Sphingosine 1-phosphate, a Novel TREM2 Ligand, Promotes Phagocytosis and Reduce Ischemic Injury


 Background: Activation of TREM2 protects against brain injury in ischemic stroke via immunoregulation. However, the endogenous ligand of TREM2 remains unknown. Here, we tested the hypothesis that S1P, an immunoregulator, functions as TREM2 ligand to promote microglial phagocytosis.Methods: SD rats, C57BL/6J mice and TREM2-/- mice were subjected to transient middle cerebral artery occlusion, and primary microglia were subjected to oxygen-glucose deprivation. Phagocytosis was investigated via immunofluorescence and two-photon microscope. LC-MS/MS, microscale thermophoresis and surface plasmon resonance were used to confirm the TREM2-S1P interaction.Results: FTY720, an analog of S1P, promoted microglial phagocytosis in ischemic stroke independent of S1PRs expressed on microglia. S1P was confirmed to be a novel endogenous ligand for TREM2 and promote cellular debris clearance. The enhanced cellular debris clearance ameliorated neurological score and infarct volume, relying on TREM2. Moreover, FTY720 was demonstrated to promote hemoglobin clearance in intracerebral hemorrhage and ameliorate hemorrhagic injury.Conlusions: The present work reveals for the first time that S1P acts as a novel endogenous ligand of TREM2 to effectively promote microglial phagocytosis, and provides a new lead compound for developing TREM2 modulator.


Background
Sphingosine is one of the most important sphingolipid metabolites, named after the Sphinx for its mysterious features. Phosphorylation of sphingosine forms the pleiotropic and bioactive lipid sphingosine-1-phosphate (S1P) (Fig. 1A). Traditionally, S1P acts not only as an intracellular second messenger, but also an extracellular rst messenger in both an autocrine and paracrine manner, via binding with S1P receptors (S1PRs) of which there are currently ve known subtypes (S1PR1-5) [1][2][3]. It has been revealed that S1P has a wide range of biological functions including regulating differentiation, survival, proliferation, angiogenesis and immune modulation [4,5]. A few studies have suggested that S1P might regulate microglial phagocytosis [6,7]. However, the involved mechanisms remain unknown.
As an important phagocytosis mediator, triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor of the Ig superfamily. It consists of an ectodomain, a transmembrane region and a short cytoplasmic tail, which transmit downstream signal by coupling with DNAX-activating protein of 12 kDa (DAP12) [8,9]. TREM2 is expressed on osteoclast, macrophage, dendritic cell, and exclusively on microglia in the brain, primary participates in phagocytosis [10][11][12][13]. Till now, the endogenous ligand of TREM2 has not been found. However, a diverse set of potential TREM2 ligands have been proposed, such as bacteria, poly-anionic [14,15], and phospholipids [16].
Stroke is the rapid development of a focal neurologic de cit caused by a disruption of blood supply to the corresponding area of brain and can either be ischemic (~ 87%) or hemorrhagic (~ 13%) [17]. Ischemic stroke is a leading cause of mortality and disability without e cient therapeutic strategy, despite thrombolysis and thrombectomy in the acute phase [18,19]. Disruption of the regional blood supply initiates ischemic cascade leading to neuronal dysfunction and subsequent death [20,21]. Brain edema and in ammation response in the sub-acute phase, contribute to the secondary injury [20,22]. The damaged and dead neurons could release nucleic acid, protein and lipid, to induce neuroin ammation and exacerbate damage [23]. In the intracerebral hemorrhagic stroke, blood products introduced from hematoma such as hemoglobin and iron can exacerbate neuronal death [24,25]. Thus, promoting phagocytic clearance of neurotoxic cellular debris is bene cial to recovery after stroke and could serve as a promising therapeutic strategy.
In the present work, we con rmed the pro-phagocytic function of S1P and a S1P analog, FTY720, that was not mediated by S1PRs. Because S1P belongs to phospholipids and as well regulates microglial phagocytosis, we speculated and used computer simulation of molecular docking to predicate that S1P might bind to TREM2. Moreover, we veri ed that S1P and FTY720p could bind to TREM2, promote microglial phagocytosis, and thereby exert neuroprotection in ischemic and hemorrhagic stroke.

