Integrated cellular 4D-TIMS lipidomics and transcriptomics for characterization of anti-inflammatory and anti-atherosclerotic phenotype of MyD88-KO macrophages

<b>Introduction:</b> Recent progress in cell isolation technologies and high-end omic technologies has allowed investigation of single cell sets across multiple omic domains and a thorough exploration of cellular function and various functional stages. While most multi-omic studies focused on dual RNA and protein analysis of single cell population, it is crucial to include lipid and metabolite profiling to comprehensively elucidate molecular mechanisms and pathways governing cell function, as well as phenotype at different functional stages. <b>Methods:</b> To address this gap, a cellular lipidomics and transcriptomics phenotyping approach employing simultaneous extraction of lipids, metabolites, and RNA from single cell populations combined with untargeted cellular 4 dimensional (4D)-lipidomics profiling along with RNA sequencing was developed to enable comprehensive multi-omic molecular profiling from the lowest possible number of cells. Reference cell models were utilized to determine the minimum number of cells required for this multi-omics analysis. To demonstrate the feasibility of higher resolution cellular multi-omics in early-stage identification of cellular phenotype changes in pathological and physiological conditions we implemented this approach for phenotyping of macrophages in two different activation stages: MyD88-knockout macrophages as a cellular model for atherosclerosis protection, and wild type macrophages. <b>Results and Discussion:</b> This multi-omic study enabled the determination of the lipid content remodeling in macrophages with anti-inflammatory and atherosclerotic protective function acquired by MyD88-KO, hence expedites the understanding of the molecular mechanisms behind immune cells effector functionality and of possible molecular targets for therapeutic intervention. An enriched functional role of phosphatidylcholine and plasmenyl/plasmalogens was shown here to accompany genetic changes underlying macrophages acquisition of anti-inflammatory function, finding that can serve as reference for macrophages reprogramming studies and for general immune and inflammation response to diseases.