S1PR3/RhoA signaling pathway in microglia mediates inflammatory activation in early brain injury after subarachnoid hemorrhage

doi:10.21203/rs.3.rs-4374501/v1

Download PDF

Research Article

S1PR3/RhoA signaling pathway in microglia mediates inflammatory activation in early brain injury after subarachnoid hemorrhage

https://doi.org/10.21203/rs.3.rs-4374501/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Spontaneous subarachnoid hemorrhage (SAH) is a serious and common cerebrovascular disease with high mortality rate and poor prognosis. The immune response caused by abnormal activation of intracranial microglia is one of the main factors contributing to early brain injury after SAH. Sphingosine 1-phosphate (S1P) signaling pathway is widely involved in immune regulation, nerve cell differentiation and other processes. It has been reported that S1P expression is increased in cerebrospinal fluid after SAH, but its role in early brain injury and neuroinflammation induced by SAH remains unclear. In the rat model of SAH established by arterial puncture, low (0.5mg/kg) or high dose (5mg/kg) of the S1P receptor inhibitor FTY720 was administered immediately or at 24 hours after surgery. Improvement of behavioral scores and brain edema symptoms after SAH was observed in immediate treatment group at high dose. In addition, activation of cortical microglia near the perforation site was observed after SAH, and this activation was significantly inhibited after 5mg/kg FTY720 treatment immediately after surgery. Further studies showed that S1P could induce activation and M1 polarization of human microglia cells in vitro. This activation may be mediated through the S1PR3-Gα_12/13-RhoA pathway. Therefore, our study highlights the important role of S1P signaling and microglia activation in SAH-induced early brain injury, and provides evidence for novel therapies targeting the neuroinflammatory process after SAH.

Subarachnoid hemorrhage

Sphingosine 1-phosphate

S1P receptor inhibitor

Microglia

Neuroinflammation

Spontaneous subarachnoid hemorrhage (SAH) is the third leading cause of stroke, with high mortality and disability rate[1]. The pathogenesis of SAH is complex, of which 85% are caused by aneurysm rupture, called aneurysmal subarachnoid hemorrhage (aSAH) [2]. Clinically, about a quarter of patients with SAH die before receiving medical care, 33% die within 48h after the initial hemorrhage, and 50% die within 30 days [3]. Patients who survive early massive hemorrhage still suffer from a series of complications, including increased intracranial pressure and delayed cerebral ischemia (DCI). Historically, many studies mainly focused on the targeted treatment of cerebral vasospasm (CVS) after SAH, but the clinical outcomes have not been improved, and the incidence and mortality of DCI have not been reduced [4]. Recently, researchers have found that early brain injury (EBI) induced by SAH may be a key factor affecting patient outcome. EBI usually occurs within 72 hours before SAH and involves a series of pathophysiological events, such as brain edema, blood-brain barrier (BBB) destruction, oxidative stress, inflammation, excitotoxicity, and impaired ion homeostasis. Among them, the activation and amplification of harmful inflammatory response play a key role in nerve injury after SAH [5]. However, how EBI occurs and how it causes delayed cerebral ischemia and neurological damage, the underlying mechanisms are still unclear.

Microglia, the resident macrophage in the central nervous system (CNS), plays an important role in the neuroinflammatory response induced by SAH[6]. Upon receiving risk-associated molecular pattern signals (DAMPs), microglia can rapidly activate, proliferate, and migrate to the site of injury, whereby they phagocytose cellular debris and harmful substances, and secrete various inflammatory cytokines and chemokines. Activated microglia also produce some anti-inflammatory cytokines and trophic factors to promote neuronal regeneration. Therefore, microglia can be classified into deleterious M1 subpopulation and beneficial M2 subpopulation according to their distinguishable phenotype. Usually, M1 microglia expressing MHCII, CD80 and CD11b secretes proinflammatory cytokines (including TNF-α, IL-1β, IL-6, COX-2, ROS and NO). In contrast, M2 microglia, which is positive for CD163 and CD206, mainly produce IL-10 and TGF-β, while phagocytosing cell debris and harmful substances. Early DAMPs signaling induce some microglia to polarize to an M2 phenotype; however, persistent brain injury may gradually replace these reparative M2 cells with a toxic M1 phenotype, resulting in prolonged inflammatory responses and subsequent impairment of neuronal regeneration[7]. Thus, the functional imbalance of activated microglia leading to an intensified immune response is one of the potential mechanisms of EBI.

