Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein-coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9-based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/β-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/β-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.

After induction in secondary lymphoid organs, a subset of antibody-secreting cells (ASCs) homes to the bone marrow (BM) and contributes to long-term antibody production. The factors determining secondary lymphoid organ residence versus BM tropism have been unclear. Here we demonstrate that in mice treated with FTY720 or that lack sphingosine-1-phosphate (S1P) receptor-1 (S1P1) in B cells, IgG ASCs are induced and localize normally in secondary lymphoid organs but they are reduced in numbers in blood and BM. Many IgG ASCs home to BM on day 3 of the secondary response and day 3 splenic ASCs exhibit S1P responsiveness, whereas the cells remaining at day 5 are unable to respond. S1P1 mRNA abundance is higher in ASCs isolated from blood compared to spleen, whereas CXCR4 expression is lower. Blood ASCs also express higher amounts of Kruppel-like factor (KLF)2, a regulator of S1P1 gene expression. These findings establish an essential role for S1P1 in IgG plasma cell homing and they suggest that differential regulation of S1P1 expression in differentiating plasma cells may determine whether they remain in secondary lymphoid organs or home to BM.

Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) in RBL-2H3 and bone marrow-derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcepsilonRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcepsilonRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcepsilonRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcepsilonRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.

In this paper, we describe the unexpected outgrowth of B lineage cells from PU.1(-/-) fetal liver cultures. The cells express all early B cell genes tested, including the putative PU.1 target genes IL-7R and EBF but not B220, and can produce immunoglobulin M. However, we observed a delay in the PU.1(-/-) B cell outgrowth and reduced precursor frequencies, indicating that although PU.1 is not strictly required for B cell commitment, it facilitates B cell development. We also ablated PU.1 in CD19-expressing B lineage cells in vivo, using a Cre-lox approach that allows them to be tracked. PU.1 excision resulted in a shift from B-2 cells to B-1-like cells, which dramatically increased with the age of the mice. Our data indicate that this shift is predominantly caused by a B-2 to B-1 cell reprogramming. Furthermore, we found that B-2 cells express substantially more PU.1 than B-1 cells, which is consistent with the idea that maintenance of the B-2 cell phenotype requires relatively high levels of PU.1, but B-1 cells require little.

S1P1 receptor expression is required for the egress of newly formed T cells from the thymus and exit of mature T and B cells from secondary lymphoid organs. In this study, we deleted the expression of the S1P1 receptor gene (S1pr1) in developing B cells in the bone marrow. Although B cell maturation within the bone marrow was largely normal in the B cell-specific S1pr1 knockout (B-S1pr1KO) mice, their newly generated immature B cells appeared in the blood at abnormally low numbers as compared with control mice. In the bone marrow of B-S1pr1KO mice, immature B cells in contact with the vascular compartment displayed increased apoptosis as compared with control mice. Forced expression of CD69, a negative regulator of S1P1 receptor expression, in developing bone marrow B cells also reduced the number of immature B cells in the blood. Attenuation of CXCR4 signaling, which is required for the proper retention of developing B cells in bone marrow, did not release immature B cells into the blood of B-S1pr1KO mice as effectively as in control mice. Our results indicate that the S1P1 receptor provides a signal necessary for the efficient transfer of newly generated immature B cells from the bone marrow to the blood.