats

Article Snippet: Antibodies used include α-CD47 (B6H12, ThermoFisher Scientific, 11–0479-42), α-Calreticulin (ThermoFisher Scientific, PA3-900), α-CD11b (Miltenyi Biotec, 130–110-554), α-CD14 (R&D Systems, FAB3832P-025), α-CD38 (R&D Systems, FAB2404P), α-CD68 (BioLegend, 333,819), α-CD80 (BioLegend, 305,220), α-CD163 (R&D Systems, FAB1607P-025), α-CD206 (R&D Systems, FAB25342P), α-CD209 (R&D Systems, FAB161P-025), Alexa Fluor 488 donkey α-rabbit IgG (Invitrogen, A21206).

Techniques: Expressing, In Vitro, Phagocytosis Assay, Labeling, Staining, Flow Cytometry, Comparison, Incubation, Control