IL-1β-mediated adaptive reprogramming of endogenous human cardiac fibroblasts to cells with immune features during fibrotic remodeling

Figures

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  Fig. 1. Identification of a subpopulation of resident hVCFs expressing dual mesenchymal and lymphoid cell surface markers with multidimensional single-cell mass cytometry. Immunophenotyping of hVCFs derived from human subjects (2 males (ID# 62122, ID# 1281202) and 1 female (ID#534282)) was performed by staining 3 × 106 cells with heavy metal-tagged antibodies, followed by mass cytometry to identify frequencies of populations in human cardiac fibroblasts. a Schematic summary of mass cytometry analysis. hVCFs were immunostained with epitope-specific antibodies conjugated to transition element isotope reporters of different masses. The cells were nebulized into single-cell droplets, followed by the acquisition of an elemental mass spectrum. Conventional flow cytometry methods were used to analyze integrated elemental reporter signals for each cell. This diagram was drawn exclusively by the authors using BioRender (Toronto, Canada) under the aegis of an academic license with Brown University. b viSNE graphs of manually gated live, nucleated, hVCF cell clusters expressing varying marker intensities and distribution for a representative donor showed a subpopulation of cells (island) distinct…

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  Fig. 2. Distribution and expression of CD4 in the right ventricle in human cardiac fibrosis. a Human right ventricular tissues from 5 autopsied donors diagnosed with other conditions (Donor A, C and E) or PH (Donor B, D and F) were stained with standard hematoxylin & eosin (H&E) and Sirius Red. Donors A-E were immunostained for CD4 and counterstained with methyl green to identify nuclei. Donor F was used as a negative control for CD4 immunostaining. The black arrows point to fibrotic areas in H&E-, Sirius Red- and CD4-stained sections. CD4+ expressing spindle-shaped cells in the RV of donor tissue was determined; arrows point to CD4+ spindle-shaped cells. b Magnified images of Sirus red staining, CD4 staining (brown) and CD4/αSMA dual-staining in human RV. Arrows indicate the spindle-shaped cells expressing CD4. Quantification of CD4+αSMA+ cells in nonfibrotic and fibrotic regions in the human RV determined by a pathologist blinded to the groups. Scale bars, 75 µm. *P < 0.05, one-way ANOVA and Tukey’s multiple comparison test. Quantification of CD4+/αSMA+ manually by a double-blinded observer from at least 15–20 sections/slide for n = 6 samples (*P < 0.05), one-way ANOVA and Tukey’s…

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  Fig. 3. Histological analysis of the right ventricle in the SUGEN/hypoxia rats. a Schematic representation of SUGEN/hypoxia for simulating PH in rats. Male Fischer rats were given a single bolus of SUGEN (20 mg/kg) and exposed to hypoxia (10% FiO2) for 3 weeks, followed by exposure to normoxia (Nx) for an additional 5 weeks (SuHx). In parallel, control animals were exposed to Nx (room air) for 8 weeks. The Nx and SuHx groups were comprised of n = 10 and n = 12 rats, respectively. Quantification of TAPSE trace measured using M-mode echocardiography is presented for Nx and SuHx rats. The Fulton index was determined from the weight of the RV and LV + septum. b (Top). Representative images of collagen content in the perivascular or interstitial RV and LV indicated by the Sirius Red-positive regions in the transverse region of Nx and SuHx rats (10 µm thickness), whole heart section cut transversely at the mid-ventral region and perivascular and interstitial regions of both Nx and SuHx animals. (Below) Representative images of Masson trichrome staining the whole heart section and perivascular and interstitial regions of both Nx and SuHx animals. Arrows suggest collagen I-rich regions. Scale…

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  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)…

