Fig. 4: IL-1β-mediated proliferation, transdifferentiation and activation of primary human ventricular cardiac fibroblasts (hVCF) into CD4-expressing cells in vitro.

Primary hVCF cells (2.0 × 10⁶ cells/mL) from donors (2 males (ID# 62122, ID# 1281202) and 1 female (ID#534282)) were cultured in Fibroblast Basal Media (FBM) for 4 days with 10 ng/mL of IL-1β or in T-cell media with CD3/CD28 T-cell activator for 13 days. a hVCF proliferation in response to IL-1β exposure was assessed by BrdU incorporation. Data are Mean ± SD for 3 biological replicates. *P < 0.05, one-way ANOVA with multiple comparisons. b Cell proliferation response of hVCF to IL-1β after 24 h, 48 h and 72 h of incubation was assessed using MTT assay. Data are Mean ± SD for 3 biological replicates (n = 3–6 technical replicates). c Cell proliferation response to IL-1β determined by Ki67 staining of nuclei. Data are Mean ± SD for 3–4 biological replicates, P = 0.1, one-way Kruskal–Wallis ANOVA with Dunn’s multiple comparisons. d Collagen content in response to IL-1β doses (1 ng/mL and 10 ng/mL) measured in the cell lysates using the Sircol assay. The data are represented as Mean ± SD values from individual subjects (n = 4) biological replicates, no significance (ns), Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons. e Left Panel: Bright-field images of live hVCF cells indicating the shifts in cellular morphology from a spindle-shaped fibroblast to a round cell with a large nucleus in response to 96 h of treatment with IL-1β (10 ng/mL). Arrows indicate a change in morphology of hVCF with IL-1β treatment, but not with vehicle treatment. Right Panel. Immunostaining of fixed hVCF with lymphoid CD4 T-cell marker (green), mesenchymal αSMA (red) and DAPI (blue) markers to characterize the transformed cells. The arrow indicates the emergence of round, multinucleated αSMA⁺ CD4⁺ expressing cells. Representative magnified images of multinucleated notch-shaped giant cells immunostained with αSMA⁺ CD4. f Immunostaining of the cardiac fibroblast cell membrane with CD68, Vimentin, DAPI and Merge showing cell clustering phenotype with IL-1B at 40x magnification. g Differentiation of cardiac fibroblast in T-cell media with T-cell activators followed for 13 days. hVCF were treated with rIL-1β (10 ng/mL) in fibroblast media for 4 days. Cells were then transferred to T-cell expansion media with T-cell activators (CD3, CD2, CD28) for 10 days. Representative bright-field images taken day 3, day 6, day 7, day 9 and day 10 are presented sequentially. Cells start clustering by day 6 and form prominent circular colonies by day 7. Arrows show multicellular clusters. h Transmission electron microscopy micrograph of ultramicroscopic subcellular structures of the vehicle and IL-1β (10 ng/mL) 24 h treated hVCF. N Nucleus, ER Endoplasmic Reticulum, MV Secreted Microvesicle, G Golgi body, M Mitochondria. Scale bar, 400 nm. n = 3 technical replicates. i Representative immunostained images of Vimentin (green) and IL-1R expression (red) and distribution across the nucleus and cytoplasm of hVCF treated with Veh or IL-1β. The white dotted lines indicate the bright red puncta of IL-1R expression on hVCFs after 24 h treatment with IL-1β. Z stack images of IL-1β treated hVCFs were projected in 3D using image J. Arrows indicate overexpression and extracellular release of IL-1R.