{"id":359667,"date":"2023-11-24T19:00:00","date_gmt":"2023-11-25T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/il-1%ce%b2-mediated-adaptive-reprogramming-of-endogenous-human-cardiac-fibroblasts-to-cells-with-immune-features-during-fibrotic-remodeling-communications-biology\/"},"modified":"2023-11-25T00:00:35","modified_gmt":"2023-11-25T05:00:35","slug":"il-1%ce%b2-mediated-adaptive-reprogramming-of-endogenous-human-cardiac-fibroblasts-to-cells-with-immune-features-during-fibrotic-remodeling-communications-biology","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/il-1%ce%b2-mediated-adaptive-reprogramming-of-endogenous-human-cardiac-fibroblasts-to-cells-with-immune-features-during-fibrotic-remodeling-communications-biology\/","title":{"rendered":"IL-1\u03b2-mediated adaptive reprogramming of endogenous human cardiac fibroblasts to cells with immune features during fibrotic remodeling – Communications Biology","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
Although several inflammatory subsets have been identified in the myocardium, the role of resident cardiac fibroblasts in the promotion of inflammation remains unclear. Recent studies in human cardiac fibroblasts isolated from patients with heart failure show that these cells are immunocompetent24<\/a><\/sup>. We sought to characterize pro-immune features of cardiac fibroblasts through immunophenotyping with multiparametric mass cytometry, a method that categorizes cell populations into lineages based on marker expression (Fig.\u00a01a<\/a>). We also independently established the identity of commercially sourced and validated human primary ventricular cardiac fibroblasts (hVCF), with multiple fibroblast markers \u03b1SMA, vimentin, collagen-1, FSP-1, PDGFR\u03b2, periostin and endothelial cell marker, VE-cadherin27<\/a><\/sup>. While >90% of the cells were positive for all fibroblast markers and negative for the endothelial cell marker, VE-cadherin, <2% PDGFR\u03b2-positive cells were negative for FSP-1, and some FSP-1-positive cells were negative for PDGFR\u03b2 (Supplementary Fig.\u00a0S1a<\/a>)28<\/a>,29<\/a>,30<\/a>,31<\/a><\/sup>. FSP-1 is also expressed by immune cells and endothelial cells and therefore is not a specific fibroblast marker32<\/a>,33<\/a>,34<\/a><\/sup>. It has been suggested that all myeloid and lymphoid cells are bone marrow-derived and play a prima facie role in inflammation, repair and tissue regeneration35<\/a><\/sup>. However, recent evidence suggests that tissue-resident cells including CCR2+<\/sup> macrophages may, in fact, initiate inflammatory processes36<\/a><\/sup>. In the setting of myocardial injury, cardiac fibroblasts may epigenetically assume a pro-inflammatory phenotype and secrete cytokines, whereas chemokines may recruit macrophages and lymphocytes before the arrival of the bone marrow immune cells3<\/a><\/sup>. By staining single-cell suspensions of 3 human male and female donors (all clinical information is provided in Supplementary Table\u00a0S1<\/a>) immune lineage markers in the CYTOF panel (Supplementary Table\u00a0S2<\/a>), we identified in all human donors a subpopulation of cardiac fibroblasts phenotypically distinct from the central cluster of homogenous cells, which expresses Vimentin, \u03b1SMA and CD4, a hallmark T-helper cell marker (Fig.\u00a01b<\/a>; red dotted lines, Supplementary Fig.\u00a0S2a\u2013d<\/a>). Manual gating of the isolated population of cells and re-application of the viSNE algorithm to qualitatively visualize the expression of markers at a single-cell level provided high-resolution maps of the cardiac myofibroblast markers, \u03b1SMA and Vimentin (Fig.\u00a01b<\/a>), along with lymphoid lineage markers, CD4, CCR6, and CD183 (Fig.\u00a01c<\/a>). Myeloid CD68+<\/sup> cells (<6%) and HLA-DR+<\/sup> dendritic cells (<0.8%) were also identified to a lesser extent (Supplementary Fig.