Sphingosine-1-phosphate (S1P) belongs to a class of lipids called sphingolipids [8], and its receptors are widely distributed in almost all tissues and organs of the human body. Studies have shown that S1P is widely involved in the regulation of vasculogenesis, angiogenesis, immune regulation, nerve cell differentiation, blood-brain barrier integrity construction and other processes by interacting with its receptor S1PRs (S1PR1-5) [9]. Its dysfunction may lead to autoimmune diseases, chronic inflammation, tumors and neurodegenerative diseases [10, 11]. In 2010, the FDA approved Fingolimod (FTY720), an S1P receptor 1 (S1PR1) inhibitor, for the treatment of multiple sclerosis, an autoimmune disease, which has triggered extensive attention to the S1P signaling pathway among researchers [12]. In addition to S1PR1, the drug can also selectively inhibit the activation of S1PR3 and S1PR5 receptor signaling pathways, reduce the number of peripheral circulating lymphocytes, and significantly prolong the survival of experimental animal transplanted organs without impairing the immune response and immune memory function to pathogens, with low toxic side effects. A series of clinical studies support its use as a good immunosuppressive agent [13]. In recent years, increasing evidence has shown that the expression levels of S1P and its receptors undergo pathological changes in diseases related to CNS injury [14], suggesting that S1P pathway may also have the ability to regulate neuroinflammation after CNS injury. However, the detail mechanism of S1P-mediated neuroinflammation is still poorly understood. Therefore, further studies will help to clarify the mechanism of S1P in CNS injury-related diseases such as SAH, and provide theoretical basis for the application of immunomodulators targeting the S1P signaling pathway in CNS injury-related diseases.

1. Cells and reagents

HMC3 cell line (CRL-3304) were obtained from ATCC. Twelve-week-old rats were purchased from Charles River. FTY720 (CAS NO. 162359-56-0) were purchased from AdooQ Bioscience, IFN-γ was purchased from Peprotech (300-02), and S1P (S45337) from Shanghai yuanye Bio-Technology Co., Ltd.. C3 exoenzyme (Amylet Scientific) was used to block RhoA activation, and Y27632 (Sigma-Aldrich) was used to inhibit ROCK activation.

2. Cell culture and treatment

HMC3 cells were maintained in RPMI1640 medium, containing 10% heat inactivated fetal bovine serum, 100 U/ml penicillin, 50U/ml streptomycin, 2mM glutamine, and 1mM sodium pyruvate at 37℃ in a 5% CO₂ humidified incubator. At a confluence of 70%, HMC3 cells were trypsinized and plated in 6 well plates. 48 hours later, IFN-γ or S1P was added to the culture medium at indicated concentrations. Cells were harvested for further analysis after a 6-hour treatment period.

3. siRNA vector construction and transfection

Pre-designed shRNA oligos targeting S1P1, S1P2, S1P3 and G_α12/13 were customized by Beijing Tsingke Biotech Co., Ltd (Online Resource Table S1). shRNA oligos were inserted into the FV055 vector containing the AmpR, GFP and puromycin sequences, then positive clones were selected and validated by sequencing. HMC3 cells were plated in 6 well plate, serum-starved for 18–24 h and transfected with plasmids expressing si-S1P1, si-S1P2, si-S1P3 and si-G_α12/13, respectively. Cells were collected for gene expression assay using QRT-PCR and western blotting.

4. QRT-PCR

Total mRNA of HMC3 cells was extracted using the RNeasy kit (Invitrogen) and the complement strand of DNA (cDNA) was synthesized using PrimeScript™ RT reagent Kit from Takara. Gene expression was determined by Real-time PCR using the TB Green® Premix Ex Taq™ II FAST qPCR kit. Primer sequences are shown in the Online Resource Table S2. Data were normalized to internal β-Actin, and fold change was determined as described previously[15] . Values for comparison for a single gene across multiple samples was determined using cycle threshold (Ct) data fitted to a standard curve. For comparison of multiple transcripts in a single sample, then the 2−ΔΔCt method was applied to the Ct value.

5. Western blot

HMC3 cell samples were lysed with RIPA buffer (20 mm Tris, 250 mm NaCl, 3 mm EDTA, 3 mm EGTA, and 20 mm βglycerophosphate) supplemented with sodium vanadate, leupeptin, aprotinin, p-nitrophenyl phosphate, and phenyl methylsulfonyl fluoride. Protein concentration of samples were measured using Bradford Protein Assay kit (Beyotime). Equal amounts of protein (20 μg) were loaded onto 4–12% 10-well or 15-well SDS-PAGE gels. Gels were transferred to PVDF membranes, and the resulting blot was probed with specific antibodies. The COX-2 antibody (Affinity#AF7003) was used at 1:500 dilution, the β-actin antibody (Cell Signaling Technology #4970) was used at 1:1000 dilution. Rabbit secondary antibody was used at 1:4000 dilution. Fold changes were determined by chemiluminescence and normalized to β-actin.

