S1PR1 Mislocalization in Lung Endothelium Regulates Innate Immune Function and Mediates Inflammatory Lung Injury

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ABSTRACT Uncontrolled accumulation of inflammatory cells in the airspace paired with vascular injury causes lethal acute lung injury (ALI). The microvascular endothelium, with which immune cells are continuously in contact with, plays a critical role in maintaining lung homeostasis basally and after infection. Sphingosine-1-phosphate receptor-1 (S1PR1), a G-protein coupled receptor (GPCR) expressed on the endothelial cell (EC) surface, is a well-known mediator of “barrier protection” and “resolution of inflammation”. Here we have discovered that S1PR1 can also trigger counterproductive signaling, thus changing the lung vascular niche. The focus of Project 1 is to identify post-translationally modified S1PR1 as a significant molecular switch that epigenetically modifies EC from an anti-inflammatory phenotype to an immune-active phenotype, leading to irreversible inflammatory lung injury. Our Supporting Data show that: 1) inflammatory cytokines (e.g., TNFα) produced during inflammatory injury phosphorylates S1PR1 on tyrosine143 (Y143-S1PR1); 2) Y143-phosphorylated S1PR1 in turn signaled receptor translocation and localization to the endoplasmic reticulum (ER), 3) the chaperone protein BiP (Binding immunoglobulin Protein) was essential for the translocation and binding of Y143-S1PR1 to the ER membrane, 4) the ER-associated Y143-S1PR1 subsequently bound to the intracellular heteromeric GTP binding protein Gi, and when activated by intracellular S1P augmented store-operated calcium entry (SOCE) in a Gi-dependent manner, and 5) rendering S1PR1 constitutively phosphorylated (by editing Y143 to D143) in EC of mice increased lung inflammatory injury. Lung EC also gained epigenetic modifications of genes inducing vascular development and myeloid cells differentiation and activation following injury by Pseudomonas aeruginosa, suggesting injury- specific modulation of EC epigenome. Based on these provocative data, Project 1 will test the hypothesis that ER-resident Y143-S1PR1 is a crucial point of confluence for key signaling events with the potential to rewire the EC epigenome, shifting EC into an inflammatory phenotype leading to intractable lung injury. Our Specific Aims are: Aim #1: to address the role of Y143 phosphorylation of S1PR1 in the ER positioning of S1PR1 and reprogramming of lung endothelium, and thereby leading to inflammatory lung injury; and Aim #2: to study the role of chaperone protein BiP interaction with Y143 phosphorylated-S1PR1 in stabilizing S1PR1 at ER membrane and mediating the shift to inflammatory phenotype in lung EC. We will use genetically modified mice generated using Crisper-Cas9 gene editing as well as EC-specific S1PR1 null mice and tools available in Cores such as epigenetic analysis (Core B), multichannel cellular imaging (Core C), and 2-photon imaging of lung EC and immune cells in living mouse (Core C) to define mechanistically how ER-resident Y143-S1PR1 in EC centralizes these signaling events to control the fate of immune cells in the lungs. We believe studies will identify novel markers of the immunologically active endothelium and targets for preventing inflammatory lung injury through manipulation of ER-S1PR1 and BiP signaling.