\u00a0S8<\/a>). Consistent with the viSNE algorithm, a uniform manifold approximation and projection (UMAP) non-linear dimensionality reduction algorithm also showed the same distinct cell population (Fig.\u00a01d<\/a>). This approach profiled human cardiac fibroblast cells in isolation as opposed to bulk transcriptomic profiling that can miss smaller populations37<\/a>,38<\/a><\/sup>. Measures were taken to ensure that the populations being analyzed were free from beads and only live, nucleated single cells were manually gated, as presented in Supplementary Fig.\u00a0S1b, c<\/a>.<\/p>\n Immunophenotyping of hVCFs derived from human subjects (2 males (ID# 62122, ID# 1281202) and 1 female (ID#534282)) was performed by staining 3 \u00d7 106<\/sup> cells with heavy metal-tagged antibodies, followed by mass cytometry to identify frequencies of populations in human cardiac fibroblasts. a<\/b> 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<\/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 from the majority of cardiac fibroblasts marked by the expression of Vimentin and \u03b1SMA+<\/sup>. c<\/b> The hVCFs expressing cluster of differentiation (CD) CD4, CCR6, and CD183 associated with lymphoid cells on hVCF. Protein expression levels are demonstrated on the secondary y<\/i>-axis scale with blue showing no expression, green, the least expression, and red, the highest expression. Each dot represents the expression profile of a single cell. The red box shows the newly identified population separated from the rest of the cell cluster. Bottom panel of (b<\/b>) and (c<\/b>) are the viSNE of viSNE after gating the newly identified population. d<\/b> UMAP-based clustering of the cells showing the distinct population of cells isolated from the main cluster of homogenous cardiac fibroblast cells. e<\/b> Representative flow cytometry histograms showing frequencies of CD4+<\/sup> subset of \u03b1SMA+<\/sup>Vim+<\/sup> hVCFs for all the human donor cells. f<\/b> Quantification of CD4+<\/sup> CD3\u2212<\/sup> cells gated from Vimentin+<\/sup>\u03b1SMA\u2212<\/sup>, Vimentin\u2212<\/sup>\u03b1SMA+<\/sup> and Vimentin+<\/sup> \u03b1SMA+<\/sup> populations is represented as a scatter plot of Mean\u2009\u00b1\u2009SD (n<\/i>\u2009=\u20093 biological replicates, 2 males and 1 female); *P<\/i>\u2009<\u20090.05, as determined using one-way ANOVA and Tukey\u2019s post-hoc multiple comparison. g<\/b> Representative hVCF cells immunostained CD4, Vimentin and DAPI antibodies showing co-expression of Vimentin and CD4. Scale bars are 20\u2009\u00b5m.<\/p>\n<\/div>\n<\/div>\n Quantification of the cells showed that 29.6% of the total cells were Vimentin+<\/sup>\u03b1SMA+<\/sup>CD4+<\/sup>, 11.91% \u00b1 2.32% cells of the total cells were Vimentin+<\/sup>\u03b1SMA\u2212<\/sup>CD4+<\/sup> resident cardiac fibroblasts, and 10.53% \u00b1 2% of the total cells were \u03b1SMA+<\/sup>Vimentin\u2212<\/sup>CD4+<\/sup> activated cardiac fibroblasts (Fig.\u00a01e, f<\/a>) indicating that this cell population expresses different levels of \u03b1SMA or Vimentin or CD4 or their combinations. The data for the remaining donors are presented in Supplementary Figs.\u00a0S4<\/a>\u2013S7<\/a>. Finally, we validated the expression of CD4 in Vimentin+<\/sup> primary human cardiac fibroblast cells by immunostaining and found a punctate staining pattern of CD4 in one or two Vimentin-positive cells (Fig.\u00a01g<\/a>). To eliminate ambiguity associated with staining artifacts, CD4 antibody specificity was tested by immunostaining of rat spleen sections and blood samples using flow cytometry (Supplementary Fig.\u00a0S3c<\/a>). To exclude the possibility of contaminating T-cells, the cells were cultured in fibroblast growth media for at least two to three passages. Besides size-based exclusion, strongly adherent cardiac fibroblasts were washed several times to ensure that any loosely adhered immune cells were removed. Based on these data, we report the presence of a resident primary human ventricular cardiac fibroblast subpopulation that co-expresses mesenchymal vimentin+<\/sup>\u03b1SMA+<\/sup> and the helper T-cell marker, CD4.