6. Flow cytometry

HMC3 cells were harvested immediately after S1P or IFN-γ treatment, and stained with FITC-conjugated CD11b antibody (Biolegend#301329), or CD80 antibody (Biolegend#375405)，or CD163 antibody (Biolegend#333617), or CD206 antibody (Biolegend#321103), or MHC-II antibody (Invitrogen#17-9956-42），or corresponding IgG isotypes controls without PMA fixation. The gates were established by fluorescence minus IgG isotype controls.

7. SAH model and treatment

All animal experiments were approved by the ethics committee of Taizhou University, performed in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health, China. SAH was performed by using an artery puncture method according to a previous study[16]. In brief, 12-week-old male rats were anesthetized with sodium pentobarbital (80 mg/kg body weight). The left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were exposed. A sharpened 4-0 monofilament nylon suture was advanced into ICA from ECA to perforate the artery at the bifurcation of the anterior and middle cerebral artery. And then the suture was immediately withdrawn to cause SAH. The procedure in the sham group was similar without the perforation.

Rats were randomly divided into 5 groups, n=5 in each group. A: sham; B: SAH + vehicle ; C: SAH + LD (0.5 mg/kg FTY720); D: SAH +HD (5 mg/kg FTY720); E: SAH + HD24 (5 mg/kg FTY720 delayed intervention). FTY720 was formulated with physiological saline at a concentration of 0.15 mg/ml or 1.5 mg/ml. Then the rats in group C, D were given with FTY720 or vehicle (physiological saline) by intraperitoneal administration at 2 h after SAH injury. The FTY720 dose was determined according to previous studies[17]. Rats in group B received intraperitoneal administration with an equal volume of physiological saline at 2 h after SAH. Rats in group E received intraperitoneal administration with 5mg/kg FTY720 at 24 h after SAH.

8. Assessment of neurological score

Neurological score was assessed at 24 h and 72 h after SAH according to previous studies by a blinded investigator[18]. In brief, animals’ neurological functions were evaluated by six tests, including symmetry in the movement of all limbs (0-3), spontaneous activity (0-3), forepaw outstretching (0-3), climbing (1-3), response to vibrissae touch (1-3) and body proprioception (1-3). The minimum neurological score was 3 (severe impairment) and the maximum was 18 (no neurological impairment).

9. Brain water content

After the assessment of neurological score, rats were sacrificed by cervical dislocation. The brains were removed and weighed immediately to obtain the wet weight. The brain was then dried in an oven at 100°C for 72 h and weighed again to obtain the dry weight. The percentage of brain water content was calculated according to the formula: [(wet weight-dry weight)/wet weight] × 100%.

10. Immunohistochemical staining

The brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin before being cut into 5-μm thick sections. Cortex adjacent to the Perforated Site (CAPS), the motor cortex (M1 cortex), and hippocampus were selected for IBA-1 staining as previously described[19]. In brief, after a xylene/ethanol dewax-rehydration series, endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide. Brain sections were then incubated for 1 h with blocking buffer comprising 2.5% goat serum, 1% bovine serum albumin (BSA), and 0.1% Triton-100. The primary antibody of IBA-1 (Thermo Fisher# PA5-21274) was applied subsequently at 4 °C overnight. Horseradish peroxidase (HRP) conjugated secondary antibody was applied for 1 h at room temperature. Diaminobenzidine (DAB) was utilized for visualization of colorimetric reaction. Three random fields were examined on each brain area respectively of each animal under microscope (Leica) at × 40 magnification.

11. Statistical analysis

IBM SPSS 23.0 software was used for statistical analysis of the data. All data were expressed as mean ± standard deviation (SD). Differences between two experimental groups were compared by the Student t-test. P<0.05 was considered to indicate a statistically significant difference.

