Background: Acute inflammation leads to increased myelopoiesis in the bone marrow followed by recruitment of immune cells to respective sites of injury. Yet, in certain cases of high demand such as acute tissue damage as in acute myocardial infarction (AMI), innate immune cells can be additionally recruited from lymphoid organs such as the spleen. Monocytes as part of the innate immune system are important in the tight control of both onset and resolution of inflammation by comprising a huge repertoire of coding and non-coding RNA molecules regulating a fast-acting immune response. Long non-coding RNAs (lncRNAs) emerge as novel regulatory cell- and species-specific molecules, which are able to influence cell physiology in numerous ways and are involved in different disease onsets.

Purpose: Characterization of the non-coding transcriptome of monocyte subpopulations from patients with AMI compared to healthy controls including different isoform usage and associated source of origin.

Methods and Results: Next generation bulk RNA sequencing has been performed from human monocytes after pre-sorting for CD14 expression and subsequent separation into three major subpopulations based on surface markers CD14 and CD16. Non-coding RNA expression shows clear differences between cells from healthy controls versus patients with AMI and among subpopulations. Intensive bioinformatics analysis revealed numerous annotated lncRNAs, antisense, pseudogene and circular RNAs displaying significant different expression profiles. Among numerous differentially expressed targets we identified linc01272 to be significantly decreased in classical and non-classical monocytes of AMI patients whereas linc02207, TNFAIP3-AS and SAP30-DT display increased expression in these monocytes.

Some lncRNAs show positive correlations with their respective coding sense gene or their convergent or divergent sense neighbouring gene, such as linc02207 vs MCTP2, SAP30-AS vs SAP30 or TNFAIP3-AS vs TNFAIP3. We also identified the existence of different isoforms of certain lncRNAs and a distinct preference thereof, such as linc02207 with its 217 isoform being most abundant in monocytes.

Real-time PCR was applied for validation of lncRNAs expression profile and their nucleocytoplasmic distribution observing preferential nuclear expression for most lncRNA. Applying nanopore sequencing of target-enriched samples from THP1 cells allows the identification of respective isoforms. In vitro assays for silencing using gapmers and overexpression approaches as well as treatment with inflammatory stimuli, RAP-MS for identifying their binding partners and in silico bioinformatics analysis will help to unravel their functionality. Additionally an attempt to associate the origin of circulating monocytes to either spleen or bone marrow is taken by mapping our bulk RNA data to deconvoluted single cell RNAseq data from publicly available human cell atlas (HCA).

Conclusions: High read depth bulk RNA sequencing revealed a human monocyte-specific long non-coding RNA transcriptome differentially expressed in three predefined subpopulations and different cardiovascular disease stages. Distinct expression of lncRNAs and associated coding genes as well as specific isoform preferences imply different functionalities. Studying these molecules, their isoforms and their interaction partners will unravel new functional targets for the development of new therapeutic approaches against cardiovascular diseases.