<\/p>\n Following the identification of a CD4+ human ventricular cardiac fibroblast (hVCF) subpopulation in vitro using mass cytometry, we determined the clinical relevance of this population by mapping the distribution and expression of \u03b1SMA+<\/sup>CD4+<\/sup> cells in autopsy specimens of patients diagnosed with RV fibrosis and PAH. The clinical history and autopsy diagnosis of all the donors designated as either the PAH group or the no PAH group are presented in Supplementary Table\u00a0S4<\/a>. Global tissue analysis with Hematoxylin and Eosin (H&E) staining showed variable amounts of cardiac myocyte hypertrophy and age-associated fibrosis in interstitial, perivascular, and subendocardial locations in all donors (Fig.\u00a02a<\/a>). Sirus red staining identified specific collagen-rich regions and bands of interstitial and subendocardial fibrosis surrounding the myocytes (Fig.\u00a02a<\/a>). The posterior papillary muscle of the RV showed interstitial fibrosis typical of fibrotic remodeling. The endocardial surfaces of the RV showed hypertrophic changes with big \u201cboxcar\u201d nuclei, hypertrophic cardiac myocytes, subendocardial and interstitial fibrosis, with entrapped cardiac myocytes in the areas of fibrosis (Fig.\u00a02a<\/a>). To determine the localization of \u03b1SMA+<\/sup>CD4+<\/sup> cells in the diseased RV, we co-stained formalin-fixed tissues using human-specific \u03b1SMA and CD4 antibodies. We noted \u03b1SMA cells with a spindle-shaped morphology and membranous CD4 staining in the perivascular connective tissue in cases with minimal interstitial fibrosis and in the dense fibrous tissue but not in non-fibrous tissue (Fig.\u00a02a<\/a>). Moreover, we found significant increases in spindle-shaped cardiac fibroblasts expressing both \u03b1SMA and CD4 in the fibrotic regions compared to the nonfibrotic regions of preserved autopsy RV tissue in humans with a clinical diagnosis of RV hypertrophy and dilation, compared to RVs from those without an autopsy diagnosis of PAH (Fig.\u00a02b<\/a>). Cells with a round shape and membranous CD4 staining typical of traditional T-cells were predominantly identified inside capillaries (Fig.\u00a02b<\/a>). Human autopsy tissue is relevant for understanding fibrotic features that phylogenetically distant rodent models fail to fully replicate39<\/a><\/sup>. We generally observed a higher frequency of cell populations expressing \u03b1SMA and CD4 in the human fibrotic tissue compared to tissue obtained from rats and mice having a smaller proportion of cardiac fibroblasts compared to humans.<\/p>\n a<\/b> 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+<\/sup> expressing spindle-shaped cells in the RV of donor tissue was determined; arrows point to CD4+<\/sup> spindle-shaped cells. b<\/b> Magnified images of Sirus red staining, CD4 staining (brown) and CD4\/\u03b1SMA dual-staining in human RV. Arrows indicate the spindle-shaped cells expressing CD4. Quantification of CD4+<\/sup>\u03b1SMA+<\/sup> cells in nonfibrotic and fibrotic regions in the human RV determined by a pathologist blinded to the groups. Scale bars, 75\u2009\u00b5m. *P<\/i>\u2009<\u20090.05, one-way ANOVA and Tukey\u2019s multiple comparison test. Quantification of CD4+<\/sup>\/\u03b1SMA+<\/sup> manually by a double-blinded observer from at least 15\u201320 sections\/slide for n<\/i>\u2009=\u20096 samples (*P<\/i>\u2009<\u20090.05), one-way ANOVA and Tukey\u2019s multiple comparison test; Nonfibrotic vs<\/i>. Fibrotic and no PH vs<\/i>. PH. Scale bars are 10\u2009\u00b5m.<\/p>\n<\/div>\n<\/div>\n Genetic lineage tracing studies show that resident cardiac fibroblasts derived from a subset of endocardial cells through endoMT mediate pressure overload-induced fibrosis in mice31<\/a><\/sup>. To determine the role of \u03b1SMA+<\/sup> CD4+<\/sup> co-expressing cells in myocardial fibrosis, we utilized Fischer rats genetically prone to develop PAH as a model of inducible cardiac fibrosis40<\/a><\/sup>. Fischer rats were treated with a single intraperitoneal injection of the VEGF inhibitor SUGEN (25\u2009mg\/kg) and then subjected to 3 weeks of hypoxia (Hx), followed by 5 weeks of normoxia (Nx) (Fig.