To test whether S1P blockage can ameliorate SAH-induced EBI, a rat SAH model was established using arterial puncture method. Compared with the sham-operated group, the SAH experimental group showed significant neurological deficits. Specifically, motor dysfunction in limb movement symmetry, forepaw outstretching, and climbing within 24 hours occurs immediately after SAH (Fig. 1A). Motor dysfunction induced by SAH worsened after 72 hours (Fig. 1B). A significant increase in brain water content, without alteration of the structural integrity of the brain (Fig. 1C) or substantial change in body weight (Online Resource Fig. S1), was observed at 72 h after SAH in the experimental group, accompanied by an increase in the number of IBA1-positive microglia in the cortex adjacent to the perforated site (CAPS), hippocampus, and motor cortex, with a more amoeboid than bifurcating morphology (Fig. 2A). These are direct evidence of early brain damage caused by the puncture-triggered arterial hemorrhage which spreads in the subarachnoid space and causes an acute inflammatory response. At 24 hours after surgery, all SAH groups, either with or without treatment, did not show significant difference for neurological scores. However, at 72 hours after SAH, in the groups that received 0.5mg/kg or 5mg/kg of FTY720 or the same volume of saline after surgery, the rats showed different responses in neurological scores. Compared with the control group (saline), 5mg/kg FTY720 administered immediately after surgery significantly alleviated neurological deficits after surgery, whereas 0.5mg/kg FTY720 administered immediately after surgery did not significantly improve neurological scores (Fig. 1A-B, Table 1). Delayed administration of 5mg/kg FTY720 at 24 hours after surgery (before neurological scoring) did improve the neurological outcomes but the effect was modest compared with the immediate-treated group. Moreover, 5mg/kg of FTY720 immediately after surgery was the only group that achieved prolonged alleviation of neurological deficits within 72 hours after SAH. Likewise, brain edema was significantly alleviated in the 5mg/kg FTY720 immediate-treated group at 72 hours after surgery, but not in the other two groups (Fig. 1C). These results confirmed that S1P receptor inhibitors could significantly improve the nerve injury and inhibit the inflammatory response caused by EBI after SAH. The blockage effects of S1P-dependent events are most likely to be maximized within 24 hours after SAH, and to be dose-dependent.

Table 1

Neurological evaluation by six behavior tests for rats with indicated operation and treatments.
Groups	Sham		SAH + vehicle		SAH + LD		SAH + HD		SAH + HD24
Time point	24 hr	72 hr	24 hr	72 hr	24 hr	72 hr	24 hr	72 hr	24 hr	72 hr
Neurological scores
Total	18	18	10.4 ± 0.5	8.6 ± 0.5	10.8 ± 0.8	9.2 ± 0.8	11.8 ± 1.3	13.2 ± 1.5	10.8 ± 0.8	10.2 ± 1.3
Symmetry in the movement of all limbs	3	3	2.4 ± 0.5	1.6 ± 0.5	2.2 ± 0.4	1.8 ± 0.4	2.2 ± 0.4	2.6 ± 0.5	2.4 ± 0.5	2.2 ± 0.4
Spontaneous activity	3	3	1.4 ± 0.5	1.2 ± 0.4	1.4 ± 0.5	1.4 ± 0.5	1.6 ± 0.5	1.8 ± 0.4	1.4 ± 0.5	1.0 ± 0.0
Forepaw outstretching	3	3	1.6 ± 0.5	1.2 ± 0.4	1.4 ± 0.5	1.0 ± 0.0	1.6 ± 0.5	2.2 ± 0.4	1.6 ± 0.5	1.4 ± 0.5
Climbing	3	3	1.0 ± 0.0	1.0 ± 0.0	1.0 ± 0.0	1.0 ± 0.0	1.4 ± 0.5	1.4 ± 0.5	1.0 ± 0.0	1.2 ± 0.4
Reaction to touch on either side of trunk	3	3	2.0 ± 0.0	1.8 ± 0.4	2.6 ± 0.5	1.8 ± 0.4	2.8 ± 0.4	3.0 ± 0.0	2.4 ± 0.5	2.4 ± 0.5
Response to vibrissae touch	3	3	2.0 ± 0.0	1.8 ± 0.4	2.2 ± 0.4	2 ± 0.0	2.2 ± 0.4	2.2 ± 0.4	2.0 ± 0.0	2.0 ± 0.0

Microglia play a pivotal role in the immune response after SAH. Activation of microglia, indicated by the IBA1 staining, is supposed to initiate at the CAPS, then spread to the distal motor cortex and to the hippocampus region deep in the brain. We showed that administration of FTY720 at 24 hours after SAH seemed to block only the remote activation of microglia while microglia located in the CAPS remained activated (Fig. 2A-B). However, immediate-treatment of FTY720 globally inhibited the microglia activation, suggesting that FTY720 may only inhibit the early activation, but not reverse the activated status of microglia.