Animal model and experimental protocol
Male Sprague Dawley (SD) rats (260 g ± 10 g, Animal Core Facility of Nanjing Medical University), C57BL/6J mice (20-25 g, Animal Core Facility of Nanjing Medical University) and TREM2 −/− mice (20-25 g, Cyagen Biosciences) were maintained with ad libitum access to standard fodder and water in a well-ventilated environment with approximately 25 °C, 50%~60% humidity and a standard 12 h light/dark cycle. Animals were assigned to Sham group, model group and FTY720 treated group randomly. Focal cerebral ischemic stroke was induced as described previously [26] and rats were intraperitoneally injected with FTY720 (2 mg/kg, selleck chemicals) or normal saline daily for 24 h, 48 h. The ICH modeling was as previously described with slight modi cation [27]. In brief, a burr hole was made after anesthesia and autologous blood (10 µl) injected at a rate of 1 µl/min using a 26-gauge needle at the coordinates: 0.2 mm anterior, 2.5 mm lateral, and 3.5 mm ventral to the bregma. The needle was removed 20 min after injection to prevent re ux. Mice were intraperitoneally injected with FTY720 (1 mg/kg) or normal saline daily for 72 h. The protocols were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.

TTC staining
Brains were removed and cut into cerebral coronary slices of 2 mm at 48 h after surgery, which were incubated in TTC (2,3,5-Triphenyltetrazolium chloride, 1%, sigma) for 5 min at 37 °C and then, placed in 4% paraformaldehyde solution (pH 7.4) at 4 °C to x up overnight. Photographies were taken and infarct size was analyzed by ImageJ software.
Neurological De cit Scoring Neurological de cit of MCAO rats was classi ed and scored referring to Bederson's scale scores at 24 h, 48 h after surgery. When following conditions occured, corresponding rats were excluded and supplemented: 0 score, massive bleeding during surgery, postoperative respiratory abnormality, early mortality and subarachnoid hemorrhage.
Neurological de cit of ICH mice were assessed at 24 h, 48 h, 72 h after ICH. All mice were scored on six neurologic tests, including body symmetry, gait, climbing, circling behavior, front limb symmetry, compulsory circling and whisker response [28]. Each test was graded from 0 to 4, establishing a maximum de cit score of 28.
Immuno uorescence and two-photon microscope After anesthetizing, rats were transcardially perfused with 37 °C saline followed by 4% paraformaldehyde (PFA). The brains were removed and post xed in 4% PFA for 24 h to 48 h at 4 °C. Then, they were embeded in para n and sectioned coronally at an interval of 5 mm. Coronal sections were processed sequently as following steps: para n melting for 60 min at 60 °C, depara nage and rehydration, endogenous peroxidase inactivation with 3% H 2 O 2 at room temperature for 20 min and washing with 0.01 M PBS for 3 times. After washing, and 0.01M citrate buffer solution was heated to 92 ~ 98 °C with microwave oven simultaneously, slices were placed into the solution for retrieval for 15 min and taken out to cool down naturally to room temperature. For the sake of blocking non-speci c antigen, slides were incubated with 10% normal goat serum for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C at the following dilutions: monoclonal rabbit anti-Iba1 (1:500, Wako), monoclonal Primary microglia and neuron co-culture system For in vitro experiments, microglial cells were separated from the mixed primary culture by apped for 15 min and then plated (2 × 10 4 cells per well) in neuron (2 × 10 5 cells per well in a 24-well plate) culture vessels with DMEM containing 10% FBS:Neuralbasal = 1:3. The cells were co-cultured for further treatment the following day.

