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Erythrocyte S1P in Health and Disease
Yang Xia1*, Tingting Xie1, Weiru Zhang1, Angelo D’ Alessandro2, Rodney E. Kellems3 and Changhan Chen11Xiangya Hospital, Central South University, China;
2University of Colorado at Denver, USA;
3University of Texas Health Science Center
Background: Erythrocytes have long been considered as the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), which is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and polarization of tissue macrophages. Currently, emerging evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O2) delivery to counteract tissue hypoxia. However, whether O2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization, inflammation and fibrosis remains unrecognized.
Aims: We sought to determine the function and metabolic and molecular bases of erythrocyte S1P in macrophage and fibrosis.
Methods: Erythrocyte SphK1 specific knockouts (eSphK1-/-) were used to determine the functional role of eSphK1 in experimental model of fibrosis. Metabolomics, isotopically labelled arginine flux analyses and cellular and molecular approaches were performed.
Results: Here we report that genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte eSphK1-/-) resulted in less O2 delivery from erythrocytes, thus leading to severe hypoxic tissue microenvironment, kidney damage and fibrosis in a well-accepted pathological hypoxic model infused with angiotensin II (a potent vasoconstrictor known to be elevated in hypertensive patients). Surprisingly, although plasma S1P levels were reduced to 50% in Ang II-infused eSphK1-/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules (one of the most hypoxic sites with Ang II infusion). Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. Further studies demonstrated no significant differences of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1-/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization in Ang II-infused eSphK1-/- mice. Instead, we revealed that reduced O2 offload from erythrocytes was previously unrecognized but essential to promote tissue M2 polarization and fibrosis by upregulating creatine shuttle to meet rapid intracellular energy needs when FAs, AAs and O2 were insufficient in Ang II-infused eSphK1-/- mice. At cellular, molecular and metabolic level, we demonstrated that hypoxia signaling via HIF-1a directly induced M2 polarization as well as expression of Slc6a8 (a gene encoding creatine transport), M2 specific marker gene and fibrotic marker gene in cultured murine BMDMs.
Conclusion: Overall, our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, tissue macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for microphage polarization and tissue fibrosis.
Speakers
Yang Xia