EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein-Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450 pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.

Activated protein C (APC) reduces mortality of severe sepsis patients but increases the risk of serious bleeding. APC exerts anticoagulant activity by proteolysis of factors Va/VIIIa. APC also exerts antiinflammatory and antiapoptotic effects and stabilizes endothelial barrier function by APC-initiated cell signaling that requires two receptors, endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR1). The relative importance of APC's various activities for efficacy in sepsis is unknown. We used protein engineering of mouse APC and genetically altered mice to clarify mechanisms for the efficacy of APC in mouse sepsis models. Mortality reduction in LPS-induced endotoxemia required the enzymatic active site of APC, EPCR, and PAR-1, highlighting a key role for APC's cytoprotective actions. A recombinant APC variant with normal signaling but <10% anticoagulant activity (5A-APC) was as effective as wild-type APC in reducing mortality after LPS challenge, and enhanced the survival of mice subjected to peritonitis induced by gram-positive or -negative bacteria or to polymicrobial peritoneal sepsis triggered by colon ascendens stent implantation. Thus, APC's efficacy in severe sepsis is predominantly based on EPCR- and PAR1-dependent cell signaling, and APC variants with normal cell signaling but reduced anticoagulant activities retain efficacy while reducing the risk of bleeding.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4 + Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ-or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7β, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4+ and γδ + T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Activated protein C (APC) reduces mortality in severe sepsis patients and exhibits beneficial effects in multiple animal injury models. APC anticoagulant activity involves inactivation of factors Va and VIIIa, whereas APC cytoprotective activities involve the endothelial protein C receptor and protease-activated receptor-1 (PAR-1). The relative importance of the anticoagulant activity of APC versus the direct cytoprotective effects of APC on cells for the in vivo benefits is unclear. To distinguish cytoprotective from the anticoagulant activities of APC, a protease domain mutant, 5A-APC (RR229/230AA and KKK191-193AAA), was made and compared with recombinant wild-type (rwt)-APC. This mutant had minimal anticoagulant activity but normal cytoprotective activities that were dependent on endothelial protein C receptor and protease-activated receptor-1. Whereas anticoagulantly active rwt-APC inhibited secondaryextended thrombin generation and concomitant thrombindependent activation of thrombin activable fibrinolysis inhibitor (TAFI) in plasma, secondary-extended thrombin generation and the activation of TAFI were essentially unopposed by 5A-APC due to its low anticoagulant activity. Compared with rwt-APC, 5A-APC had minimal profibrinolytic activity and preserved TAFI-mediated anti-inflammatory carboxypeptidase activities toward bradykinin and presumably toward the anaphlatoxins, C3a and C5a, which are well known pathological mediators in sepsis. Thus, genetic engineering can selectively alter the multiple activities of APC and provide APC mutants that retain the beneficial cytoprotective effects of APC while diminishing bleeding risk due to reduction in APC's anticoagulant and APC-dependent profibrinolytic activities.

No abstract