Sphingosine kinase-2 prevents macrophage cholesterol accumulation and atherosclerosis by stimulating autophagic lipid degradation

Atherosclerosis is the major cause of ischemic coronary heart diseases and characterized by the infiltration of cholesterol-accumulating macrophages in the vascular wall. Although sphingolipids are implicated in atherosclerosis as both membrane components and lipid mediators, the precise role of sphingolipids in atherosclerosis remains elusive. Here, we found that genetic deficiency of sphingosine kinase-2 (SphK2) but not SphK1 aggravates the formation of atherosclerotic lesions in mice with ApoE deficiency. Bone marrow chimaera experiments show the involvement of SphK2 expressed in bone marrow-derived cells. In macrophages, deficiency of SphK2, a major SphK isoform in this cell type, results in increases in cellular sphingosine and ceramides. SphK2-deficient macrophages have increases in lipid droplet-containing autophagosomes and autolysosomes and defective lysosomal degradation of lipid droplets via autophagy with an impaired luminal acidic environment and proteolytic activity in the lysosomes. Transgenic overexpression of SphK1 in SphK2-deficient mice rescued aggravation of atherosclerosis and abnormalities of autophagosomes and lysosomes in macrophages with reductions of sphingosine, suggesting at least partial overlapping actions of two SphKs. Taken together, these results indicate that SphK2 is required for autophagosome- and lysosome-mediated catabolism of intracellular lipid droplets to impede the development of atherosclerosis; therefore, SphK2 may be a novel target for treating atherosclerosis.

In OxLDL-loaded macrophages, cytoplasmic lipid droplets (LDs) are isolated into the autophagosomes, delivered to lysosomes, and hydrolysed by lysosomal acid lipase, which leads to the release of free cholesterol. The cholesterol is then transported into the cell exterior by the ABC family transporters located on the plasma membrane. Autophagy is an essential process for LD breakdown in macrophages and serves a protective role in cholesterol accumulation in macrophages and consequently atherosclerosis [3][4][5] .
Sphingolipids may also be involved in atherosclerosis 6 . Sphingomyelin, which is the most abundant sphingolipid species in the cell membrane, has a high affinity to cholesterol and accumulates in advanced atheroma 7 . Sphingomyelin and other sphingolipids located in the plasma membrane and the cell organelles are converted to ceramides, sphingosine and sphingosine-1-phosphate (S1P) by the sequential actions of the sphingolipid-metabolizing enzymes. S1P is established as the blood-borne, pleiotropic lipid mediator that acts through binding to the S1P-specific, cell surface G protein-coupled receptors S1PR1-S1PR5 8,9 . In mouse models of atherosclerosis, genetic knockout (KO) studies showed that S1PR2 and S1PR3 facilitate atherosclerosis 10,11 , whereas pharmacological stimulation of S1PR1diminishes atherosclerosis 12,13 . S1P is produced from sphingosine by the actions of sphingosine kinase-1 (SphK1) and sphingosine kinase-2 (SphK2), which are solely responsible for generation of S1P in vivo 14 . Neither Sphk1-KO (Sphk1 −/− ) nor Sphk2-KO (Sphk2 −/− ) mice display any phenotypic abnormality under basal conditions, whereas double KO (Sphk1 −/− ; Sphk2 −/− ) mice are embryonic lethal and have almost complete absence of S1P in the fetal tissues 15 , suggesting that sphingolipid metabolism via SphKs is indispensable for embryonic development. In addition to synthesizing the lipid mediator S1P, SphKs are implicated in endocytosis 16,17 ; lysosomal functions including Ca 2+ release and regulation of the transcription factor TFEB 18 ; and autophagy 19 , for which the detailed mechanisms are not fully understood.
