ABSTRACT
Endothelial-cell surface localized sphingosine 1 phosphate receptor 1 (S1PR1) is known to promote anti-inflammatory and barrier enhancing niche upon ligating S1P. Recently we showed that S1P and TNFalpha, later being a well-known inflammatory agonist, phosphorylate S1PR1 at tyrosine143 (Y143 ) which functions as an endoplasmic reticulum (ER) import signal (Anwar et al, 2021). ER-retained S1PR1 instructs barrier disruptive signaling but the mechanism remains unclear. Here, we generated S1PR1 knock-in mice using CRISPR-Cas9 strategy to edit endogenous S1PR1 into Y143D-S1PR1 (phospho mimicking) or Y143F-S1PR1 (phosphodefective) to test the hypothesis that ER-localized S1PR1 subverts EC from anti-inflammatory to pro-inflammatory EC leading to vascular injury. Because EC constitutes about 50% of cells in the lungs, we assessed if knock-in of Y143 DS1PR1 impaired lung homeostasis. We show that editing of S1PR1 into Y143D- or Y143F-S1PR1 did not alter total S1PR1 expression. Interestingly, Y143D-S1PR1 knock-in mice showed marked vascular leak at homeostasis along with increased neutrophil influx and inflammatory cytokine generation including TNFalpha, IL1beta and MiP2 as compared to Y143F-S1PR1 or WT mice. We next challenge these mice with intratracheal LPS. LPS-induced non-resolvable vascular inflammatory injury in Y143D-S1PR1 mice. Surprisingly, Y143F-S1PR1 knock-in mice did not develop vascular inflammatory injury. Furthermore, NFkappaB activity, a predominant transcription factor inducing inflammatory EC phenotype, was increased in EC transducing Y143D-S1PR1 mutant as compared to WT. However, TNFalpha failed to induce NFkB activity in EC transducing Y143F-S1PR1 mutant. Together, these results show that ER-resident S1PR1 program endothelial niche into immune-active niche by activating NFkB pathway leading to irreparable lung injury. Further experiments are being done to assess epigenetic changes (ATACseq and ChIP-Seq) in EC to address the concept the ER- resident S1PR1 controls the fate of immune cells in the lungs. We believe that understanding how ER-resident S1PR1 programs EC into inflammatory phenotype would allow development of new targets for treating the inflammatory vascular diseases including lung injury, ARDS, and COVID-19 .