To further explore the mechanism by which S1P regulates neuroinflammation, we first selected an immortalized human microglia cell line, HMC3, for in vitro studies. Similar to primary microglia, HMC3 can be induced and activated by IFN-γ to secrete inflammatory factors such as IL-6 and TGFβ, and upregulate M1 markers such as MHCII, CD80 and CD11b. If cultured in the presence of neuronal precursor cells or astrocytes, the expression of M2 markers (including CD163, CD206, etc.) is up-regulated [20]. We found that S1P activates HMC3 cells in a dose-dependent manner. At a concentration above 50nM, S1P induces induced an IFN-γ-induced phenotype in HMC3 and a marker of M1 polarization (Fig. 3A, Online Resource Fig. S2). In contrast, S1P had no effect on the expression of several CD163 and CD206 associated with M2 polarization. HMC3 is derived from embryonic microglia, and the level of secreted TNF-α is very low even in the activated state, which is not easy to detect [21]. In contrast, the mRNA expression of several major inflammatory factors, including IL-6, TNF-α, and TGF-β1, was significantly upregulated in the activated state after receiving IFN-γ. Our results showed that S1P (50nM) induced an up-regulation of IL-6 and TNF-α expression at the mRNA level, but did not change the expression of anti-inflammatory factor TGF-β1 (Fig. 3B). In addition, expression of the inflammation-related expression product cyclooxygenase-2 (COX2) was significantly increased after S1P induction (Fig. 4A). All these results suggest that S1P induces HMC3 activation and M1 polarization, but not M2 polarization.

S1P activates downstream signals through its receptors, including S1PR1-S1PR5. Among them, S1PR1-3 were the most studied. To further elucidate the mechanism by which S1P activates HMC3, siRNA vectors targeting S1pr1, S1pr2 and S1pr3 were constructed. The results of QRT-PCR showed that the mRNA of S1pr1, S1pr2 and S1pr3 were all detected in HMC3 cells. After 48 hours of transfection with si-S1P1, si-S1P2 or si-S1P3 vectors, the mRNA expression of S1pr1, S1pr2 and S1pr3 in HMC3 cells was down-regulated by 62%, 63% and 58%, respectively (Online Resource Fig S3). Meanwhile, western blot showed that the expression of COX2 was still up-regulated after S1P induction in HMC3 cells transfected with si-S1P1 and si-S1P2. In contrast, the expression level of COX2 in HMC3 cells transfected with si-S1P3 was not responsive to S1P induction (Fig. 4B). Thus, S1P acts primarily through S1PR3, but not S1PR1 and S1PR2, in HMC3 cells.

When bound by S1P ligands, S1P receptors transmit signals downstream by activating their corresponding G-coupled protein receptors, where S1PR1 only couples G_i/o, whereas S1PR2 and S1PR3 couple G_i/o, G_q or G_12/13[22]. It has been reported that S1PR3 can activate downstream inflammatory signals and play a role in microglia-mediated inflammatory response [23], while S1P activates inflammatory astrocytes mainly through the S1PR3-G_12/13-RhoA pathway [24]. To further demonstrate whether S1P activates microglia via G_12/13-RhoA, we used selective inhibitors of RhoA/ROCK or knockdown of Gα_12/13. The S1P-induced increase in COX2 expression was significantly inhibited by the addition of FTY720, the RhoA inhibitor C3, the ROCK inhibitor (Y27632) to the culture medium, or by transfection of HMC3 with siRNA targeting G_α12/13 (Fig. 4C). It should be noted that expression of G_α12 and G_α13 in HMC3 cells was reduced by 72% and 64% after transfection with vectors expressing si-GNA12/13 mixture, respectively (Online Resource Fig. S3). These results suggest that S1P can activate the pro-inflammatory phenotype of HMC3 by activating the S1PR3-G_12/13-RhoA/ROCK signaling pathway.

There is increasing evidence that neuroinflammation occurs rapidly after SAH and is an important factor in causing EBI [25]. S1P regulates a variety of cell functions through its receptors S1PR1-5 and is considered to be involved in the regulation of inflammatory process [22]. FTY720 is the first approved S1P receptor inhibitor. Reduction in circulating lymphocytes and limiting inflammatory cell migration into the CNS are considered to be its two most important pharmacological mechanisms [26]. However, unlike other immunosuppressants, FTY720 is lipophilic and therefore easily penetrates the blood-brain barrier, acting at the site of CNS injury through sphingosine kinase activation secreted by neuronal cells. Several preclinical and clinical studies have demonstrated the inhibitory effect of FTY720 on neuroinflammation caused by CNS injury [17, 27–29]. Our findings are consistent with literature reports that FTY720 plays a protective role in the CNS injury caused by SAH. Meanwhile, our evidence further strengthens the notion that S1P signaling is an important molecular mechanism responsible for CNS neuroinflammation.