In a mouse model of atherosclerosis, pharmacological inhibition of SphKs by a non-selective SphK inhibitor reduced plasma S1P concentration and exhibited both atherogenic and anti-atherogenic properties 20 . As a consequence, the compound had no significant effect on atherosclerotic lesion formation. However, it is possible that SphK1 and SphK2 have distinct, isoform-specific roles in atherosclerosis. For example, a recent study 21 showed a SphK2-specific role in the regulation of ABCA1-mediated cholesterol efflux. In this study, we analysed atherosclerosis in Western diet (WD)-fed Sphk1 −/− mice and Sphk2 −/− mice, both with an ApoE-deficient background and found that Sphk2 −/− mice, but not Sphk1 −/− mice, show aggravation of atherosclerosis because of defective autophagic breakdown of LDs in macrophages. These observations indicate that SphK2 is a novel key factor essential for autophagosome-and lysosome-mediated LD catabolism and may be a target in the development of new therapies for atherosclerosis.

Results
Genetic disruption of Sphk2 aggravates atherosclerosis in mice. To investigate roles of the two SphK isoforms in atherosclerosis, we fed four mice groups with different SphK genotypes, i.e. Sphk1 +/+ ; Sphk2 +/+ (hereafter abbreviated as Sphk2 +/+ ), Sphk1 +/+ ; Sphk2 +/− (Sphk2 +/− ), Sphk1 +/+ ; Sphk2 −/− (Sphk2 −/− ), and Sphk1 −/− ; Sphk2 +/+ (Sphk1 −/− ) in the Apoe −/− background, on a WD for 12 weeks, followed by determinations of the aortic plaque lesion areas. The plaque lesions in the spread aortae, as evaluated with Oil Red O (ORO) staining, were increased by approximately 60% in Sphk2 −/− mice compared with control Sphk2 +/+ mice, whereas those in Sphk1 −/− and Sphk2 +/− mice were similar to those in control mice (Fig. 1a). Total intimal lesion size and OROpositive area in the cross-sections of the aortic sinus, a site frequently affected by atherosclerosis, were greater in Sphk2 −/− mice than control mice (Fig. 1b). The ORO-positive cross-sectional area of the abdominal aorta in Sphk2 −/− mice was also increased compared with control mice (Supplemental Fig. S1). Plasma concentrations of total cholesterol and triglyceride, plasma lipoprotein profiles, liver histology and cardiovascular parameters were all similar between Sphk2 −/− mice and control mice (Supplemental Figs. S2, S3a,b). Both groups of mice had similar body weights after 12 weeks of WD feeding, although the basal body weight of Sphk2 −/− mice at 8 weeks was slightly lower than that of control mice (Supplemental Fig. S3c). These results suggest that SphK2 has a protective role in atherosclerotic lesion formation in the aorta without affecting a plasma lipid profile.

SphK2 deletion in bone marrow (BM)-derived cells aggravates atherosclerosis.
We investigated SphK2 gene expression in various mouse tissues by performing 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) staining of tissues from Sphk2 −/− mice that harbor the β-galactosidase (LacZ) gene at the SphK2 gene locus 15 . Macroscopically, Sphk2 −/− mouse aortae showed much more intense blue color in X-gal staining compared with Sphk2 +/+ mouse aortae (Fig. 2a, left). Microscopically, X-gal-positive cells (arrowheads in Fig. 2a) were scattered in the intima of aortic plaques. The double immunostaining of aortic plaques using anti-monocyte/macrophage marker LAMP2/Mac3 and anti-LacZ antibodies showed LacZ immunoreactivity in LAMP2-positive macrophages (Fig. 2b). Moreover, anti-SphK1 and anti-SphK2 immunostaining of aortic plaques showed that SphK2 was stained as fine and coarse puncta and larger dots with the strong colocalization of SphK2 and the lysosomal membrane protein LAMP2, whereas SphK1 was stained in a coarse punctate pattern in LAMP2-positive cells with modest colocalization between SphK1 and LAMP2 (Fig. 2c). The intimal cells were positive for the macrophage marker F4/80 (Supplemental Fig. S4). These observations suggested that macrophages in the plaque expressed SphK2. Therefore, we studied a role of SphK2 in BM-derived cells by generating chimeric mice in which Sphk2 +/+ host mice had received transplantation of BM cells from Sphk2 −/− or control donor mice (Fig. 2d). The ORO-stained atherosclerotic lesion area in the spread aortae was greater in mice that received Sphk2 −/− BM compared with mice that received Sphk2 +/+ BM. These observations suggest that SphK2 in BM-derived cells has a protective role in atherosclerosis.
