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Neutropenia with Impaired Immune Response to Streptococcus pneumoniae in Ceramide Kinase-Deficient Mice

Christine Graf^*, Barbara Zemann^*, Philipp Rovina^*, Nicole Urtz^*, Andrea Schanzer^*, Roland Reuschel^*, Diana Mechtcheriakova^*, Matthias Müller, Evelin Fischer, Claudia Reichel^*, Susanna Huber^*, Janet Dawson, Josef G. Meingassner^*, Andreas Billich^*, Satoru Niwa^*, Rudolf Badegruber, Paul P. Van Veldhoven, Bernd Kinzel, Thomas Baumruker^* and Frédéric Bornancin^1,*

^* Novartis Institutes for BioMedical Research and Nabriva Therapeutics, Vienna, Austria; Novartis Institutes for BioMedical Research, Basel, Switzerland; and Katholieke Universiteit Leuven, Faculteit Geneeskunde, Departement Moleculaire Celbiologie, Afdeling Farmacologie, Leuven, Belgium

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Dr. Frédéric Bornancin, Novartis Institutes for BioMedical Research, Brunner Strasse 59, 21/373, Vienna A-1235, Austria. E-mail address: frederic.bornancin{at}novartis.com

² Abbreviations used in this paper: CerK, ceramide kinase; C1P, ceramide-1-phosphate; cPLA₂, cytosolic phospholipase A₂; LC/MS, liquid chromatography followed by mass spectrometry; BMMC, bone marrow-derived mast cell; VEGF, vascular endothelial growth factor; BMDM, bone marrow-derived macrophage; NBD, N-[7-(4-nitrobenzo-2-oxa-l,3-diazole)]; WT, wild type; MRM, multiple reaction monitoring; mBSA, methylated BSA; KO, knockout.

This article has been cited by other articles:

				C. Graf, M. Klumpp, M. Habig, P. Rovina, A. Billich, T. Baumruker, B. Oberhauser, and F. Bornancin Targeting Ceramide Metabolism with a Potent and Specific Ceramide Kinase Inhibitor Mol. Pharmacol., October 1, 2008; 74(4): 925 - 932. [Abstract] [Full Text] [PDF]