Sphingosine-1-phosphate receptor-1 (S1P1) is expressed by lymphocytes, dendritic cells, and endothelium and modulated during inflammatory bowel disease

The sphingosine-1-phosphate receptor-1 (S1P1) agonist ozanimod ameliorates ulcerative colitis, yet its mechanism of action is unknown. Here, we examine the cell subsets that express S1P1 in intestine using S1P1-eGFP mice, the regulation of S1P1 expression in lymphocytes after administration of dextran sulfate sodium (DSS), after colitis induced by transfer of CD4+CD45RBhi cells, and by crossing a mouse with TNF-driven ileitis with S1P1-eGFP mice. We then assayed the expression of enzymes that regulate intestinal S1P levels, and the effect of FTY720 on lymphocyte behavior and S1P1 expression. We found that not only T and B cells express S1P1, but also dendritic (DC) and endothelial cells. Furthermore, chronic but not acute inflammatory signals increased S1P1 expression, while the enzymes that control tissue S1P levels in mice and humans with inflammatory bowel disease (IBD) were uniformly dysregulated, favoring synthesis over degradation. Finally, we observed that FTY720 reduced T-cell velocity and induced S1P1 degradation and retention of Naïve but not effector T cells. Our data demonstrate that chronic inflammation modulates S1P1 expression and tissue S1P levels and suggests that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on DC migration and vascular barrier function

In Vitro and In Vivo Antagonism of a G Protein-Coupled Receptor (S1P3) with a Novel Blocking Monoclonal Antibody

Background: S1P 3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtypeselective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist. Methodology/Principal Findings: Specific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts. Conclusions/Significance: We have developed the first-reported monoclonal antibody that selectively recognizes a lipidactivated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsi

Interrelationship between Dendritic Cell Trafficking and Francisella tularensis Dissemination following Airway Infection

Francisella tularensis, the etiological agent of the inhalation tularemia, multiplies in a variety of cultured mammalian cells. Nevertheless, evidence for its in vivo intracellular residence is less conclusive. Dendritic cells (DC) that are adapted for engulfing bacteria and migration towards lymphatic organs could serve as potential targets for bacterial residence and trafficking. Here, we focus on the in vivo interactions of F. tularensis with DC following airway infection of mice. Lethal airway infection of mice with the live vaccine strain (LVS) results in trafficking of a CD11bhigh/CD11cmed/autofluorescencelow DC subset from the respiratory tract to the draining mediastinal lymph node (MdLN). Simultaneously, a rapid, massive bacterial colonization of the MdLN occurs, characterized by large bacterial foci formation. Analysis of bacteria in the MdLN revealed a major population of extracellular bacteria, which co-exists with a substantial fraction of intracellular bacteria. The intracellular bacteria are viable and reside in cells sorted for DC marker expression. Moreover, in vivo vital staining experiments indicate that most of these intracellular bacteria (∼75%) reside in cells that have migrated from the airways to the MdLN after infection. The correlation between DC and bacteria accumulation in the MdLN was further demonstrated by manipulating DC migration to the MdLN through two independent pathways. Impairment of DC migration to the MdLN, either by a sphingosine-1-phosphate receptor agonist (FTY720) or by the D prostanoid receptor 1 agonist (BW245C), resulted in reduced bacterial colonization of MdLN. Moreover, BW245C treatment delayed the onset of morbidity and the time to death of the infected mice. Taken together, these results suggest that DC can serve as an inhabitation niche for F. tularensis in the early stages of infection, and that DC trafficking plays a role in pathogen dissemination. This underscores the therapeutic potential of DC migration impairing drugs in tularemia treatment

CD4 blockade directly inhibits mouse and human CD4(+) T cell functions independent of Foxp3(+) Tregs.

CD4(+) helper T cells orchestrate protective immunity against pathogens, yet can also induce undesired pathologies including allergies, transplant rejection and autoimmunity. Non-depleting CD4-specific antibodies such as clone YTS177.9 were found to promote long-lasting T cell tolerance in animal models. Thus, CD4 blockade could represent a promising therapeutic approach for human autoimmune diseases. However, the mechanisms underlying anti-CD4-induced tolerance are incompletely resolved. Particularly, multiple immune cells express CD4 including Foxp3(+) regulatory T cells (Tregs) and dendritic cells (DCs), both controlling the activation of CD4(+)Foxp3(-) helper T cells. Utilizing mixed leukocyte reactions (MLRs) reflecting physiological interactions between T cells and DCs, we report that anti-CD4 treatment inhibits CD4(+)Foxp3(-) T cell proliferation in an IL-2-independent fashion. Notably, YTS177.9 binding induces a rapid internalization of CD4 on both CD4(+)Foxp3(-) T cells and Foxp3(+) Tregs. However, no expansion or activation of immunosuppressive CD4(+)Foxp3(+) Tregs was observed following anti-CD4 treatment. Additionally, cytokine production, maturation and T cell priming capacity of DCs are not affected by anti-CD4 exposure. In line with these data, the selective ablation of Foxp3(+) Tregs from MLRs by the use of diphtheria toxin (DT)-treated bacterial artificial chromosome (BAC)-transgenic DEREG mice completely fails to abrogate the suppressive activity of multiple anti-CD4 antibodies. Instead, tolerization is associated with the defective expression of various co-stimulatory receptors including OX40 and CD30, suggesting altered signaling through the TCR complex. Consistent with our findings in mice, anti-CD4 treatment renders human CD4(+) T cells tolerant in the absence of Tregs. Thus, our results establish that anti-CD4 antibodies can directly tolerize pathogenic CD4(+)Foxp3(-) helper T cells. This has important implications for the treatment of human inflammatory diseases

Heterogeneous expression of the adhesion receptor CD226 on murine NK and T cells and its function in NK-mediated killing of immature dendritic cells.

The adhesion receptor CD226 (DNAM-1) is a member of the Ig superfamily possessing two extracellular V-like domains. In humans, CD226 was shown to be expressed by NK as well as T cells. During T cell priming, CD226-mediated costimulatory signals may skew the subsequent differentiation into the Th1 pathway. In addition, CD226 expressed on NK and cytotoxic T cells is engaged by its counter-receptor CD155, present on target cells, thereby triggering their elimination. We established mAb specifically recognizing mCD226, demonstrating that CD226 is expressed by precursor and mature but not developing T cells. In contrast, NK cells are distinguished by a rather heterogeneous CD226 expression profile. In addition, expression of CD226 appears coupled to that of other NK cell receptors, as high expression of CD226 was found to correlate with decreased proportions of Ly49D and H positive NK cells. Upon injection into mice, the anti-CD226 antibodies caused selective depletion of CD8(+) T cells. Moreover, these antibodies as well as a naturally occurring CD226 splice variant lacking the outermost V-like domain were instrumental in determining that CD226 adheres to CD155 via its first domain. In addition, antibodies were identified as capable of blocking the CD226/CD155 interaction and to prevent NK-driven killing of immature DC. CD226 is thus the first mNK receptor identified to be essential for the elimination of this particular cell type