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Sphingosine 1-phosphate receptor subtype 3 (S1P₃) contributes to brain injury after transient focal cerebral ischemia via modulating microglial activation and their M1 polarization.

ABSTRACT:

Background

The pathogenic roles of receptor-mediated sphingosine 1-phosphate (S1P) signaling in cerebral ischemia have been evidenced mainly through the efficacy of FTY720 that binds non-selectively to four of the five S1P receptors (S1P_1,3,4,5). Recently, S1P₁ and S1P₂ were identified as specific receptor subtypes that contribute to brain injury in cerebral ischemia; however, the possible involvement of other S1P receptors remains unknown. S1P₃ can be the candidate because of its upregulation in the ischemic brain, which was addressed in this study, along with underlying pathogenic mechanisms.

Methods

We used transient middle cerebral artery occlusion/reperfusion (tMCAO), a mouse model of transient focal cerebral ischemia. To identify S1P₃ as a pathogenic factor in cerebral ischemia, we employed a specific S1P₃ antagonist, CAY10444. Brain damages were assessed by brain infarction, neurological score, and neurodegeneration. Histological assessment was carried out to determine microglial activation, morphological transformation, and proliferation. M1/M2 polarization and relevant signaling pathways were determined by biochemical and immunohistochemical analysis.

Results

Inhibiting S1P₃ immediately after reperfusion with CAY10444 significantly reduced tMCAO-induced brain infarction, neurological deficit, and neurodegeneration. When S1P₃ activity was inhibited, the number of activated microglia was markedly decreased in both the periischemic and ischemic core regions in the ischemic brain 1 and 3 days following tMCAO. Moreover, inhibiting S1P₃ significantly restored the microglial shape from amoeboid to ramified microglia in the ischemic core region 3 days after tMCAO, and it attenuated microglial proliferation in the ischemic brain. In addition to these changes, S1P₃ signaling influenced the proinflammatory M1 polarization, but not M2. The S1P₃-dependent regulation of M1 polarization was clearly shown in activated microglia, which was affirmed by determining the in vivo activation of microglial NF-?B signaling that is responsible for M1 and in vitro expression levels of proinflammatory cytokines in activated microglia. As downstream effector pathways in an ischemic brain, S1P₃ influenced phosphorylation of ERK1/2, p38 MAPK, and Akt.

Conclusions

This study identified S1P₃ as a pathogenic mediator in an ischemic brain along with underlying mechanisms, involving its modulation of microglial activation and M1 polarization, further suggesting that S1P₃ can be a therapeutic target for cerebral ischemia.

SUBMITTER: Gaire BP

PROVIDER: S-EPMC6180378 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Project description:Hyperglycemia aggravates hepatic ischemia/reperfusion injury (IRI), but the underlying mechanism for the aggravation remains elusive. Sphingosine-1-phosphate (S1P) and sphingosine-1-phosphate receptors (S1PRs) have been implicated in metabolic and inflammatory diseases. Here, we discuss whether and how S1P/S1PRs are involved in hyperglycemia-related liver IRI. For our in vivo experiment, we enrolled diabetic patients with benign hepatic disease who had liver resection, and we used streptozotocin (STZ)-induced hyperglycemic mice or normal mice to establish a liver IRI model. In vitro bone marrow-derived macrophages (BMDMs) were differentiated in high-glucose (HG; 30 mM) or low-glucose (LG; 5 mM) conditions for 7 days. The expression of S1P/S1PRs was analyzed in the liver and BMDMs. We investigated the functional and molecular mechanisms by which S1P/S1PRs may influence hyperglycemia-related liver IRI. S1P levels were higher in liver tissues from patients with diabetes mellitus and mice with STZ-induced diabetes. S1PR3, but not S1PR1 or S1PR2, was activated in liver tissues and Kupffer cells under hyperglycemic conditions. The S1PR3 antagonist CAY10444 attenuated hyperglycemia-related liver IRI based on hepatic biochemistry, histology, and inflammatory responses. Diabetic livers expressed higher levels of M1 markers but lower levels of M2 markers at baseline and after ischemia/reperfusion. Dual-immunofluorescence staining showed that hyperglycemia promoted M1 (CD68/CD86) differentiation and inhibited M2 (CD68/CD206) differentiation. Importantly, CAY10444 reversed hyperglycemia-modulated M1/M2 polarization. HG concentrations in vitro also triggered S1P/S1PR3 signaling, promoted M1 polarization, inhibited M2 polarization, and enhanced inflammatory responses compared with LG concentrations in BMDMs. In contrast, S1PR3 knockdown significantly retrieved hyperglycemia-modulated M1/M2 polarization and attenuated inflammation. In conclusion, our study reveals that hyperglycemia specifically triggers S1P/S1PR3 signaling and exacerbates liver IRI by facilitating M1 polarization and inhibiting M2 polarization, which may represent an effective therapeutic strategy for liver IRI in diabetes.