\u00a03a<\/a>). We noted significant increases in the expression of Collagen I\/III in both perivascular and interstitial regions in rat RV and LV sections, as determined by Sirius red staining and Masson trichrome staining (Fig.\u00a03b<\/a>). Control animals were injected with vehicle and housed in room air or Nx for 8 weeks. The SUGEN\/Hypoxia rats had lower body weights compared to control rats (Fig.\u00a03c<\/a>). Fulton index measured from the RV and LV weights using the formula (RV\/LV+S) was not significantly increased (Fig.\u00a03d<\/a>), however, tricuspid annular plane systolic excursion (TAPSE) shows a trend toward reduction in SUGEN\/Hypoxia-treated rats (Fig.\u00a03e<\/a>). None of the other echocardiographic parameters measured, such as cardiac output, ejection fraction, and mean pulmonary arterial pressure, differed significantly from controls. Further, changes in cardiac fibroblast number and localization in response to SuHx treatment were determined using the fibroblast marker FSP-1 (Supplementary Fig.\u00a0S10a, b<\/a>).<\/p>\n a<\/b> Schematic representation of SUGEN\/hypoxia for simulating PH in rats. Male Fischer rats were given a single bolus of SUGEN (20\u2009mg\/kg) and exposed to hypoxia (10% FiO2<\/sub>) 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<\/i>\u2009=\u200910 and n<\/i>\u2009=\u200912 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<\/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\u2009\u00b5m 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 bars are 10\u2009\u00b5m. c<\/b> Body weight of Nx and SuHx in grams is represented. d<\/b> Fulton Index of Nx and SuHx is represented. Values are mean\u2009\u00b1\u2009SD (n<\/i>\u2009=\u200910 rats), unpaired t<\/i>-test. e<\/b> TAPSE of Nx and SuHx animals measured using echocardiography. Values shown as the mean\u2009\u00b1\u2009SD (n<\/i>\u2009=\u200910), unpaired t<\/i>-test. f<\/b> 2D plots depicting cell cytometry of CD3\u2212<\/sup>CD4\u2212<\/sup>, CD3+<\/sup>CD4\u2212<\/sup>, CD3\u2212<\/sup>CD4+<\/sup>, and CD3+<\/sup>CD4+<\/sup> in cardiac fibroblasts following induction of hypoxia in the RV and LV of Nx and SuHx animals, with CD3 shown on the vertical axis and CD4 shown on the horizontal axis. The specific CD3+<\/sup>CD4\u2212<\/sup> population is encircled by an interrupted red line on each graph. g<\/b> Quantification of CD3+<\/sup>CD4\u2212<\/sup> cells in the RV and LV under Nx and SuHx conditions derived from cell cytometry is shown. h<\/b> \u03b1SMA+<\/sup>CD4+<\/sup> cardiac fibroblast cells following induction of hypoxia protocol in the right and left ventricle of Nx and SuHx animals. i<\/b> High magnification of the perivascular region of Nx, SuHx, and SuHx (x 40) RV tissue expressing \u03b1SMA+<\/sup> (red), CD4+<\/sup> (green), and DAPI (blue). Collagen I staining shows the collagen expression in the fibrotic regions of Nx and SuHx ventricle (Right panel). Scale bar is 10\u2009\u03bcm.<\/p>\n<\/div>\n<\/div>\n To determine the percentage of cardiac fibroblasts expressing CD4 in the right and left ventricle of rat hearts in response to SUGEN\/Hypoxia (SuHx), we quantified CD3+<\/sup> CD4\u2212<\/sup> and CD3+<\/sup>CD4+<\/sup> primary cardiac fibroblasts populations isolated from the RV and LV of Nx and SuHx rats using flow cytometry (Fig.\u00a03f<\/a>). CD3+<\/sup>CD4\u2212<\/sup> cardiac fibroblast populations were elevated in RV under both Nx (16.39% \u00b1 14.06%) and SuHx conditions (17.24% \u00b1 15.46%) compared to CD3+<\/sup>CD4+<\/sup> and were found to be less prominent in LV, under both Nx (2.10% \u00b1 0.71%) and SuHx conditions (9.41% \u00b1 6.04%) (Fig.