The mechanism by which sphingosine-1-phosphate receptor-1 (S1P1) acts to promote lymphocyte egress from lymphoid organs is not defined. Here, we showed that CCR7-deficient T cells left lymph nodes more rapidly than wild-type cells did, whereas CCR7-overexpressing cells were retained for longer. After treatment with FTY720, an agonist that causes downmodulation of lymphocyte S1P1, CCR7-deficient T cells were less effectively retained than wild-type T cells. Moreover, treatment with pertussis toxin to inactivate signaling via G alpha i-protein-coupled receptors restored egress competence to S1P1-deficient lymphocytes. We also found that T cell accumulation in lymph node cortical sinusoids required intrinsic S1P1 expression and was antagonized by CCR7. These findings suggest a model where S1P1 acts in the lymphocyte to promote lymph node egress by overcoming retention signals mediated by CCR7 and additional G alpha i-coupled receptors. Furthermore, by simultaneously upregulating S1P1 and downregulating CCR7, T cells that have divided multiple times switch to a state favoring egress over retention.

The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth in vitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S. Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.

Lymphocyte egress from lymph nodes (LNs) is dependent on sphingosine-1-phosphate (S1P), but the cellular source of this S1P is not defined. We generated mice that expressed Cre from the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) locus and that showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report that mice with Lyve-1 CRE-mediated ablation of sphingosine kinase (Sphk) 1 and lacking Sphk2 have a loss of S1P in lymph while maintaining normal plasma S1P. In Lyve-1 Cre+ Sphk-deficient mice, lymphocyte egress from LNs and Peyer's patches is blocked. Treatment with pertussis toxin to overcome Gαi-mediated retention signals restores lymphocyte egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial cadherin distribution at cell–cell junctions. Our data provide evidence that lymphatic endothelial cells are an in vivo source of S1P required for lymphocyte egress from LNs and Peyer's patches, and suggest a role for S1P in lymphatic vessel maturation.

Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell G i -coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models. IntroductionSphingosine-1-phosphate (S1P), a lipid phosphate produced in the course of sphingosine metabolism in all cell types (1), promotes endothelial cell spreading and barrier function in cell culture (2-5) and in vivo (6, 7). S1P can regulate cell behavior via 5 GPCRs, designated S1P receptor 1 (S1pr1) through S1pr5 (also known as S1P 1 -S1P 5 ) (1,4,8). Models of receptor-dependent roles for S1P in regulating endothelial barrier function have focused on S1P produced by the endothelial cells themselves, casting S1P as a downstream, autocrine/paracrine mediator of the barrier-protective effects of other agents such as activated protein C (9, 10) and angiopoietin (7). However, S1P is present at high concentrations in plasma (11), and the importance of this source of S1P in regulating vascular integrity has not been examined. In addition, GPCR-independent S1P signaling mechanisms and cell-autonomous metabolic effects of disrupting sphingosine conversion to S1P have been reported and may affect vascular integrity (1-5, 7, 12, 13). Central to understanding the physiological roles of S1P in regulating blood vessel function are identification of the sources of S1P that are important for barrier protection in vivo as well as determination of the importance of S1P from blood acting in trans on endothelial cells by receptor-dependent mechanisms ver-

The cellular dynamics of lymphocyte egress from lymph nodes are poorly defined. Here, we visualized the branched organization of lymph node cortical sinuses and found that after entry some T cells were retained while others returned to the parenchyma. Sphingosine-1-phosphate receptor 1 (S1P1)-deficient T cells probed the sinus surface but failed to enter. In some sinuses T cells became rounded and moved in a unidirectional fashion. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress where cortical sinus probing is followed by S1P1-dependent entry, capture of cells in a sinus region with flow and transport to medullary sinuses and the efferent lymph.

Lymphocytes egress from lymphoid organs in response to sphingosine-1-phosphate (S1P); minutes later they migrate from blood into tissue against the S1P gradient. The mechanisms facilitating cell movement against the gradient have not been defined. Here we show that G-protein coupled receptor kinase-2 (GRK2) functions in down-regulation of S1P receptor-1 (S1PR1) on blood-exposed lymphocytes. T- and B-cell movement from blood into lymph nodes is reduced in the absence of GRK2 but is restored in S1P-deficient mice. In the spleen, B-cell movement between the blood-rich marginal zone and follicles is disrupted by GRK2-deficiency and by mutation of an S1PR1 desensitization motif. Moreover, delivery of systemic antigen into follicles is impaired. Thus, GRK2-dependent S1PR1 desensitization allows lymphocytes to escape circulatory fluids and migrate into lymphoid tissues.

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Experimental infection with mouse cytomegalovirus (MCMV) has been used to elucidate the intricate host-pathogen mechanisms that determine innate resistance to infection. Linkage analyses in F(2) progeny from MCMV-resistant MA/My (H2 (k)) and MCMV-susceptible BALB/c (H2 (d)) and BALB.K (H2 (k)) mouse strains indicated that only the combination of alleles encoded by a gene in the Klra (also called Ly49) cluster on chromosome 6, and one in the major histocompatibility complex (H2) on chromosome 17, is associated with virus resistance. We found that natural killer cell-activating receptor Ly49P specifically recognized MCMV-infected cells, dependent on the presence of the H2 (k) haplotype. This binding was blocked using antibodies to H-2D(k) but not antibodies to H-2K(k). These results are suggestive of a new natural killer cell mechanism implicated in MCMV resistance, which depends on the functional interaction of the Ly49P receptor and the major histocompatibility complex class I molecule H-2D(k) on MCMV-infected cells.