However, several issues still require further discussion. First of all, the inflammatory response after SAH is complex and seems to get involved in both the acute phase and the post-CVS period[30, 31]. Aneurysm rupture causes blood leakage throughout the subarachnoid space, followed by a sharp increase in intracranial pressure (ICP), a decrease in perfusion pressure (CPP), transient ischemia of brain tissue and death of nerve cells [25]. At this time, the blood-brain barrier rapidly breaks down, causing massive inflammatory cells (mainly neutrophils and macrophages) infiltration and microglia activation. After an acute response period, these inflammatory cells may remain trapped at the site of injury, undergo apoptosis, and cause a secondary and chronic inflammatory response [32]. Our study provides evidence that FTY720 directly inhibits the initial microglial activation within 24 hours after SAH, but prolonged observation of FTY720-treated SAH rats was absent due to the limited experiment animals in this study. Further studies are needed to elucidate whether and how the S1P pathway is involved in subsequent events of this complex process, including amplification of the inflammatory cascade and later neural repair. Therefore, the therapeutic strategy targeting S1P pathway still faces some challenges in CNS injury, including how to choose the intervention window, to optimize administration dose and frequency, and to develop personalized dosing regimens according to specific conditions of CNS injury.

S1PR3 is widely expressed in heart, lung, kidney, spleen and other organs, and is involved in the maintenance of endothelial barrier, vascular tone and immune response. Different from S1PR1, which mainly functions through JAK2 and PI3K pathways [33, 34], S1PR3 can extensively affect pathways including G_α12/13-RhoA-ROCK, VEGF, PI3K, STAT3 [35]. Among them, the G_α12/13-RhoA-ROCK pathway is considered to be closely related to the migration and M1 polarization of macrophages. In mouse peritoneal macrophages, the expression level of S1PR3 is lower than that of S1PR1 and S1PR2. However, LPS stimulation can up-regulate the expression of S1PR3 and stimulate S1PR3-mediated inflammatory activation, while the expression of S1PR1 is down-regulated [23]. Consistent with this, our in vitro studies showed that microglial activation in the CNS was dependent on S1PR3 but not S1PR1. However, this conclusion cannot be simply translated to in vivo studies because FTY720 is a broad-spectrum S1PR inhibitor with known substrates including S1PR1, S1PR3, and S1PR5 among others [26]. The widespread expression of S1PR is related to several adverse reactions such as slow heart rate and hypertension caused by FTY720 in clinical studies [28]. The pathologic consequences of systemic inhibition of the S1PR3 pathway are unclear. Therefore, detailed studies on different types of S1PR are necessary to develop more specific S1PR receptor inhibitors and optimize the benefit-risk balance of S1P pathway-based therapies.

In summary, our study highlights the role of the S1P pathway in the inflammatory response after SAH, deepening our understanding of the complex inflammatory response process after SAH. In addition, our study also suggests that therapeutic strategies targeting S1P may play a protective role in attenuating CNS injury after SAH.

Ethics Approval

All animal experiments in this study was approved by the Ethics Committee of Taizhou University.

Funding

This study was supported by Taizhou social development science and technology Program project (Grant No. 21ywa31).

Competing Interests

The authors declare that they have no conflict of interest.