\u00a03g<\/a>). In the LV, the percentages of CD3+<\/sup> CD4\u2212<\/sup> cardiac fibroblasts were higher in the SuHx conditions and trending toward significance compared to Nx conditions (2.10% \u00b1 0.71% vs 9.41% \u00b1 6.04%, P<\/i>\u2009=\u20090.053). Percentages of CD3+<\/sup>CD4+<\/sup> populations expressing cardiac fibroblast cells showed some increase in RV (5.79% \u00b1 4.92%) under SuHx conditions in comparison to Nx, (5.50% \u00b1 5.75%) although it did not reach significance (Fig.\u00a03g<\/a>). In the RV, there is no change in the percentage of CD3+<\/sup> CD4\u2212<\/sup> cardiac fibroblast population after treatment with SuHx. In the LV, there is an increase in the percentage of CD3+<\/sup> CD4\u2212<\/sup> cardiac fibroblast population after treatment with SuHx (Fig.\u00a03g<\/a>). Next, we quantified the number of cardiac fibroblasts co-expressing \u03b1SMA+<\/sup>CD4+<\/sup> cells that accompanied fibrotic changes in the RV of Nx and SuHx rats using confocal microscopy (Fig.\u00a03h<\/a>). We found three cell populations: T-cells (\u03b1SMA\u2212<\/sup> CD4+<\/sup>), cardiac fibroblasts (\u03b1SMA+<\/sup>CD4\u2212<\/sup>), and cardiac fibroblasts expressing CD4 (\u03b1SMA+<\/sup>CD4+<\/sup>) in rat RV (Fig.\u00a03i<\/a>). Two distinct morphologies of conventional CD4 cells, round \u03b1SMA+<\/sup>CD4+<\/sup> and spindle-shaped \u03b1SMA+<\/sup>CD4+<\/sup> were noted in the RV regions, both which were significantly increased in SuHx compared to Nx rats (Fig.\u00a03i<\/a>). The number of \u03b1SMA+<\/sup>CD4+<\/sup> cells tended to be higher in the perivascular fibrotic regions of the RV of SuHx rats compared to control.<\/p>\n Identification of CD4-expressing cardiac fibroblasts in both human and rat RV prompted us to explore possible mechanisms underlying the induction of CD4 expression in cardiac fibroblasts. PAH patients have high levels of plasma IL-1\u03b2 that correlate with the severity of PAH41<\/a>,42<\/a><\/sup>, and reduction of IL-1\u03b2 reduces inflammation and improves right heart function42<\/a><\/sup>. Based on these and related findings43<\/a>,44<\/a>,45<\/a>,46<\/a><\/sup>, we postulated that the pro-inflammatory cytokine, IL-1\u03b2, contributes to the induction of CD4 expression in resident cardiac fibroblasts. Our single-cell mass cytometry profiles showed an increase in immunocompetent cardiac fibroblast sub-populations with recombinant IL-1\u03b2 suggesting a role of IL-1\u03b2 in cardiac fibroblast re-phenotyping (Supplementary Fig.\u00a0S8a<\/a>). Further, CD4 expression varied with IL-1\u03b2 treatment specifically for male donors (Supplementary Fig.\u00a0S8b<\/a>). The non-redundancy scores suggested that CD4 is expressed at levels similar to those of vimentin and \u03b1SMA, markers of cardiac fibroblasts (Supplementary Fig.\u00a0S8c<\/a>). The percentages of HLA-DR+<\/sup> dendritic cell, CD4+<\/sup> lymphocyte, and CD68+<\/sup> monocyte populations also shifted in response to IL-1\u03b2 treatment (Supplementary Fig.\u00a0S8d<\/a>). \u03b1SMA+<\/sup> and CD4+<\/sup> expressing cardiac fibroblast populations increased with IL-1\u03b2 treatment, as seen in the normalized density distribution plots (Supplementary Fig.\u00a0S8e<\/a>). The changes in the expression of specific immune cell lineage markers with IL-1\u03b2 treatment were shown as a heatmap for all three human donors (yellow is high expression and blue is low expression). The vertical pink and green boxes highlighted increases in marker expression in association with IL-1\u03b2. Based on the pattern of marker expression (X<\/i>-axis) for all proteins tested, a cell identity can be defined on the Y<\/i>-axis. As the pattern of expression has not previously been described in human cardiac fibroblasts, we have not labeled the lineages on the Y<\/i>-axis47<\/a><\/sup> (Supplementary Fig.\u00a0S9a<\/a>). However, identified the more common patterns for lymphoid lineages, myeloid lineages and memory T-cells and expressed our data in the form of SPADE visualization48<\/a><\/sup> (Supplementary Fig.