Microsoft Word-macrophageSphkKOfinalRev2.docx

Sphingosine kinases (Sphk), which catalyze the formation of sphingosine 1-phosphate (S1P) from sphingosine (Sph), have been implicated as essential intracellular messengers in inflammatory responses. Specifically, intracellular Sphk1derived S1P was reported to be required for NFκB induction during inflammatory cytokine action. To examine the role of intracellular S1P in inflammatory response of innate immune cells, we derived murine macrophages that lack both Sphk1 and Sphk2 (mø Sphk dKO). Compared to WT counterparts, mø Sphk dKO cells showed marked suppression of intracellular S1P levels while sphingosine and ceramide levels were strongly upregulated. Cellular proliferation and apoptosis were similar in mø Sphk dKO cells compared to WT counterparts. Treatment of WT and Sphk dKO mø with inflammatory mediators TNFα or E. coli LPS resulted in similar NFκB activation and cytokine expression. Furthermore, LPS-induced inflammatory responses, mortality and thioglycollateinduced macrophage recruitment to the peritoneum were indistinguishable between mø Sphk dKO and littermate control mice. Interestingly, autophagic markers were constitutively induced in bone marrowderived macrophages from Sphk dKO mice. Treatment with exogenous sphingosine further enhanced intracellular sphingolipid levels and autophagosomes. Inhibition of autophagy resulted in caspase-dependent cell death. Together, these data suggest that attenuation of Sphk activity, particularly Sphk2, leads to increased intracellular sphingolipids and autophagy in macrophages. __________________________________________