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  Fig. 5. Primary human cardiac fibroblasts secrete cytokines, chemokines, immunomodulatory proteins in response to IL-1β. a Chemokine CCR2 and cytokine IL-1R gene expression performed in adult rat ventricular cardiac fibroblasts treated with Veh or IL-1β (10 ng/mL) after 24 h using quantitative PCR (qPCR). n = 3, *P < 0.05. b IL-8 and pro-differentiation TGFβ gene expression levels in primary hVCF treated with Veh or IL-1β (10 ng/mL) after 24 h using quantitative PCR (qPCR). n = 3, *P < 0.05. c Heatmap representation of secreted cytokines and chemokines (IL6, IL8, MCP-1, MCP-3, IL12p70, IL17A, IL10, 1L1a, IL1β, IL3, TNFa, VEGF, MIP1b, MIP1a and IL1RA) in the conditioned media after 24 h of treatment with Veh or IL-1β (10 ng/mL) profiled using a milliplex bead-based detection method. The values in the heatmap are expressed in Raw Z-score, ranging from blue (-2) to orange (+2). d Scatterplots of cytokines and chemokines (IL-6, IL-8, IL-10 and MCP-3) quantified in the condition media from Veh and IL-1β treated cardiac fibroblasts. The scatterplots represent Mean ± SD values (n = 3 biological replicates, *P < 0.05) by unpaired-student t-test with Mann–Whitney post hoc test. e GO Function…

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  Fig. 6. Genetic characterization of differentiated CD4+cardiac fibroblast cells. Primary hVCFs from 3 human donors (males and female) were differentiated for 10 days in T-cell media with or without IL-1 β (10 ng/mL) and flow-sorted into CD4+ and CD4− populations, followed by next-generation RNA sequencing using the Illumina platform. a Box plot of shifts in CD4+ human cardiac fibroblast population in response to IL-1 β. b Heatmaps showing the expression pattern of genes uniquely identified in Veh CD4+, Veh CD4−, IL-1 β CD4+ and IL-1β CD4− populations. c Volcano plot showing the number of significantly upregulated and downregulated genes for each of the comparisons. False discovery rate (FDR) (adjusted P-value < 0.01). d Venn diagram showing the number of common genes among the Veh and IL-1β among the unique genes identified for all the populations. e Summary of gene ontology hits using a database for annotation, visualization, and integrated discovery (DAVID) online platform showing the enrichment of genes involved in secretion, pluripotency, development, metabolism and T-cell activation and differentiation. Benjamini–Hochberg score of false discovery rate. f Validation of expression of…

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  Fig. 7. Lineage tracing of cell fate for endogenous cardiac fibroblasts following induction via IL-β administration. Lineage tracing of Pdgfrα+ cardiac fibroblasts was performed to confirm that these explicit cells become CD4-expressing cells in association with IL β1 induction. a Generation of the lineage tracing mouse model in which tamoxifen-dependent Cre recombinase is expressed under the influence of a Pdgfra promoter. Combined with a Cre-dependent tdTomato (red fluorescence) expressing reporter, the allele permanently labels all resident cardiac fibroblasts. b Experimental scheme in which PdgfraCreERT2/+; Rosa26tdT/tdT mice are given tamoxifen chow for two weeks prior to harvesting to tag cardiac fibroblasts in vivo. c Flow cytometric sorting of tdTomato labeled cardiac fibroblasts. Bracket shows the tdTomato positive population that is sorted and cultured. d Live red fluorescence imaging of cultured fibroblasts. Throughout all of the steps of the culturing protocol, tdTomato signal has been maintained due to the permanent recombination of the reporter allele. (Scale bar is 100 mm). e Flow cytometry analysis showing CD45+ and CD4+ phenotype acquisition of tdTomato+ fibroblasts…

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  Fig. 8. Proposed role of endogenous fibroblast transdifferentiation in the pathogenesis of right-sided heart failure due to myocardial fibrosis. We propose that resident cardiac fibroblasts undergo staged polarization/phenotypic switching in response to pro-inflammatory signals, such as IL-1β, to facilitate tissue repair and cell survival. This results in the transdifferentiation of endogenous fibroblasts to a secretory cell with a lymphoid cell-like phenotype that participates in the amplification and regulation of the inflammatory response through the secretion of immunomodulatory proteins, metabolites, cytokines, and chemokines. Enhancing IL-1β expression may enhance the participation of resident cardiac fibroblasts in the processes of cardiac inflammation and repair. This diagram was drawn exclusively by the authors using BioRender (Toronto, Canada) under the aegis of an academic license with Brown University.