Project description:BACKGROUND:Lysophosphatidic acid receptor 1 (LPA1) is in the spotlight because its synthetic antagonist has been under clinical trials for lung fibrosis and psoriasis. Targeting LPA1 might also be a therapeutic strategy for cerebral ischemia because LPA1 triggers microglial activation, a core pathogenesis in cerebral ischemia. Here, we addressed this possibility using a mouse model of transient middle cerebral artery occlusion (tMCAO). METHODS:To address the role of LPA1 in the ischemic brain damage, we used AM095, a selective LPA1 antagonist, as a pharmacological tool and lentivirus bearing a specific LPA1 shRNA as a genetic tool. Brain injury after tMCAO challenge was accessed by determining brain infarction and neurological deficit score. Role of LPA1 in tMCAO-induced microglial activation was ascertained by immunohistochemical analysis. Proinflammatory responses in the ischemic brain were determined by qRT-PCR and immunohistochemical analyses, which were validated in vitro using mouse primary microglia. Activation of MAPKs and PI3K/Akt was determined by Western blot analysis. RESULTS:AM095 administration immediately after reperfusion attenuated brain damage such as brain infarction and neurological deficit at 1?day after tMCAO, which was reaffirmed by LPA1 shRNA lentivirus. AM095 administration also attenuated brain infarction and neurological deficit at 3?days after tMCAO. LPA1 antagonism attenuated microglial activation; it reduced numbers and soma size of activated microglia, reversed their morphology into less toxic one, and reduced microglial proliferation. Additionally, LPA1 antagonism reduced mRNA expression levels of proinflammatory cytokines and suppressed NF-?B activation, demonstrating its regulatory role of proinflammatory responses in the ischemic brain. Particularly, these LPA1-driven proinflammatory responses appeared to occur in activated microglia because NF-?B activation occurred mainly in activated microglia in the ischemic brain. Regulatory role of LPA1 in proinflammatory responses of microglia was further supported by in vitro findings using lipopolysaccharide-stimulated cultured microglia, showing that suppressing LPA1 activity reduced mRNA expression levels of proinflammatory cytokines. In the ischemic brain, LPA1 influenced PI3K/Akt and MAPKs; suppressing LPA1 activity decreased MAPK activation and increased Akt phosphorylation. CONCLUSION:This study demonstrates that LPA1 is a new etiological factor for cerebral ischemia, strongly indicating that its modulation can be a potential strategy to reduce ischemic brain damage.

Dataset Information

Sphingosine 1-phosphate receptor subtype 3 (S1P₃) contributes to brain injury after transient focal cerebral ischemia via modulating microglial activation and their M1 polarization.

Background

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Sphingosine 1-phosphate receptor subtype 3 (S1P3) contributes to brain injury after transient focal cerebral ischemia via modulating microglial activation and their M1 polarization.

Background

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Sphingosine 1-phosphate receptor subtype 3 (S1P₃) contributes to brain injury after transient focal cerebral ischemia via modulating microglial activation and their M1 polarization.