Transfection: knock down of TREM2
Microglia cells in the co-culture system or in culture vessels (6 × 10 5 cells per well in a 6-well plate) were transfected using siRNA-mate (Genepharma, Shanghai, China) according to the manufacturer's instructions.
Phagocytosis assay TREM2-DAP12 cDNA was generated as previously described [13]. CHO cells were transfected with the construct to generate a stable cell line that express TREM2-DAP12 chimera. The parental cells or transfected CHO cells were seeded in 24-well plates at the density of 1 × 10 4 cells per well and incubated overnight. After two washes with PBS, cells were dyed with 5 mM Cell Tracker™ Green (Thermo scienti c) for 20 min, followed by washing and incubation in Opti-MEM medium containing 3 µl/100 µl of pHrodo Red zymosan bioparticles and/or 20 µM S1P and/or 10 µg/ml LPS, or S1P and 2 µM CytoD. The treated cells were examined at 2 h and 4 h by uorescent microscope.

Oxygen and glucose deprivation/Reperfusion (OGD/R)
To initiate OGD, the culture medium was removed, rinsed with phosphate buffered saline (PBS) and replaced with Opti-MEM (Gibco). The cultured cells were placed into the hypoxia chamber (Thermo scienti c) with a premixed gas (1% O 2 , 94% N 2 , 5% CO 2 ) at 37℃ for 3 h. After OGD, the cells were perfused by 10% FBS-DMEM medium or 10% FBS-DMEM medium : Neurobasal = 1 : 3 and transferred to a 5% CO 2 -95% O 2 air incubator for relative time. Control cells were incubated under normal conditions throughout the procedure.

LC-MS/MS
BV2 cells were grown to 80%-90% con uency in two dishes and treated with 20 µM S1P or not. After 2 h, the cells were washed thrice with PBS and collected with 400 µl homogenate buffer per dish (250 mM sucrose, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, NaOH, pH to 7.4). The collected cells were frozen at -80 ℃ and underwent 5 freezing and thaw cycles to facilitate lysis. Then the buffer was homogenized further with bead mill. After centrifugation at 12000 rpm for 15 min at 4 ℃, the protein of cell lysates was acquired and incubated with 3 µl anti-TREM2 (abcam, ab125117) overnight at 4 ℃ on a rotating device, followed by adding 100 µl proteinA + G beads/ml lysate overnight to capture the conjugated polymersat 4 ℃ on a rotating device. Immunoprecipitates were collected by centrifugation at 8000 rpm for 2 min at 4 ℃ and washed thrice with 1 ml homogenate buffer, then resuspended in 50 mM NH 4 HCO 3 twice the volume of beads. After boiling, the supernatant was added 200 µl chromatographic grade ethaol, blended and centrifugated at 12000 rpm, 4 ℃ for 30 min to discard precipitation. The solution obtained was puri ed and concentrated with Amicon Ultra-0.5 ml Centrifugal Filter Units (Millipore), and detected by Analysis and Testing Center of Nanjing Medical University.

Preperation of recombinant TREM2
The plasmids encoding human TREM2, rat TREM2, mouse TREM2 as well as hTREM2(R47A), hTREM2(S65A), hTREM2(R77A) were purchesed from Public Protein/Plasmid Library. TREM2 expression for binding assay was performed as described previously [29]. In brief, TREM2 was expressed in freestyle 293F cells and puri ed using Ni-NTA resin and AKTA for further puri cation. The protein was stored in PBS and used to measure binding a nity rapidly.

Microscale thermophoresis (MST)
The above-mentioned obtained fusion proteins were labelled using Monolith His-Tag Labeling Kit (NanoTemper Technologies, Munich, Germany). The recombinant TREM2 and S1P were prepared at the concentration of 250 nM and 2 mM, respectively. The binding a nity was detected with Monolith NT.115 (NanoTemper Technologies, Munich, Germany).

Surface Plasmon Resonance (SPR)
The obtained fusion proteins and S1P were prepared as described above. The fusion proteins were attached to Sensor chip NTA (Biacore). The binding a nity was detected with GE Biacore T200 (GE, USA).

