Cardiac resident macrophages (CRMs) have recently gained recognition as a pivotal cell population in the heart, playing key roles in cardiovascular health and disease. Among various immune cell types, macrophages stand out as crucial regulators of cardiac remodelling and drivers of heart failure. While macrophages are known for their involvement in inflammation, the precise functions of CRMs remain elusive. Emerging evidence suggests their role in cardiac conduction, angiogenesis, and cardiomyocyte hypertrophy. While long non-coding RNAs (lncRNAs) have been recognized for their important role in resident macrophages in various organs, no functional analysis or in-depth characterization of lncRNAs in CRMs has been reported to date.
This study aims to elucidate the functional significance of lncRNAs in CRMs in health and disease. We selected 30 candidate lncRNAs based on their abundance in CRMs, their deregulation in mouse disease models, and their conservation in the human genome. To study these lncRNAs simultaneously, we employ CRISPR-droplet sequencing (CROP-Seq), a pooled CRISPR screen that allows to distinguish cells at single cell resolution based on their expressed sgRNA. LncRNAs that cause a significant macrophage phenotype are further validated using cell-based in vitro assays followed by in vivo experiments using mouse knockout (KO) strains and disease models.
We successfully performed three CROP-Seq experiments, which revealed seven candidates with intriguing transcriptomic phenotypes. One lncRNA, NIP16, exhibited a striking regulatory effect on members of the Clec4 gene family, pattern recognition receptors (PRRs) that detect microbial and damage-associated sugars, leading to immune system activation. RNA-seq analysis of NIP16 knockdown macrophages confirmed the pronounced deregulation of Clec4 genes, alongside other immune-relevant genes. Following stimulation with a mycobacterial-derived CLEC4D ligand, NIP16 KD macrophages demonstrated a reduced inflammatory response.
While originally identified for their role in antimicrobial defense, recent studies underline the role of CLEC4D and CLEC4E in sterile inflammation and inflammatory heart disease, including myocardial infarction. Therefore, ongoing work investigates the role of NIP16 in such contexts using KO mouse models. Given its conservation in human, we investigate NIP16’s therapeutic potential in hiPSC-derived macrophages. To this end, we established an iPSC differentiation platform and co-culture system with hiPSC-derived cardiac organoids, where macrophages promote regenerative phenotypes, such as enhanced endothelial cell formation. Upcoming experiments will assess how NIP16 deficiency alters regenerative processes within these organoids
In summary, we successfully established a high-throughput CROP-Seq platform that identified seven functionally relevant lncRNAs in macrophages. Among them, NIP16 emerged as a key regulator of the inflammatory response, especially regulating a cluster of C-type lectins, key to inflammatory signaling. NIP16 and other promising candidates will undergo further analysis, including in vivo- and iPSC-macrophage studies, to develop therapeutic strategies targeting lncRNAs for cardiovascular disease.