Recent studies 3-5 reported a protective role of autophagy in limiting cholesterol accumulation in macrophages and attenuation of autophagy in plaque macrophages. Moreover, a homeostatic role of sphingosine kinases in autophagy and lysosomal functions in macrophages and other cell types was suggested 19,22 . Deficiency of SphK2 or both SphK1 and SphK2 in macrophages resulted in enhancement of autophagic vesicles, which might be due to abnormal lysosomes or enhanced autophagy 22 . Therefore, we studied possible impairment of autophagy in Sphk2 −/− mice. In tissue lysates from the aortae of WD-fed Sphk2 −/− mice, the expression of p62 (Sqstm1), a ubiquitin-binding scaffold protein which recruits ubiquitinated autophagosomal contents to the phagophore isolation membrane by binding to phagophore-bound LC3 3 , was increased compared with control mice (Fig. 2e), suggesting impaired progress of autophagic degradation processes in the aortae of Sphk2 −/− mice. Furthermore, double immunofluorescence staining of aortic sections showed enhanced accumulation of LAMP2-positive macrophages and an increase of p62 expression in LAMP2-positive plaque macrophages (yellow) in Sphk2 −/− mice compared with control mice (Fig. 2f). Previous studies [16][17][18][19] showed that both SphK1 and SphK2 were localized in the cytosol and cell organelles, which include lysosomes and early and late endosomes. Immunostaining of peritoneal macrophages showed that both SphK1 and SphK2 were highly co-localized in LAMP2-positive lysosomes in macrophages (Fig. 2g, arrowheads). These observations together suggest that aggravation of atherosclerosis in SphK2 −/− mice may be accompanied by defective autophagy in plaque macrophages.
Uptake of modified lipids by scavenger receptors and cholesterol efflux by ABC family transporters affect lipid deposition in macrophages. Sphk2 −/− macrophages have elevated basal cellular lipids compared with Sphk2 +/+ macrophages, and Sphk2 +/+ macrophages had 2.4-fold increase in cellular lipids after 4 h incubation with oxLDL whereas Sphk2 −/− macrophages had no increase in cellular lipid content (Supplemental Fig. S7a). The cholesterol efflux in the presence of 2% serum or HDL was not different between Sphk2 −/− and Sphk2 +/+ macrophages (Supplemental Fig. S7b). OxLDL loading induced increases in the protein expression of cholesterol transporters ABCA1 and ABCG1. However, the extents of their expression were similar in Sphk2 −/− and Sphk2 +/+ macrophages with oxLDL loading (Supplemental Fig. S7c).
To investigate the mechanisms underlying the altered autophagic LD catabolism in Sphk2 −/− macrophages, we analyzed the gene expression in macrophages freshly harvested from WD-fed mice. Interestingly, Sphk2 −/− macrophages showed 20-60% increases in the expression of lysosome-related genes, including the H + -ATPase Atp6vod2, the lysosomal acid lipase LIPA and the lysosomal glycosylated membrane protein Lamp2, as well levels of these proteins were normalized to those of GAPDH (n = 8 per group) (bottom). (f) Immunostaining of aortic root sections after 12weeks of WD feeding (n = 9-10 per group). Scale bars, 50 μm. MFI: Mean fluorescent intensity. (g) Subcellular distribution of SphK1 and SphK2 in peritoneal macrophages from control SphK2 +/+ male mice. The arrowheads indicate the localization of SphK1 and SphK2 in LAMP2-positive lysosomes. Scale bars, 5 μm. *P < 0.05 and ****P < 0.0001. (2019) 9:18329 | https://doi.org/10.1038/s41598-019-54877-6 www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ as the autophagy-related genes LC3B and p62 and the lipid oxidase stress-activated transcription factor NRF2 (Supplemental Fig. S9b). Concomitantly, mRNA expression of the lysosome-and autophagy-regulating master transcription factor TFEB 26 was elevated in Sphk2 −/− macrophages. These findings indicate that the expression of lysosome-and autophagosome-related genes was not decreased but upregulated in Sphk2 −/− macrophages. Despite that, Sphk2 −/− macrophages had dysfunctions at the lysosomes/autolysosomes and impaired LD degradation. Previous studies 26,27 showed that the phosphorylation status regulates the activity of TFEB: the dephosphorylated form of TFEB is active and translocates into the nucleus to stimulate transcription of its target genes. The major phosphorylating kinase of TFEB is mTOR. Indeed, the mTOR inhibitor Torin abolished phosphorylation of TFEB in macrophages (Supplemental Fig. S9c). However, the activity of mTOR appeared to be rather increased in Sphk2 −/− macrophages compared with Sphk2 +/+ macrophages, as evaluated by phosphorylation of the endogenous mTOR substrate 4E-BP1. Nevertheless, the ratio of dephosphorylated TFEB to total TFEB was not different between Sphk2 −/− and Sphk2 +/+ macrophages (Supplemental Fig. S9c,d). Therefore, it is possible that SphK2 could be involved in the regulation of TFEB activity in a manner not directly dependent on the phosphorylation event of TFEB.

