Abstract P3094: Cell-autonomous Immune Signaling In Human Cardiac Fibroblasts Modulates Fibrosis

Abstract

Cardiac fibrosis is the pathophysiological hallmark of injury in an adult heart. Immune response in the injury niche modulates fibrosis. However, how cell-autonomous immune signaling in cardiac fibroblasts modulates fibrosis is unknown. In this report, we demonstrate that cardiac fibroblasts express TLR4 receptors, and inhibition of TLR4 signaling inhibits fibrotic changes such as proliferation and myofibroblast conversion in human cardiac fibroblasts. Using FACS, and single-cell data of cardiac fibroblasts from Colagen1 GFP mouse, we demonstrate that TLR4 is the predominant TLR and approximately 90 % of human cardiac fibroblasts express TLR4 receptors. Using EdU labeling, cardiac fibroblasts taking up EdU have reduced from 91.65 ± 3.89% from control conditions to 71.15 % ± 2.05 % (mean ± SEM, n=3, p< 0.05) in TLR4 inhibited condition. Ki67 staining shows a similar reduction from 93.1 ± 2.34 % to 59.3% ± 3.54%. TLR4 inhibition in TGF-β treated human cardiac fibroblasts reduced the expression of Periostin expression by 2.6 ± 0.58 and Sma1 by 1.46-± 0.18(mean ± SEM, n=3)fold. Further, migration of cardiac fibroblasts demonstrated a 76.3± 10.23 % (mean ± SEM, n=3, p< 0.05) reduction in the presence of TLR4 inhibitor. Transcriptome analysis of TLR4-inhibited cardiac fibroblasts shows downregulation of proliferation-related genes and pathways. Ingenuity pathway analysis (IPA) of differentially expressed genes in TLR4-inhibited TGF-β -treated cells compared to TGF-β alone shows a reduced expression of a subset of target genes of TGF-β signaling. Further, compared to healthy controls, single-cell data of heart failure patients show significant elevation of Histone 3 and Histone 4 in the fibroblast cluster along with chromatin readers, Brd4 and Nox4. Inhibition of TLR4 in human cardiac fibroblasts reduced the expression of Histone 3 and Histone 4, with Nox4 modulating the chromatin architecture required for fibroblast activation. Thus our data demonstrate that cardiac fibroblast act as an immune cell having cell-autonomous immune signaling, regulating fibrotic changes. Ongoing experiments in 3D human cardiac organoid injury models will reveal how TLR4 signaling regulates fibrotic activation of human cardiac fibroblasts in a heterogeneous system.