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Sphingosine Kinase-2 inhibition enhances macrophage polarization toward the M2 phenotype
shobha Thangada, Phd1, Mallika Ghosh, PhD1, Linda Shapiro, PhD1, Fernando Ferrer, MD2.
1Uconn Health, Farmington, CT, USA, 2Uconn Health and Connecticut Children Hospital, Farmington and Hartford, CT, USA.

Introduction: Metabolites of the sphingolipids, primarily ceramide and sphingosine-1-phosphate (S1P), are bioactive lipids that initiate signal transduction cascades to control cellular functions critical to inflammation and inflammatory disorders. S1P is the product of phosphorylation of sphingosine by either of two closely related sphingosine kinases, SphK1 or SphK2. Both isoforms are significantly active at baseline and are responsible for maintaining intracellular and circulating S1P levels. S1P released extracellularly has been shown to trigger downstream signaling via the S1PR1 receptors to polarize macrophages toward a pro-healing M2 phenotype. In the present study using genetic and pharmacological manipulation we demonstrate that deletion or inhibition of sphingosine kinase 2 enhances the polarization of macrophages toward the M2 phenotype.
Materials and Methods: Two cohorts of 6 to 7 week old male mice, consisting of: 1) untreated WT and SphK2 knockout animals or 2) WT mice treated with the novel SK2 inhibitor {SK2i, (S)-2-[3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl] pyrrolidine-1-carboximidamide, 3mg/kg dose} or vehicle control, were subjected to complete unilateral ureteral obstruction. Obstructed and unobstructed kidneys from control, knockout and inhibitor treated cohorts were harvested at day 3 post surgery and kidney cells isolated by collagenase digestion. Immune cell profiles of the isolated cells were analyzed by flow cytometry using inflammatory markers including specific markers of M2 macrophages such as CD301 and CD206. Bone marrow cells isolated from Sphk2-/- and wild type mice were differentiated into macrophages in vitro by incubation in mCSF-containing medium and subsequently polarized to M1 macrophages with the addition of IFNγ or to the M2 phenotype with IL-4 or IL-13. Expression profiles of these populations were analyzed by RNAseq and confirmed by quantitative real-time polymerase chain reaction (QRT-PCR) analysis.
Results: Flow cytometry results indicated that the percentage of M2 macrophages (F4/80+ CD206+ or F4/80+ CD301+) was significantly increased in the kidneys of both Sphk2-/- and SK2i treated mice when compared to control animals, suggesting an overall pro-healing environment at the injury site. This observation was further supported by diminished expression of the pro-inflammatory cytokines MCP-1, TNF α, CXCL2 and IL-1β. RNAseq and QRT-PCR data obtained from in vitro polarized bone marrow derived macrophages indicated that expression levels of genes specifically enriched in M2 macrophages, Arg1, Retnla, Mgl2 and Clec7a were increased 2-3 fold in the absence or inhibition of sphingosine kinase 2.
Conclusions: The results demonstrate that in a murine model of ureteropelvic junction obstruction, inhibition of sphingosine kinase-2 mitigates the pro-inflammatory environment in response to injury by promoting the polarization of pro-healing M2 phenotype macrophages. We hypothesize that the diminished renal injury and fibrosis that we observe in sphingosine kinase 2 knockout and SK2i treated mice may be due in part to an SK2-dependent enhancement of pro-healing M2 macrophage polarization. Therefore, inhibition of SK2 may present a novel therapeutic strategy to prevent or reduce renal injury.


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