Discussion
Here, we demonstrate that lipid-loaded Sphk2 −/− but not Sphk1 −/− mice have larger atherosclerotic lesions than control mice. Mechanistically, Sphk2 −/− macrophages have increased lipid-containing autophagosomes/autolysosomes with the lysosomal dysfunctions, which include a reduced proteolytic activity and luminal acidification, suggesting impaired lysosomal degradation of the autophagic bodies. Moreover, the effects of genetic Sphk disruption on plaque formation are SphK isoform-specific, which is likely due to the predominant expression of the SphK2 isoform in macrophages because transgenic overexpression of SphK1 rescued the phenotypes caused by SphK2 deletion. Collectively, SphK2 plays a crucial role in the LD catabolism by autophagy and lysosomes in macrophages and has a protective role in atherogenesis.
In lipid-loaded mice, macrophages and smooth muscle in the vascular subendothelial layer uptake OxLDL and store OxLDL-derived cholesterol as esterified cholesterol in the intracellular LDs to become foam cells, which are a key component of atherosclerotic lesions. SphKs were previously shown to regulate hepatic lipid metabolism and adipogenesis 30,31 . However, SphK2 deficiency did not affect plasma lipoprotein profiles in this study. Notably, SphK2 deficiency resulted in increases in lipid-containing autophagosomes/autolysosomes in macrophages; nevertheless, cholesterol efflux was not altered by the SphK2 deficiency (Supplemental Fig. S7). Moreover, the cellular expression of p62 and LC3 proteins, which are autophagosome/autolysosomes-associated and consumed structures in macrophages from SphK2 +/+ and SphK2 −/− male mice (n = 9-10 per group). Representative images (top). Scale bars, 20 μm. MFI of anti-LAMP2 signals, as well as Bodipy-and anti-LAMP2 double signals (bottom). (g) Histogram analysis of the sizes of LAMP2 + vesicles in macrophages. *P < 0.05, **P < 0.01 and ***P < 0.001. www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ OxLDL is endocytosed and after oxLDL-loaded endosomes fuse with lysosomes, OxLDL is degraded by lysosomal enzymes. Released free cholesterol is transported into the endoplasmic reticulum and processed into LDs, which is released into the cytoplasm. The present study showed that lipid processing in the autophagic pathway is impaired in SphK2 −/− macrophages very likely due to lysosomal dysfunctions, resulting in more accumulation of lipids. The lysosomal dysfunctions in SphK2 −/− macrophages suggest that lysosomal degradation of endocytosed oxLDL may be impaired. Several recent studies 16,19 suggested the involvement of SphK1 in endocytic process. However, it is currently unknown whether SphK2 is involved in the regulation of endocytosis of oxLDL and its lysosomal processing.
