Dectin-1/Syk signaling triggers neuroinflammation after ischemic stroke in mice

doi:10.21203/rs.2.17199/v1

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Dectin-1/Syk signaling triggers neuroinflammation after ischemic stroke in mice

https://doi.org/10.21203/rs.2.17199/v1

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published 11 Jan, 2020

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Background: Dendritic cell-associated C-type lectin-1 (Dectin-1) receptor has been reported to be involved in neuroinflammation in Alzheimer's disease and traumatic brain injury. The present study was designed to investigate the role of Dectin-1 and its downstream target spleen tyrosine kinase (Syk) in early brain injury after ischemic stroke using a focal cortex ischemic stroke model.

Methods: Adult male C57BL/6J mice were subjected to a cerebral focal ischemia model of ischemic stroke. The neurological score, adhesive removal test, and foot-fault test were evaluated on days 1, 3, 5, and 7 after ischemic stroke. Dectin-1, Syk, phosphorylated (p)-Syk, tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression was analyzed by western blotting in ischemic brain tissue after ischemic stroke and in BV2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro. The brain infarct volume and Iba1-positive cells were evaluated using Nissl’s and immunofluorescence staining, respectively. The Dectin-1 antagonist laminarin (LAM) and a selective inhibitor of Syk phosphorylation (piceatannol, PIC) were used for intervention.

Results: Dectin-1, Syk, and p-Syk expression was significantly enhanced on days 3, 5, and 7 and peaked on day 3 after ischemic stroke. The Dectin-1 antagonist LAM or Syk inhibitor PIC decreased the number of Iba1-positive cells and TNF-α and iNOS expression, reduced the brain infarct volume and improved neurological functions on day 3 after ischemic stroke. In addition, the in vitro data showed that Dectin-1, Syk, and p-Syk expression was increased following the 3-hour OGD followed by 0, 3, and 6 hours of reperfusion in BV2 microglial cells. LAM and PIC also reduced TNF-α and iNOS expression 3 hours after OGD/R induction.

Conclusion: Dectin-1/Syk signaling plays a crucial role in inflammatory activation after ischemic stroke, and further investigation of Dectin-1/Syk signaling in stroke is warranted.

Neurobiology of Disease

Dectin-1

Syk

inflammation

ischemic stroke

In recent decades, ischemic stroke has become one of the most common causes of disability and mortality worldwide. Although the pathophysiology of cerebral ischemia injury is multifactorial, increasing studies have suggested that the inflammatory response plays a crucial role in stroke progression [1, 2]. Inflammation in the brain parenchyma after cerebral ischemia is mediated by neurons, endothelial cells, microglia and other immune cells [3], and inhibition of inflammatory responses has been shown to improve the outcome after stroke [4, 5]. However, the detailed mechanisms by which the inflammatory response is triggered after stroke remain largely unknown.

Dendritic cell (DC)-associated C-type lectin–1 (Dectin–1) has been identified as an immune-receptor tyrosine-based activation motif (ITAM)-coupled C-type lectin receptor (CLR). Dectin–1 recognizes various danger-associated molecular patterns and triggers inflammatory signals by recruiting its downstream molecular tyrosine kinase (spleen tyrosine kinase, Syk) [6–8]. Dectin–1 is a type II transmembrane receptor that is expressed on different cell types during inflammation, including DCs, neutrophils, monocytes, T cells, and epithelial cells [9–12]. Previous studies have reported that immune receptors that initiate the inflammatory response play an important role in stroke progression [13–16]. However, whether the immune receptor Dectin–1 is involved in the inflammatory response after stroke has not been investigated.

Syk is a nonreceptor protein tyrosine kinase that is found extensively in hematopoietic and nonhematopoietic cells [17, 18]. It possesses tandem N-terminal src homology–2 (SH2) domains that can bind to ITAMs [19, 20]. Incomplete ITAMs, referred to as hemITAMs, can interact with Syk and mediate its activation [21]. A number of C-type lectin receptors, such as Dectin–1, C-type lectin-like receptor 2 (CLEC2), and C-type lectin domain containing 9A (CLEC9A), possess hemITAMs and transduce signals through Syk [22]. Syk has been reported to play an indispensable role in acute and chronic inflammation, and inhibition of Syk impedes brain tissue damage after ischemic stroke [23–26]. However, the specific role of Dectin–1 and its downstream target Syk in ischemic stroke has not yet been investigated.

The present study reveals that the Dectin–1/Syk pathway is involved in neuroinflammation following ischemic stroke. Changes in Dectin–1, Syk, p-Syk, TNF-α and iNOS expression following ischemic stroke were evaluated in vivo and in vitro. In addition, the present study investigated whether the Dectin–1 antagonist laminarin (LAM) or the Syk inhibitor piceatannol (PIC) could partly reverse neuroinflammation following ischemic stroke in mice. The working model for this study is available in figure 1.

Cerebral focal ischemia. Adult male C57BL/6J mice (weight, 22–30 g) were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). The cerebral focal ischemia model was induced by photothrombotic ischemia as previously described [27]. The main steps are as follows. The mice were injected with 1% Rose Bengal [100 mg/kg intraperitoneally (i.p.) dissolved in 0.9% saline] (Sigma-Aldrich; Germany) after induction of anesthesia with 10% chloral hydrate [300 mg/kg (i.p.) dissolved in 0.9% saline]. After 10 min, the sensorimotor region, which is located ~2 mm lateral to the bregma, was exposed for 15 min to cold light. To investigate the optimal ischemia time, the mice were made ischemic at 6 h and 1, 3, 5 and 7 days. The animals were randomly divided into the following four groups: i) sham group; ii) ischemic group with vehicle treatment (ischemia + saline/DMSO group); iii) ischemia group; and iv) Dectin–1 antagonist treatment group/Syk inhibitor treatment group (ischemia + LAM/PIC group).