\u00a0S9b\u2013f<\/a>).<\/p>\n To determine the mechanisms of IL-1\u03b2 mediated phase shifts in cardiac fibroblast phenotypes to immune cells, we studied IL-1\u03b2-mediated cell proliferation and differentiation. Inflammatory processes involving immune cell proliferation, recruitment, reprogramming, secretion of cytokines and chemokines are critical to the IL-1\u03b2-linked biological responses49<\/a><\/sup>. Previous reports suggest that IL-1\u03b2 is a paracrine growth factor for fibroblasts during intestinal fibrosis50<\/a><\/sup> and regulates collagenase expression51<\/a><\/sup>. To determine the proliferative and collagen regulation of IL-1\u03b2, hVCFs were treated with vehicle or recombinant IL-1\u03b2 for 24\u2009h, followed by BrdU and MTT assays and Ki67 staining. IL-1\u03b2 treatment resulted in an increase in cardiac fibroblast proliferation in a dose-dependent and time-dependent manner (Fig.\u00a04a, b<\/a>). IL-1\u03b2 similarly affected the proliferation rates of primary hVCF cells isolated from males and females (Fig.\u00a04c<\/a>). IL-1\u03b2 treatment tended to stimulate the deposition of Collagen I\u03b1, as determined from total collagen in the lysates (Fig.\u00a04d<\/a>) and confocal microscopy (Supplementary Fig.\u00a0S9g<\/a>); however, these effects were not statistically different. To determine whether IL-1\u03b2 differentiates cardiac fibroblasts and induces phenotypic changes, human hVCFs were treated with IL-1\u03b2 (10\u2009ng\/mL) for 96\u2009h, which resulted in the induction of significant morphological changes in hVCF cells by day 4 at this dose and time, including increased detachment and rounding of cells (Fig.\u00a04e<\/a>). Therefore we used IL-1\u03b2 dose of 10\u2009ng\/mL and 96\u2009h of treatment for all subsequent experiments. Round cells with nonhomogeneous DAPI-stained nuclei appeared with 96\u2009h of IL-1\u03b2 treatment (Fig.\u00a04e<\/a>). The round cells, but not the surrounding cells, were positive for both \u03b1SMA and CD4 T-cells (Fig.\u00a04e<\/a>). The round cells were viable and not apoptotic, as determined using trypan blue staining. Additionally, myeloid-specific CD68 positive clusters were also seen in IL-1\u03b2 hVCF but not in Veh (Fig.\u00a04f<\/a>). We sought to expand the number of cardiac fibroblasts with T-cell features by growing the cells in T-cell expansion media for 10 days with added IL-1\u03b2 (Supplementary Fig.\u00a0S14<\/a>). We noted the formation of cell clusters in all the human donors, suggesting that IL-1\u03b2 induced cell clustering from day 7 onward (Fig.\u00a04g<\/a>). We next characterized the subcellular phenotypic switching of cardiac fibroblasts with IL-1\u03b2 after 96\u2009h using transmission electron microscopy. Transmission electron microscopy showed more prominent endoplasmic reticulum and Golgi apparatus, suggesting activation and secretory transformation of cardiac fibroblasts in the presence of IL-1\u03b2 (Fig.\u00a04h<\/a>). Increased intracellular and budding extracellular microvesicles were seen in the IL-1\u03b2-treated cells immunostained with IL-1\u03b2 receptor (IL-1R) (Fig.\u00a04i<\/a>). Our data demonstrated that cardiac fibroblasts have the capacity to assume a secretory cell phenotype in the presence of IL-1\u03b2.<\/p>\n<\/a><\/div>\n
Distribution and expression of spindle-shaped \u03b1SMA+<\/sup> CD4+<\/sup>-co-expressing cells in the fibrotic right ventricle (RV) of patients with pulmonary arterial hypertension (PAH)<\/h3>\n
<\/a><\/div>\n
Distribution and expression of spindle-shaped \u03b1SMA+<\/sup> CD4+<\/sup> co-expressing cells in the fibrotic RV in rats treated with SUGEN\/hypoxia<\/h3>\n
<\/a><\/div>\n
Shifts in human cardiac fibroblast cell population lineages in response to recombinant IL-1\u03b2<\/h3>\n
IL-1\u03b2 proliferates and differentiates primary human cardiac fibroblasts into immunocompetent cells<\/h3>\n