SphK2-deficient macrophages accumulated sphingosine and ceramides within cells, which led to a disturbance in the membrane lipid composition. A previous study 22 showed that SphK2-deficiency induced a compensatory autophagic response, which eliminated sphingolipids that accumulated in the cell membrane due to SphK2 deficiency. In our experimental condition, macrophages were exposed to further stress of increased lipid-burden due to hyperlipidemia. Despite that SphK2-deficient macrophages sought to compensate for lipid-overloading by enhancing an autophagic response through the mechanisms involving the upregulation of TFEB, the master transcription factor for expression of autophagy-and lysosome-regulating genes, enhancement of autophagic degradation was not sufficient to prevent accumulation of the LDs in SphK2-deficient macrophages. Thus, our data imply that disturbed sphingolipid metabolism due to SphK2 deficiency compromises the autophagosome-lysosomal functions at least in macrophages, leading to excess accumulation of lipids in macrophages. A recent study 32 showed that SphK2 but not SphK1 is involved in preconditioning-induced protection from brain injury through an autophagy-dependent mechanism. Therefore, it is an intriguing possibility that SphK2 may modulate various pathological conditions through stimulating autophagy.
Sphingosine, which has the amino group in the molecule, is recognized as a lysosomotropic substance 18,19 . In the lysosomes where the bulk of sphingosine is generated by sphingolipid degradation 9 , SphK2 deficiency results in sphingosine accumulation, which probably increases lysosomal membrane permeability like the cases of increased production of reactive oxygen species and stimulation with exogenous inflammatory cytokines, leading to lysosomal dysfunctions 33,34 . Once the lysosomal membrane is disrupted, the lysosomes release cathepsins, leading to further damages of the lysosomes and other organelles through a vicious cycle 35 . Sphingosine accumulation in the lysosomes was also shown to affect the luminal Ca 2+ concentration and Ca 2+ release in the lysosomes, which may change the activation of TFEB 18,34,36 . In addition, defective autophagy was reported to lead to activation of inflammasomes, which could enhance the production of inflammatory cytokines 28 . Consistent with this notion, we observed enhancement of lipopolysaccharide-induced IL-1β release in SphK2 −/− mice, which may contribute to aggravation of atherosclerosis.
Previous studies showed that pharmacological interventions to target the biosynthesis and actions of sphingolipids and genetic manipulations exerted beneficial effects on atherosclerosis. Myriocin is a potent inhibitor of serine palmitoyltransferase, which is the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway. Administration of myriocin decreased the concentrations of blood sphingolipids including ceramides and sphingomyelin and substantially inhibited atherosclerosis 7,37 . FTY720 (fingolimod), which was originally derived from myriocin, is a prodrug and its phosphorylated form acts as an agonist for all S1P receptor subtypes but S1PR2. FTY720 was found to reduce circulating lymphocytes via S1PR1, exerting immunosuppressive effects, and is now employed for treatment of multiple sclerosis 38 . In a murine model of atherosclerosis, chronic administration of FTY720 and S1PR1-selective agonists reduced atherosclerotic lesions 12,13,39,40 , and a more recent study 41 showed that endothelial-specific genetic deletion of S1pr1 exacerbated atherosclerosis, indicating that endothelial S1PR1 limits atherosclerosis. In addition, genetic deletion of either S1pr2 or S1pr3 diminished atherosclerosis associated with suppression of macrophage infiltration in a murine model 10,11 , indicating that S1PR2 and S1PR3 facilitate atherosclerosis.
Very recently, low density lipoprotein receptor (LDLR)-deficient chimera mice that had been transplanted with bone marrow of Sphk2 −/− mice were reported to show attenuated atherosclerosis 42 . The chimera mice with Sphk2 −/− bone marrow exhibited a moderate increase (1.5-to 2.0-fold) in plasma S1P concentration compared with wild-type bone marrow-transplanted control mice. These authors suggested that increased endogenous S1P levels exerted anti-atherogenic effects that were mediated by favorable modulation of endothelial functions. In our study, plasma S1P concentration in Sphk2 −/− mice was approximately 4.0-fold higher compared with wild-type mice. It could be possible that the higher S1P levels might exert enhanced anti-atherogenic effects via S1PR1. The higher S1P levels might also exert enhanced pro-atherogenic effects via S1PR2 and S1PR3. These anti-atherogenic and pro-atherogenic effects might cancel each other out. Further, since overexpression of SphK1 in Sphk2 −/− mice rescued both the macrophage lysosomal dysfunctions and aggravation of atherosclerosis in the present study, it is unlikely that slightly enhanced activation of S1PR2 and S1PR3 by the elevated plasma S1P could account for aggravated atherosclerosis in Sphk2 −/− mice. It is also possible that the different atherogenic mouse models employed in these two studies (LDLR-KO vs. ApoE-KO) might affect the discrepancies of the experimental results. Further studies including analysis of macrophage-specific Sphk2 KO mice are required for fully understanding a role of SphK2 in atherosclerosis.