Apoptosis assay
The TUNEL Apoptosis Assay Kit was purchased from KeyGEN bioTECH (Jiangsu, China) and performed following the manufacturer's instructions.

Statistical analysis
The obtained data are presented as mean ± SEM of at least two independent experiments. The relationship between two factors was analyzed using Pearson correlation analysis and groups were compared using a two-way ANOVA with post hoc Bonferroni's multiple comparisons test. All of the data were analyzed with GraphPad Prism 6.0 software. A value of p < 0.05 indicated that the difference was statistically signi cant.

FTY720 promoted microglial phagocytosis in ischemic stroke rats
We rst investigated the pro-phagocytic effects of FTY720 in ischemic rats via NeuN-labeled neuron, Iba1-labeled microglia and CD68-labeled phagocytic microglia. We found that supplement with FTY720, an analog of S1P after phosphorylation, could promote microglia to phagocytose debris indicating by the increased proportions of (Iba + +NeuN + ) cells/total Iba1 + cells (Fig. 1, B and C), (Iba + +NeuN + ) cells/total NeuN + cells (Fig. 1, B and D), CD68 + area per microglia (Fig. 1, E and F). We used two-photon microscope to further con rm that FTY720 signi cantly enhanced the microglial phagocytosis of neuronal debris appeared at 48 h after MCAO (Fig. 1G). These data demonstrate the pro-phagocytic function of FTY720. S1P or FTY720p enhances phagocytosis via TREM2 rather than S1PRs In order to demonstrate whether S1PRs mediate the pro-phagocytosis of S1P and FTY720 in microglia, we knocked down S1PR2 and S1PR4, the two major subtypes expressed on the microglia (Fig. S1A). The results showed that S1PR2 and S1PR4 knockdown did not affect the pro-phagocytosis of S1P ( Fig. S1B and C), indicating certain receptor other than S1PRs should mediate microglial phagocytosis.
TREM2 is an important phagocytosis mediator that exclusively expressed on microglia in the CNS. Its endogenous ligand has not been found, but phospholipids was proposed as one of candidates. Since S1P shares structural similarities with phospholipids, we speculated that S1P might be an endogenous ligand for TREM2. Hence, we used computer simulation of molecular docking to predicate the potential interactions between S1P or FTY720p (FTY720p is structurally similar to S1P) and TREM2. The predicated results showed that human TREM2 (hTREM2) could bind to S1P and FTY720p via H-bonds at 47R, 65S and 77R, as well as via salt bridge at 47R and 77R ( Fig. 2A).
Furtherly, we constructed CHO cell expressing TREM2-DAP12 (T/D CHO cells, Fig. 3A) to identify the prophagocytic functions of S1P on TREM2-DAP12 complex. As shown in Fig. 3B, CHO cells did not phagocytose pHrodo Red zymosan bioparticles with or without S1P or LPS treatment. Importantly, S1P or LPS, increased phagocytosis of T/D CHO cells in a time-dependent pattern (Fig. 3, B and C), which was cancelled by phagocytosis inhibitor CytoD. Collectively, our data demonstrate that S1P or FTY720p functions as TREM2 ligand to enhance phagocytosis. R47, S65 and R77 are the crucial residues for the binding of S1P and TREM2 To further determine the binding site, we induced the point mutation according to computer prediction data (Fig. 2I). R47A, S65A or R77A variants caused slightly lower a nity of S1P to hTREM2 (111.76 ± 21.85 µM, 93.92 ± 21.35 µM, 95.93 ± 18.81 µM, respectively) (Fig. 2, J to L). However, R47A/S65A/R77A variant of hTREM2 remarkably lowered the binding a nity to 239.87 ± 12.20 µM (Fig. 2M). These data con rmed the prediction results by computer simulation of molecular docking, and revealed the importance roles of R47, S65 and R77 residues in the binding of S1P to TREM2.
