Erythrocytes increase endogenous sphingosine 1-phosphate levels as an adaptive response to SARS-CoV-2 infection

Abstract

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Keywords: SARS-CoV-2; endothelial cell barrier; erythrocytes; high-density lipoprotein; hypoxia; infection.

Figure 1. S1P levels in plasma and serum

Shown are medians with interquartile ranges of S1P levels in plasma and serum from 17 healthy volunteers and 23 severe Covid-19 patients. Statistical significance was tested with unpaired Student’s t test with nonparametric correction using the Mann–Whitney U-test; **P<0.01, ***P<0.001.

Figure 2. Distribution of S1P in different lipoprotein and lipoprotein-free compartments

(A) Total plasma S1P concentrations separated according to the association with VLDL and chylomicrons, LDL, HDL and SA. (B) Plasma S1P concentrations associated with either 1 mg HDL or 1 g SA. (A,B) Shown are medians with interquartile ranges of S1P concentrations from 17 healthy volunteers and 23 severe Covid-19 patients. Statistical significance was tested with unpaired Student’s t test with nonparametric correction using the Mann–Whitney U-test, *P<0.05, **P<0.01, ***P<0.001.

Figure 3. S1P levels in RBC

Shown are medians with interquartile ranges of S1P levels in RBCs from 17 healthy volunteers and 23 severe Covid-19 patients. Statistical significance was tested with unpaired Student’s t test with nonparametric correction using the Mann–Whitney U-test, **P<0.01.

Figure 4. Phosphorylation and release of S1P by RBC

(A) Release of C17-S1P by RBCs into PBS supplemented with 50 mg/ml SA. (B) Phosphorylation of C17-Sph by sphingosine kinases (SPHK) in RBC. (A,B) Shown are means ± SD of freshly isolated RBCs from five healthy volunteers and four severe Covid-19 patients. Statistical significance was tested with unpaired Student’s t test with nonparametric correction using the Mann–Whitney U-test; *P<0.05; ns, not significant.

Figure 5. Blood cell expression of S1P-generating and metabolizing enzymes, S1P receptors and enzymes of the glycolytic pathway

mRNA expression of (A) the extracellular S1P producing enzyme sphingosine kinase 1 (SPHK1) and (B) the S1P exporter SPNS2, (C) SPHK2 and (D) the lyase SGPL1, both involved in blood-borne S1P degradation (25), the S1P receptors (E) S1PR1, (F) S1PR5, (G) S1PR2, (H) S1PR3, and (I) S1PR4, and the glycolytic enzymes GAPDH (J), ALDOA (K) and PGK1 (L), all normalized to HPRT1. (A–L) Shown are means ± SEM for 17 healthy volunteers and 18 severe Covid-19 patients. Statistical analysis was performed using the pairwise fixed reallocation randomization test (23), ***P<0.001; ns, not significant.