Abstract
Background
Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs, long noncoding RNAs (lncRNAs) are implicated in inflammation control. To obtain further information on the role of lncRNAs in the context of atherosclerosis, we analyzed transcriptome maps of circulating immune cells (PBMCs) of post-MI patients in whom the lncRNA NEAT1 was suppressed. Here, we report immune disturbances in murine NEAT1 knockout models with wildtype or ApoE−/− genetic background.
Methods and results
RNA-sequencing (RNA-seq) of PBMCs from post-MI patients revealed profound transcriptome disturbances compared to healthy controls. Among these, NEAT1 suppression was notable since it affected the most highly expressed lncRNA as part of a molecular circuit also encompassing chemokines and interleukins.
We used NEAT1−/− mice to evaluate whether NEAT1 depletion per se may cause immune dysfunction. NEAT1−/− splenocytes displayed enhanced baseline ROS production, and RNA-seq identified anomalous expression and regulation of chemokines/ receptors, innate immunity genes, TNF and caspases. FACS revealed displayed anomalous Treg and TH cell differentiation in NEAT1−/− spleens vs. wildtype (WT).
Beyond grossly altered transcriptome, NEAT1−/− bone marrow derived macrophages (BMDMs) responded to LPS with increased (p<0.001) ROS production, enhanced baseline phagocytic activity (p<0.001), and attenuated proliferation (p=0.001). FACS revealed deregulated monocyte-macrophage differentiation in NEAT1−/− bone marrow and blood.
Further, NEAT1−/− mice displayed aortic wall CD68+ cell infiltration and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals. This observation suggests that even stochastic activation of the highly unstable NEAT1−/− immune system may trigger uncontrolled pathogenic cascades, explaining the survival disadvantage of NEAT1−/− mice.
In addition to these studies on homozygous NEAT1−/− deficiency in WT background, we obtained data on mice with partial i.e. heterozygous NEAT1−/+ deficiency on ApoE−/− background. Analysis of this new NEAT1−/+ ApoE−/− strain indicates that even partial NEAT1 deficiency leads to systemic inflammation with high IFN-gamma levels, when the animals are exposed to immune stress e.g. high LDL cholesterol.
Conclusions
Regarding the monocyte-enriched NEAT1 suppressed in post-MI PBMCs, the data from NEAT1−/− and NEAT1−/+ ApoE−/− mice document NEAT1 as a key immune system coordinator whose deficiency affects monocyte-macrophage and T cell differentiation and functions and renders the immune system unstable and highly vulnerable to immune stress. Since in patients NEAT1 is part of a molecular circuit persistently deregulated post-MI, too, it appears reasonable to further search for new therapeutic targets within this circuit, taking advantage of the described genetic animal models.
RNA-Seq Bayesian Network Exploration of Immune System in Bovine