Claassen J, Park, S. 2022. Spontaneous subarachnoid haemorrhage. Lancet. https://doi.org/10.1016/S0140-6736(22)00938-2.
Macdonald RL, Schweizer, T.A. 2017. Spontaneous subarachnoid haemorrhage. Lancet. https://doi.org/10.1016/S0140-6736(16)30668-7.
Song HC, Yuan, S., Zhang, Z.W., Zhang, J.Y., Zhang, P., Cao, J., Li, H.Y., Li, X., Shen, H.T., Wang, Z., Chen, G. 2019. Sodium/Hydrogen exchanger 1 participates in early brain injury after subarachnoid hemorrhage both in vivo and in vitro via promoting neuronal apoptosis. Cell Transplant. https://doi.org/10.1177/0963689719834873.
Hoh BL, Ko, N.U., Amin-Hanjani, S., H-Y, Chou S., Cruz-Flores, S., Dangayach, N.S., Derdeyn, C.P., Du, R., Hänggi, D., Hetts, S.W., Ifejika, N.L., Johnson, R., Keigher, K.M., Leslie-Mazwi, T.M., Lucke-Wold, B., Rabinstein, A.A., Robicsek, S.A., Stapleton, C.J., Suarez, J.I., Tjoumakaris, S.I., Welch, B.G. 2023. Guideline for the Management of Patients With Aneurysmal Subarachnoid Hemorrhage: A Guideline From the American Heart Association/American Stroke Association. Stroke. https://doi.org/10.1161/STR.0000000000000436.
Jin J, Duan, J., Du, L.Y., Xing, W.L., Peng, X.C., Zhao, Q.J. 2022. Inflammation and immune cell abnormalities in intracranial aneurysm subarachnoid hemorrhage (SAH): Relevant signaling pathways and therapeutic strategies. Front Immunol. https://doi.org/10.3389/fimmu.2022.1027756.
Wang XY, Wu, F., Zhan, R.Y., Zhou, H.J. 2022. Inflammatory role of microglia in brain injury caused by subarachnoid hemorrhage. Front Cell Neurosci. https://doi.org/10.3389/fncel.2022.956185.
Kumar A, Alvarez-Croda, D., Stoica, B.A., Faden, A.I., Loane, D.J. 2016. Microglial/Macrophage Polarization Dynamics following Traumatic Brain Injury. J Neurotrauma. https://doi.org/10.1089/neu.2015.4268.
Gault CR, Obeid, L.M., Hannun, Y.A. 2010. An overview of sphingolipid metabolism: from synthesis to breakdown. Adv Exp Med Biol. https://doi.org/10.1007/978-1-4419-6741-1_1.
Cartier A, Hla, T. 2019. Sphingosine 1-phosphate: Lipid signaling in pathology and therapy. Science, 366:eaar5551. https://doi.org/10.1126/science.aar5551.
Tsai HC, Nguyen, K., Hashemi, E., Engleman, E., Hla, T., Han, M.H. 2019. Myeloid sphingosine-1-phosphate receptor 1 is important for CNS autoimmunity and neuroinflammation. J Autoimmun. https://doi.org/10.1016/j.jaut.2019.06.001.
Motyl J, Strosznajder, J.B. 2018. Sphingosine kinase 1/sphingosine-1-phosphate receptors dependent signalling in neurodegenerative diseases. The promising target for neuroprotection in Parkinson's disease. Pharmacol Rep. https://doi.org/10.1016/j.pharep.2018.05.002.
Strader CR, Pearce, C.J., Oberlies, N.H. 2011. Fingolimod (FTY720): a recently approved multiple sclerosis drug based on a fungal secondary metabolite. J Nat Prod. https://doi.org/10.1021/np2000528.
McGinley MP, Cohen J.A. 2021. Sphingosine 1-phosphate receptor modulators in multiple sclerosis and other conditions. Lancet. https://doi.org/10.1016/S0140-6736(21)00244-0.
Liu J, Sugimoto K, Cao Y, Mori M, Guo L, Tan G. 2020. Serum Sphingosine 1-Phosphate (S1P): A Novel Diagnostic Biomarker in Early Acute Ischemic Stroke. Front Neurol. https://doi.org/10.3389/fneur.2020.00985.
Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. https://doi.org/10.1038/nprot.2008.73.
Li Z, Han X. 2018. Resveratrol alleviates early brain injury following subarachnoid hemorrhage: possible involvement of the AMPK/SIRT1/autophagy signaling pathway. Biol Chem. https://doi.org/10.1515/hsz-2018-0269.
Rolland WB, Lekic T, Krafft PR, Hasegawa Y, Altay O, Hartman R, Ostrowski R, Manaenko A, Tang J, Zhang JH. 2013. Fingolimod reduces cerebral lymphocyte infiltration in experimental models of rodent intracerebral hemorrhage. Exp Neurol. https://doi.org/10.1016/j.expneurol.2012.12.009.