The pro-phagocytosis and neuroprotective effects of S1P/FTY720 in stroke are reduced when TREM2 was knockdown or knockout In order to further con rm the crucial roles of TREM2 in S1P/FTY720-induced microglial phagocytosis, we used TREM2 knockout (TREM2 −/− ) mice to investigate the effect of FTY720. Consistant with previous results, FTY720 signi cantly promoted phagocytosis, as indicated by increased proportions of phagocytic microglia (Fig. 4, A and B) and CD68 + area per microglia (Fig. 4, A and C). However, the prophagocytosis effect of FTY720 was abolished in TREM2 −/− mice (Fig. 4, A to C). In vitro data showed similar results. We co-cultured normal or TREM2 knockdown microglia with neurons, followed by OGD/R treatment. Phagocytosis by normal microglia signi cantly increased at 5 h after reperfusion, peaked at 7 h and decreased thereafter (Fig. 4, D to G). S1P or FTY720 treatment dramatically promoted phagocytosis at 3 h. However, TREM2 knockdown obviously inhibited the effect. These data highlight the importance of TREM2 in S1P/FTY720-induced pro-phagocytosis.
The expression of S1P in the penumbra of ischemic stroke rats were decreased at 24 and 48 h after reperfusion (Fig. 5A), indicating the insu cient S1P in acute phase of cerebral ischemia. FTY720 treatment could supplement the shortage (Fig. 5A), promote cellular debris clearance (Fig. 1) and thereby play the neuroprotective effects, indicated by decreased infarct volume (Fig. 5, B and C) and alleviated neurological de cits (Fig. 5D). However, the neuroprotection was not observed in TREM2 −/− mice treated with FTY720 after MCAO (Fig. 5E). We further assessed the protective effects of S1P/FTY720 via microglia-neuron co-culture system. At 24 h after OGD/R, S1P or FTY720 treatment maintained the length of longest neurite of neurons, which was signi cantly shortened when knockdown of TREM2 in microglia without affecting the number of neurites (Fig. 6, A to C). Consistently, S1P or FTY720 dramatically decreased apoptotic cells, which was abolished by TREM2 knockdown or CytoD (Fig. 6, D and E). These data show that TREM2 is pivotal in FTY720/S1P induced phagocytosis and protection.
Considering the signi cance of phagocytosis in hemorrhagic stroke, which is characterized by accumulated blood products like hemaglobin (Hb) in the CNS, rstly, we investigated the effect of S1P on the microglial clearance of Hb. After 4 h of Hb treatment, primary microglia showed nearly no Hb clearance. Microglia barely phagocytosed Hb when treated with low dose of S1P (250 nM), which is able to activate S1PRs [30]. However, high dose of S1P (5 µM) signi cantly promoted the phagocytosis of Hb, which was almost completely abolished by cytoD (Fig. S2A). In the hemorrhagic stroke mice, FTY720 treatment dramatically increased Hb clearance at 72 h after ICH (Fig. S2B), ameliorated injury as indicated by reduced hematoma volume ( Fig. S2C and D) and lower neurological score (Fig. S2E). Taken together, our data suggest that S1P/FTY720 could promote phagocytosis and alleviated stroke-induced injury via TREM2.

