Lysophospholipids induce innate immune transdifferentiation of endothelial cells, resulting in prolonged endothelial activation - PubMed (original) (raw)

Lysophospholipids induce innate immune transdifferentiation of endothelial cells, resulting in prolonged endothelial activation

Xinyuan Li et al. J Biol Chem. 2018.

Abstract

Innate immune cells express danger-associated molecular pattern (DAMP) receptors, T-cell costimulation/coinhibition receptors, and major histocompatibility complex II (MHC-II). We have recently proposed that endothelial cells can serve as innate immune cells, but the molecular mechanisms involved still await discovery. Here, we investigated whether human aortic endothelial cells (HAECs) could be transdifferentiated into innate immune cells by exposing them to hyperlipidemia-up-regulated DAMP molecules, i.e. lysophospholipids. Performing RNA-seq analysis of lysophospholipid-treated HAECs, we found that lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) regulate largely distinct gene programs as revealed by principal component analysis. Metabolically, LPC up-regulated genes that are involved in cholesterol biosynthesis, presumably through sterol regulatory element-binding protein 2 (SREBP2). By contrast, LPI up-regulated gene transcripts critical for the metabolism of glucose, lipids, and amino acids. Of note, we found that LPC and LPI both induce adhesion molecules, cytokines, and chemokines, which are all classic markers of endothelial cell activation, in HAECs. Moreover, LPC and LPI shared the ability to transdifferentiate HAECs into innate immune cells, including induction of potent DAMP receptors, such as CD36 molecule, T-cell costimulation/coinhibition receptors, and MHC-II proteins. The induction of these innate-immunity signatures by lysophospholipids correlated with their ability to induce up-regulation of cytosolic calcium and mitochondrial reactive oxygen species. In conclusion, lysophospholipids such as LPC and LPI induce innate immune cell transdifferentiation in HAECs. The concept of prolonged endothelial activation, discovered here, is relevant for designing new strategies for managing cardiovascular diseases.

Keywords: RNA-Seq; atherosclerosis; endothelial cell; immunometabolism; inflammation; lysophosphatidylcholine; lysophosphatidylinositol; lysophospholipid; metabolism; transdifferentiation.

© 2018 Li et al.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.

Figure 1.

RNA-Seq analysis reveals that LPI induces both transient and sustained endothelial cell activation. Human aortic ECs were treated with either vehicle control (Ctr) or LPI (10 μ

m

) for 18 h, and RNA-Seq experiments were performed. n = 3 in each group. A, volcano plot showing log(-fold change (FC)) and −log10(p value) of control versus LPI treatment. Red genes indicate genes significantly changed by more than 1.4-fold by LPI. B, heat map of genes that are significantly changed by more than 1.4-fold by LPI in ECs. C, the LPC–up-regulated genes from the top regulated pathway, cellular infiltration by leukocytes, are shown. Genes that are related to innate immunity are boxed. D–H, GSEA of the gene signatures that are significantly enriched in the LPI-treated EC group. I, representative gene expression changes in different categories corresponding to the GSEA plots.

Figure 2.

Figure 2.

LPI reprograms endothelial cell metabolism extensively besides up-regulating adhesion molecules and cytokines/chemokines in human aortic endothelial cells. Human aortic ECs were treated with either vehicle control or LPI (10 μ

m

) for 18 h, and RNA-Seq experiments were performed. The transcript level in units of transcripts per million (tpm) of the genes related to different categories, including EC adhesion molecules, cytokines/chemokines, and metabolic regulators, are shown. Box and whisker plot is shown for each sample (n = 100 bootstrap replicates from Kallisto). Lower and upper whiskers (error bars) indicate 25th and 75th percentiles, respectively.

Figure 3.

Figure 3.

RNA-Seq analysis reveals that LPC induces both acute and sustained endothelial cell activation. Human aortic ECs were treated with either vehicle control or LPC (10 μ

m

) for 18 h, and RNA-Seq experiments were performed. n = 3 in each group. A–E, GSEA of the gene signatures that are significantly enriched in the LPC-treated EC group. F, representative gene expression changes in different categories corresponding to the GSEA plots.

Figure 4.

Figure 4.

LPC positively regulates genes downstream of master regulator of lipid metabolism SREBP2. HAECs were challenged with LPC (10 μ

m

) for 18 h and RNA-Seq with IPA was performed. A, top regulated diseases or functions annotation of the genes that are significantly changed by LPC in HAECs. B, gene set enrichment analysis from the top LPI-regulated pathway “steroid biosynthesis” was shown. C, top upstream regulator analysis of the genes that are changed by LPC in HAECs predicted by the IPA. D, the 20 SREBP2-regulated genes that are significantly changed by LPC in HAECs. The red genes are induced by LPC and the blue genes are decreased by LPC.

Figure 5.

Figure 5.

LPI and LPC similarly activate mitochondrial ROS, cytosolic calcium, and acute EC activation marker genes but regulate largely distinct gene programs in human aortic endothelial cells. A and B, HAECs were treated with either LPC (10 μ

m

) or LPI (10 μ

m

) for 1 h. Flow cytometry analysis with mitochondrial ROS (A) and cytosolic calcium (B) probes was performed afterward. C, GSEA of the LPI–up-regulated genes in the “calcium-mediated signaling” collection. D, principal component analysis showing the global transcription profile relationship among control, LPI, and LPC. E, heat map showing the similarities and differences between LPC-regulated and LPI-regulated genes. F, Venn diagram showing the number of LPC and LPI co-up-regulated genes. G, top enriched pathways of the LPI and LPC co-up-regulated genes (58 genes from F) as determined by Ingenuity Pathway Analysis. H, the LPC and LPI co-up-regulated genes from their top regulated pathway, attraction of leukocytes (in G), are shown. I, comparison of LPS- and lysophospholipid-induced endothelial activation. Red numbers indicate gene expression -fold changes that are greater than 1.4-fold. For all panels, data are expressed as mean ± S.D. (error bars). **, p < 0.01; ***, p < 0.001.

Figure 6.

Figure 6.

A new working model. Left, during acute inflammation, a danger signal from pathogen or virus infection induces transient endothelial cell activation as characterized by two features, up-regulated adhesion molecule expression and increased secretion of cytokines and chemokines. Right, in the process of chronic metabolic inflammation during cardiovascular disease development, constant stimulation from endogenous metabolic DAMPs, such as lysophospholipids (during hyperlipidemia), glucose (during hyperglycemia), and homocysteine (during hyperhomocysteinemia), transform endothelial cells into innate immune cells and induce prolonged endothelial cell activation. The innate reprogramming of endothelial cells is characterized not only by up-regulation of cytokine/chemokines and adhesion molecule gene expression but also by up-regulation of additional DAMP receptors, such as caspase-1 and CD36, as well as up-regulation of costimulatory molecules and MHC class II molecules.

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