Chen S, Ma Q, Krafft PR, Hu Q, Rolland W, 2nd, Sherchan P, Zhang J, Tang J, Zhang JH. 2013. P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH. Neurobiol Dis. https://doi.org/10.1016/j.nbd.2013.06.011.
Zheng ZV, Lyu H, Lam SYE, Lam PK, Poon WS, Wong GKC. 2020. The Dynamics of Microglial Polarization Reveal the Resident Neuroinflammatory Responses After Subarachnoid Hemorrhage. Transl Stroke Res. https://doi.org/10.1007/s12975-019-00728-5.
Dello Russo C, Cappoli N, Coletta I, Mezzogori D, Paciello F, Pozzoli G, Navarra P, Battaglia A. 2018. The human microglial HMC3 cell line: where do we stand? A systematic literature review. J Neuroinflammation. https://doi.org/10.1186/s12974-018-1288-0.
Janabi N, Peudenier S, Heron B, Ng KH, Tardieu M. 1995. Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen. Neurosci Lett. https://doi.org/10.1016/0304-3940(94)11792-h.
Obinata H, Hla T. 2019. Sphingosine 1-phosphate and inflammation. Int Immunol. https://doi.org/10.1093/intimm/dxz037.
Heo JY, Im DS. 2019. Pro-Inflammatory Role of S1P(3) in Macrophages. Biomol Ther (Seoul). https://doi.org/10.4062/biomolther.2018.215.
Dusaban SS, Chun J, Rosen H, Purcell NH, Brown JH. 2017. Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes. J Neuroinflammation. https://doi.org/10.1186/s12974-017-0882-x.
Rass V, Helbok R. 2019. Early Brain Injury After Poor-Grade Subarachnoid Hemorrhage. Curr Neurol Neurosci Rep. https://doi.org/10.1007/s11910-019-0990-3.
Zhang L, Wang H. 2020. FTY720 in CNS injuries: Molecular mechanisms and therapeutic potential. Brain Res Bull. https://doi.org/10.1016/j.brainresbull.2020.08.013.
Miron VE, Schubart A, Antel JP. 2008. Central nervous system-directed effects of FTY720 (fingolimod). J Neurol Sci. https://doi.org/10.1016/j.jns.2008.06.031.
Fu Y, Hao J, Zhang N, Ren L, Sun N, Li YJ, Yan Y, Huang D, Yu C, Shi FD. 2014. Fingolimod for the treatment of intracerebral hemorrhage: a 2-arm proof-of-concept study. JAMA Neurol. https://doi.org/10.1001/jamaneurol.2014.1065.
Zhu Z, Fu Y, Tian D, Sun N, Han W, Chang G, Dong Y, Xu X, Liu Q, Huang D, Shi FD. 2015. Combination of the Immune Modulator Fingolimod With Alteplase in Acute Ischemic Stroke: A Pilot Trial. Circulation. https://doi.org/10.1161/CIRCULATIONAHA.115.016371.
de Oliveira Manoel AL, Macdonald RL. 2018. Neuroinflammation as a Target for Intervention in Subarachnoid Hemorrhage. Front Neurol. https://doi.org/10.3389/fneur.2018.00292.
Roa JA, Sarkar D, Zanaty M, Ishii D, Lu Y, Karandikar NJ, Hasan DM, Ortega SB, Samaniego EA. 2020. Preliminary results in the analysis of the immune response after aneurysmal subarachnoid hemorrhage. Sci Rep. https://doi.org/10.1038/s41598-020-68861-y.
Pradilla G, Chaichana KL, Hoang S, Huang J, Tamargo RJ. 2010. Inflammation and cerebral vasospasm after subarachnoid hemorrhage. Neurosurg Clin N Am. https://doi.org/10.1016/j.nec.2009.10.008.
Zhao S, Adebiyi MG, Zhang Y, Couturier JP, Fan X, Zhang H, Kellems RE, Lewis DE, Xia Y. 2018. Sphingosine-1-phosphate receptor 1 mediates elevated IL-6 signaling to promote chronic inflammation and multitissue damage in sickle cell disease. FASEB J. https://doi.org/10.1096/fj.201600788RR.
Cianciulli A, Porro C, Calvello R, Trotta T, Lofrumento DD, Panaro MA. 2020. Microglia Mediated Neuroinflammation: Focus on PI3K Modulation. Biomolecules. https://doi.org/10.3390/biom10010137.
Li Q, Li Y, Lei C, Tan Y, Yi G. 2021. Sphingosine-1-phosphate receptor 3 signaling. Clin Chim Acta. https://doi.org/10.1016/j.cca.2021.03.025.

No competing interests reported.

Onlineresource.pdf

Download PDF

Version 1

posted

You are reading this latest preprint version

S1PR3/RhoA signaling pathway in microglia mediates inflammatory activation in early brain injury after subarachnoid hemorrhage

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Results

Discussion

Statements and Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1