Journal: Scientific Reports

Article Title: Suppression of CSF2RA macrophage polarisation impacts pathological cardiac remodelling in mice

doi: 10.1038/s41598-025-33936-1

Figure Lengend Snippet: CSF2RA inhibition suppresses CSF2 signalling and associated macrophage behaviour. (A) Quantification and representative images of human monocyte-derived macrophages polarised with CSF2 (20 ng/mL) and treated for 4-hours with a CSF2RA peptide inhibitor (E21R) at 0.1, 1, and 10 µg/mL for pSTAT5. Comparative mRNA expression of (B) MMP12 and (C) MMP7 in CSF2 polarised macrophages treated for 24-hours with a CSF2RA peptide inhibitor (E21R) at 0.1, 1, and 10 µg/mL. Quantification and representative images of CSF2-polarised macrophages treated for 24-hours with a CSF2RA peptide inhibitor (E21R) at 0.1 µg/mL for: (D) invasion as assessed through invasion within Matrigel-coated transwells, (E) FasL-induced apoptosis, as assessed through cleaved caspase-3 immunocytochemistry (green), and (F) proliferation, as assessed through EdU incorporation (green). All groups are n = 4. Statistical significance is reported as * P < 0.05, ** P < 0.01, or *** P < 0.001, using an ordinary one-way ANOVA in panels A-C, and paired Student’s t-test in panels D-F. Scale bar represents 200 μm and is applicable to panels E and F.

Article Snippet: Monocytes were cultured for 7 days with the appropriate CSFs: 40 ng/mL CSF1 (M-CSF) (catalogue number 130-096-491, Miltenyi Biotec) or 20 ng/mL CSF1 (M-CSF) and 20 ng/mL CSF2 (GM-CSF) (catalogue number 130-095-372, Miltenyi Biotec), to differentiate into CSF1 (M-) or CSF2 (GM-) macrophage subsets, respectively.

Techniques: Inhibition, Derivative Assay, Expressing, Immunocytochemistry

Journal: Scientific Reports

Article Title: Suppression of CSF2RA macrophage polarisation impacts pathological cardiac remodelling in mice

doi: 10.1038/s41598-025-33936-1

Figure Lengend Snippet: CSF2 polarised macrophages sustain a cardiac myofibroblast phenotype, alongside increased migratory and contractile capacity. Human cardiac fibroblasts and human primary + CSF2 or -CSF2 macrophages (green) were co-cultured for 24 h, and fibroblast activation hallmarks were assessed. Quantification and representative images of (A) cardiac fibroblast proliferation, assessed through EdU incorporation (red; n = 5); (B) activation marker, α-SMA (red; n = 6). Human cardiac fibroblasts were treated with macrophage secretome with or without CSF2 polarisation (CM) for 24 h, unless otherwise stated. Quantification and representative images of cardiac fibroblast proliferation, assessed through (C) EdU incorporation (green; n = 5), (D) phospho-RB and cyclin D1 protein expression ( n = 3), and (E) CCND1 copy number ( n = 5); activation marker, α-SMA, assessed through (F) immunocytochemistry (red; n = 7), (G) α-SMA protein expression ( n = 6), and (H) ACTA2 copy number ( n = 3). (I) Cardiac fibroblast contraction was assessed through collagen gel contraction ( n = 4), and (J) migration was assessed through scratch-wound assay for 48 h ( n = 4). Statistical significance is reported as * P < 0.05 or ** P < 0.01, using paired Students t-test. White scale bar represents 200 µM and is applicable to panels A-C and F; yellow scale bar represents 1 mm and is applicable to panel J; panel I shows 24-well plates.

Article Snippet: Monocytes were cultured for 7 days with the appropriate CSFs: 40 ng/mL CSF1 (M-CSF) (catalogue number 130-096-491, Miltenyi Biotec) or 20 ng/mL CSF1 (M-CSF) and 20 ng/mL CSF2 (GM-CSF) (catalogue number 130-095-372, Miltenyi Biotec), to differentiate into CSF1 (M-) or CSF2 (GM-) macrophage subsets, respectively.

Techniques: Cell Culture, Activation Assay, Marker, Expressing, Immunocytochemistry, Migration, Scratch Wound Assay Assay

Journal: Scientific Reports

Article Title: Suppression of CSF2RA macrophage polarisation impacts pathological cardiac remodelling in mice

doi: 10.1038/s41598-025-33936-1

Figure Lengend Snippet: Loss of CSF2 macrophage polarisation promotes CXCL10 expression and CXCR3-induced cardiac fibroblast dedifferentiation, while CSF2-macrophage CTSZ degrades CXCL10. Validation of CTSZ and CXCL10 expression in (A) + CSF2 or -CSF2 macrophage secretome ( n = 7 and 4), and (B) cell lysates ( n = 7 and 8). Quantification and representative images of α-SMA assessed through immunocytochemistry (red in panel C, green in panel F): (C) supplementing CXCL10 protein (50 ng/mL) in the + CSF2 secretome ( n = 10); (D) effect of CXCL10 protein (50 ng/mL) and AMG487 (1 µM), a CXCR3 inhibitor, on naïve cardiac fibroblasts ( n = 4); (E) effect of CXCR3 inhibition, through AMG487 (1 µM) on -CSF2 secretome effect on cardiac fibroblast α-SMA expression ( n = 4); (F) inhibition of CTSZ protein (4 µg/mL) in the + CSF2 secretome ( n = 6, IgG n = 4); (G) effect of CTSZ protein (6 ng/mL) ( n = 3) or (H) inhibition of CTSZ protein (4 µg/mL) on naïve cardiac fibroblasts ( n = 3); (I) supplementing CTSZ protein (6 ng/mL) in macrophage secretome ( n = 4). (J) CTSZ cleavage of CXCL10 protein ( n = 4) and (K) effect on cardiac fibroblast α-SMA expression, assessed through immunocytochemistry ( n = 5). (L) Proposed CTSZ/CXCL10-mediated mechanism in crosstalk between pro-inflammatory (CSF2) or pro-fibrotic (CSF1) macrophages and cardiac fibroblasts; generated using bioRender ( https://www.biorender.com ). Statistical significance is reported as * P < 0.05, ** P < 0.01, or *** P < 0.001, using paired Students t-test to panels A, B, and J; ordinary one-way ANOVA, to panels C-F, and I, or repeated measures one-way ANOVA, to panel K, with Tukey’s multiple comparisons post-hoc. Scale bar represents 200 µM and is applicable to panels C and F.

Article Snippet: Monocytes were cultured for 7 days with the appropriate CSFs: 40 ng/mL CSF1 (M-CSF) (catalogue number 130-096-491, Miltenyi Biotec) or 20 ng/mL CSF1 (M-CSF) and 20 ng/mL CSF2 (GM-CSF) (catalogue number 130-095-372, Miltenyi Biotec), to differentiate into CSF1 (M-) or CSF2 (GM-) macrophage subsets, respectively.

Techniques: Expressing, Biomarker Discovery, Immunocytochemistry, Inhibition, Generated