Discussion
Recent ndings reveal the importance of rapid clearance of cellular debris after ischemic stroke [31,32]. In the present study, we reveal for the rst time that S1P acts as a novel endogenous ligand of TREM2 to effectively promote microglial phagocytosis, and thereby play neuroprotective effects in stroke.
As is known, S1P can be secreted outside cells and activate S1PRs to regulate differentiation, survival, proliferation, angiogenesis and immune modulation [4,5]. A few studies have suggested that S1P could regulate microglial phagocytosis [6,7] via unknown mechanisms. We found that S1P and its analog FTY720 could enhance microglial phagocytosis. However, S1PR2 and S1PR4, main S1PRs expressed on microglia, did not participated in the pro-phagocytic function, indicating certain receptor other than S1PRs mediates the effect.
TREM2 is exclusively expressed on microglia in the brain whose endogenous ligand is still unknown.
Since S1P structurally resembles phospholipids, we speculated the potential interaction between S1P and TREM2 to promote phagocytosis. Firstly, computer simulation of molecular docking predicated that S1P or FTY720p could bind to TREM2. For veri cation, LC-MS/MS, MST and SPR were performed and identi ed the a nity. The results showed the binding a nity of S1P to hTREM2, rTREM2 and mTREM2 were 62.59 ± 11.93 µM, 56.80 ± 13.96 µM and 64.62 ± 16.64 µM, respectively. FTY720p has higher a nity to hTREM2 and rTREM2, but lower a nity to mTREM2. TREM2-DAP12 transfected CHO cells were used to further demonstrate the pro-phagocytic function of S1P via acting on TREM2. Furthermore, the point mutation analysis suggested that R47, S65 and R77 are the crucial residues for the binding of S1P and TREM2. Overall, our results reveal that S1P is a novel endogenous ligand for TREM2 to promote phagocytosis, and that S1P can be uses as a lead compound for modi cation to increase its a nity and effects.
Emerging evidence suggest that TREM2 is essential for disease associated microglia (DAM) [33,34] and microglial neurodegenerative phenotype (MGnD) [35] transition, [36]. Whether S1P activates TREM2 to facilitate microglia transition to these phenotypes, and then promote phagocytosis deserves further investigation. The neuroprotection of FTY720 in MCAO models has been proved [37,38]. However, the contribution of FTY720 induced cellular debris clearance to its protective effects has not been reported. We found that S1P in the penumbra decreased in the acute phase of ischemic stroke, accompanied by insu cient phagocytic function of microglia. Supplemented with FTY720 could signi cantly enhance microglial phagocytose. We further investigated the crucial role of TREM2 in mediating phagocytosis using TREM2 −/− mice and TREM2 knockdown microglia. As expected, the pro-phagocytic function of FTY720 and S1P was abolished in TREM2 −/− mice and TREM2-de cient microglia, resulting in reduced protective effect. These data con rm that S1P or FTY720 acts on TREM2 to induce phagocytosis and protection.
In addition to cerebral ischemia, phagocytosis also exerts critical role in hemorrhagic stroke. Rapid clearance of Hb should be protective in the hemorrhagic stroke. However, increased activity and expression of S1P-lyase occur after hemorrhage result in a reduction of 60% in S1P level [39]. FTY720 treatment signi cantly reduced the hematoma volume and neurological de cit [40][41][42] which could not be abolished by blockage of central S1PRs [43]. Previous studies have uncovered that S1P forms a complex with Hb and promotes deoxy-Hb anchoring to the membrane [30]. On the basis of our ndings, we supposed that FTY720 may function as S1P analog to mediate TREM2 recognition of Hb for phagocytosis. To prove the point, we investigated the Hb clearance by isolated microglia after Hb treatment. Hb was not be cleared by microglia cultured in normal culture medium or low dose of S1P (250 nM), but signi cantly be phagocytized by microglia treated with high dose of S1P (5 µM). These data also suggested S1P-induced phagocytosis mediated by TREM2 rather than S1PRs. The prophagocytosis effect of FTY720 was further observed in the peri-hematoma following ICH.
Collectively, our results reveal for the rst time that S1P is a novel endogenous ligand for TREM2. S1P or its analog FTY720p, binding to TREM2, promote microglial phagocytosis and exerts neuroprotection. Our ndings provide a new lead compound for develop TREM2 agonist and a promising strategy for modulating microglia to treat ischemic stroke.

Conclusion
In summary, we reveal that S1P is a novel endougenous TREM2 ligand that promotes microglial phagocytosis and debris clearance. Expression of S1P decreases in the penumbra within 48 h after cerebral ischemia. Targeting TREM2-S1P interaction is a promising strategy to alleviate damaged and dead neurons induced damage, and to exert neuroprotection. These ndings provide a new lead compound for developing TREM2 modulator.

Availability of data and materials
Results generated and analyzed during the current study are included in this article and its supplementary les.