Activation of the aryl hydrocarbon receptor is essential for mediating the anti-inflammatory effects of a novel low-molecular-weight compound.

VAF347 is a low-molecular-weight compound that inhibits allergic lung inflammation in vivo. This effect is likely the result of a block of dendritic cell (DC) function to generate proinflammatory T-helper (Th) cells because VAF347 inhibits interleukin (IL)-6, CD86, and human leukocyte antigen (HLA)-DR expression by human monocyte-derived DC, 3 relevant molecules for Th-cell generation. Here we demonstrate that VAF347 interacts with the aryl hydrocarbon receptor (AhR) protein, resulting in activation of the AhR signaling pathway. Functional AhR is responsible for the biologic activity of VAF347 because (1) other AhR agonists display an identical activity profile in vitro, (2) gene silencing of wild-type AhR expression or forced overexpression of a trans-dominant negative AhR ablates VAF347 activity to inhibit cytokine induced IL-6 expression in a human monocytic cell line, and (3) AhR-deficient mice are resistant to the compound's ability to block allergic lung inflammation in vivo. These data identify the AhR protein as key molecular target of VAF347 and its essential role for mediating the anti-inflammatory effects of the compound in vitro and in vivo.

Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice.

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk-/- mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk-/- macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk-/- animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk-/- animals. When tested in a model of fulminant pneumonia, Cerk-/- animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.