Bodipy-and anti-LAMP2 double signals (right). (g) Staining of macrophages from SphK2 −/− and SphK2 −/− ; SphK1-Tg male mice with DQRed-BSA and Bodipy (n = 5-6 mice per group, total cell number = 119-128). Representative images (left). Scale bars, 20 μm. MFI of DQRed-BSA signals (right). (h) Immunoblot analysis of the autophagy-related protein expression in peritoneal macrophages. In (b-h), macrophages were freshly harvested from mice of the indicated genotypes, which were fed a WD for two weeks. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. NS, not significant. (2019) 9:18329 | https://doi.org/10.1038/s41598-019-54877-6 www.nature.com/scientificreports www.nature.com/scientificreports/ In the present study, the effects of genetic deletion of Sphk2 and Sphk1 on atherosclerosis were clearly different. Previous studies on SphK-deficient mice showed that the two SphK isoforms possess partially redundant functions in organismal development 15 . The SphK isoforms also play differential roles in various pathological conditions, including cancer 43,44 , inflammatory diseases 45 , and ischemic diseases 46 . SphK1 and SphK2 are expressed ubiquitously in a variety of tissues, but with different tissue abundance 47 . The abundance of the two SphKs at cellular levels is also variable 48 . In macrophages, the SphK2 mRNA level is higher than the SphK1 mRNA level (Supplemental Fig. S5c), which was consistent with the results in the previous report 22 . Although the subcellular localization of the two SphK isoforms is reportedly different, we found that both SphK1 and SphK2 are best colocalized with LAMP2-positive puncta, among the various organelles examined in macrophages (Fig. 2e), which is consistent with the fact that the bulk of sphingosine is generated by sphingolipid degradation in lysosomes 49 . Furthermore, Sphk2 deletion resulted in the accumulation of sphingosine and ceramides in macrophages (Supplemental Fig. S5a), indicating that endogenous SphK1 fails to compensate for SphK2 deficiency in macrophages. However, transgenic overexpression of SphK1 corrected the phenotypes, including cellular accumulation of sphingosine and ceramides, which were caused by SphK2 deficiency (Fig. 5), suggesting at least partial functional redundancy of SphK1 and SphK2 in the LD catabolism. Based on these results, we reasoned that the predominant expression of SphK2 leads to its distinct role in autophagy-mediated LD catabolism in macrophages, although partial qualitative non-redundancy between the actions of SphK1 and SphK2 may exist.
In the present investigation, we studied male mice to avoid the effects of estrogen. Previous reports [50][51][52] showed variable effects of sex difference on atherosclerosis in the studies that explored roles and effects of various endogenous molecules and exogenous substances on atherosclerosis. Further studies using both male and female mice are required to reveal the possible effects of sex difference on the role of SphK2 in atherosclerosis.
In conclusion, we demonstrated that macrophage SphK2 plays a critical role in autophagy-lysosomal-mediated LD catabolism to protect from atherosclerotic lesion formation. Rescuing dysregulated sphingolipid metabolism and organelle dysfunctions in macrophages may be a novel therapeutic target for atherosclerosis and other autophagy-involving pathological conditions.

Material and Methods
Mice. Sphk1 −/− mice and Sphk2 −/− mice 15 were backcrossed into the C57BL/6J background more than 10 times. Sphk2 −/− mice carried LacZ-Neo cassette containing an internal ribosomal entry sequence at exon 4 of the Sphk2 gene locus and expressed Sphk2-LacZ hybrid transcript, which was driven by endogenous Sphk2 promoter elements 15 . Sphk1-Tg mice with the C57BL/6J background were previously described 53 . Apoe −/− mice (B6.129P2-Apoetm1Unc/J, Stock Number:002052) were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in a temperature-controlled conventional facility (24 °C) under a 12-h light/12-h dark cycle with ad libitum. Experiments were performed with male knockout mice and appropriate male wild-type littermate control. For atherosclerotic lesion analysis, mice were fed a western diet (1.25% cholesterol, 7.5% cocoa butter, 7.5% casein (%fat/kcal 36%); Oriental Yeast Co., Ltd (Tokyo, Japan)) and analyzed for atherosclerotic lesion size after 12-weeks of WD feeding. For BM transplantation for generating chimeric mice, the recipient SphK2 +/+ mice were irradiated with 2 doses of 4.0 Gy, 3-4 h apart, and reconstituted with unfractionated BM cells (around 2 × 10 7 cells/recipient) from donor mice. Engraftment efficiency was about 94-95% confirmed by flow cytometry. Four weeks later, WD was started. All experiments using mice were performed according to the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University, which strictly conforms to NIH guidelines, and were approved by the Committee on Animal Experiments in Kanazawa University.
Isolation and analyses of macrophages. Peritoneal macrophages were harvested from mice 3-5 days after intraperitoneal injection of 4% thioglycolate solution (#225640, BD Difco). After 2 h incubation with 10% FBS/DMEM on plastic dishes, nonadherent cells were removed by PBS wash and adherent cells were used for further experiments. For determinations of modified LDL uptake, macrophages were incubated with human OxLDL (50 μg/ml, #RP-049, Intracel Resources) in 10% FBS/DMEM for 4 h followed by Bodipy 493/503 staining (1:1000, #D3922) (Molecular Probes) for visualization of LDs. For cholesterol efflux assays, cells were labeled with 2 μCi/ml [1,2-3 H(N)]-cholesterol (Perkin Elmer) overnight. After washing with PBS three times, cells were equilibrated 2 h by DMEM plus 0.2% fatty acid free bovine albumin (DMEM/BSA). The cells were then incubated with fresh DMEM/BSA in the absence or presence of 2% serum or HDL (50 μg/ml) at 37 °C for 4 h. The media were collected and counted for radioactivity by liquid scintillation counter. The cells were solubilized by 0.1 N NaOH and the residual radioactivity was determined. The percent efflux was calculated as [(cpm in media)/ (cpm in media + cpm in cells)] × 100. Cholesterol contents were measured in cell extracts using the Amplex Red Cholesterol assay kit (A12216, Molecular Probes) and adjusted by cell protein contents determined with Bradford method (#500-0006) (Bio-Rad). For amino acid starvation, cells were incubated by 10% FBS/DMEM without amino acid (#048-33575) (WAKO, Osaka, Japan) after OxLDL uptake in the normal growth medium. To evaluate LDs and lysosomal functions, Bodipy 493/503, DQRed-BSA (#D12051) (Molecular Probes) and LysoTracker Red (#L5728) (Molecular Probes) were used according to the instruction manuals from the manufacturers.
Resultant plasma was removed carefully, aliquoted and stored at −80 °C until analysis. Total cholesterol and triglycerides were determined with AutoAnalyzer (SRL Inc.). For lipoprotein fraction analysis, HPLC system with 2 tandem gel permeation columns was used to evaluate the size distribution of plasma lipoprotein particles (Skylight Biotech Inc). Plasma S1P concentration and sphingolipid profile of mouse macrophages were determined by HPLC and LC-MS/MS, respectively as described before 57,58 . Enzyme-linked immunosorbent assay. The murine plasma IL-1β was measured by DuoSet ELISA kit (DY401-05, R&D systems) according to the manufacturer's instructions. 1-Step ™ Ultra TMB-ELISA (Thermo scientific, cat# 34028) was used for final horseradish peroxidase reaction and terminated by 1 M sulfuric acid. Statistical analysis. All data are represented as the means ± s.e.m. At least, 2 independent experiments were performed unless otherwise indicated. Statistical significance was determined using one-way ANOVA with Turkey's post hoc test for pairwise comparison or the unpaired two-tailed Student's t-test with Prism version 6 software (GraphPad Software Inc.). P < 0.05 was considered significant different and Welch's correction was applied when variances are significantly different in the Student's t-test.

Data availability
The data that support the findings of this study are available from the corresponding authors on reasonable request.