Neurological function tests. In all animals, the adhesive removal and foot-fault tests were performed and the modiﬁed neurological severity score (mNSS) was obtained before stroke and at 1, 3, 5 and 7 days after stroke with or without LAM/PIC treatment. The mNSS is a composite that is used to assess neurological functions based on motor, sensory, balance, and reﬂex measures, which are graded on a scale of 0 to 18 (normal score, 0; maximal deﬁcit score, 18); higher scores imply greater neurological injury [28, 29].

Analysis of the brain infarct volume. Formaldehyde-fixed specimens were cut into 50-μm-thick sections and subjected to Nissl’s staining. The sections were placed in 100%, 95%, and 80% ethanol for 30 seconds each and then treated with FD Cresyl Violet Solution^TM (FD Neoro Technologies, Columbia, MD, USA) for 2 minutes. After washing in distilled water, the sections were dehydrated through an alcohol series, cleared in xylene and coverslipped with neutral resin. We scanned the stained sections and measured the infarct areas using the ImageJ software. The total infarct volume was obtained by indirect methods as previously described [27].

Drug administration in vivo and in vitro. The Dectin–1 receptor antagonist LAM (Sigma-Aldrich; Merck) was diluted to 10 mg/ml in vehicle (saline) according to the manufacturer’s instructions. Then, LAM at 300 mg/kg/day or the same volume of vehicle (saline) was injected i.p. 1 h after ischemic stroke and once daily for the 2 subsequent days after stroke. For the in vitro experiment, the cells were preincubated with LAM for 1 h before induction with oxygen-glucose deprivation/reoxygenation (OGD/R) or lipopolysaccharide (LPS, 1,000 ng/ml). The optimal treatment time was selected for the subsequent in vivo experiments. For the in vitro experiments, LAM (300 μg/ml) was added to BV2 microglial cells for 1 h, followed by stimulation with or without OGD/R or LPS.

Similarly, the mice were treated with either the Syk inhibitor (PIC; Selleck Chemicals, Houston, TX, USA) or vehicle (DMSO) once daily after ischemic stroke. For the in vitro experiment, the PIC dose was 20 mg/kg/day. Next, the optimal treatment time was selected for the subsequent in vivo experiments. For the in vitro experiments, BV2 cells were pretreated with 25 μM PIC for 1 h before treatment with or without OGD/R or LPS.

Cell culture and experimental protocols. BV2 microglial cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose supplemented with 10% fetal bovine serum, 100 U/ml of penicillin and 100 mg/l of streptomycin at 37°C in a 5% CO₂ incubator. Mouse primary microglial cells were prepared as described previously [30, 31]. Briefly, brains were removed from mice at post-natal day 1–3 and the cortices were triturated into single cells. Mixed glial were plated in 25T culture flasks coated with poly-D-lysine and grown in DMEM/F12 with 10% fetal bovine serum, GlutaMAX (Invitrogen) and 1% penicillin/streptomycin in a 5% CO₂/37 °C incubator, changing medium after 7 days, for a total of 2 weeks. To harvest primary microglial cells, the flask was shaken at 150 rpm for 2 h. The fluid medium was subsequently collected and centrifuged at 1000 rpm for 10 min. The cell pellets were resuspended to plate 5 × 10⁵ cells per well onto 6-well plates, and subjected to various treatments within 24 h of harvest. Cells were pretreated with LAM or PIC for 1 h and then subjected to OGD/R or LPS treatment. For OGD/R, the cells were washed twice with PBS, placed in glucose-free DMEM and then exposed to hypoxia (94% N₂, 5% CO₂, and 1% O₂) at 37°C in a humidified chamber (Thermo Fisher Scientific, Inc.) for 3 h. Then, OGD was terminated, and the cells were exposed to normal culture conditions (37°C, 95% air, and 5% CO₂) for 3 h. For LPS treatment, the cells were exposed to LPS after 1 h of LAM or PIC treatment, followed by incubation under normal culture conditions for 24 h. Then, the BV2 cells were randomly divided into the following groups: i) normal control group (control group); ii) LAM-pretreated control group (control + LAM group); iii) OGD/R or LPS group; iv) LAM-pretreated OGD/R or LAM-pretreated LPS group (LAM + OGD/R or LPS group); v) PIC-pretreated control group (control + PIC group); and vi) PIC-pretreated OGD/R or LPS group (PIC + OGD/R or LPS group).

RNA interference experiment. The siRNA gene silencing in BV2 cells was performed as previously described[32, 33]. Briefly,BV2 Cells were seeded into a six-well plate (Corning, Corning, NY, USA) at a density of 1.5 × 10⁵cells/well 24 h prior to transfection. BV2 cells were transfected by Lipofectamine 2000 (Invitrogen) with siRNAs targeting to Dectin–1 (sense: 5’-GGGAAGAGCUGUUACCUAUTT–3’; antisense: 5’-AUAGGUAACAGCUCUUCCCTT–3’), Syk (sense: 5’-CCUGCUGCACGAAAGGGAAATT–3’; antisense: 5’- UUUCCCUUCGUGCAGCAGGTT–3’) or control siRNA (sense: 5’-UUCUCCGAACGUGUCACGUTT–3’; antisense: 5’-ACGUGACACGUUCGGAGAATT–3’) as negative control. Two days after transfection, the knockdown efficiency of siRNA was determined by Western-blot analysis. We assigned the cells into the following groups: i) control siRNA group; ii) Dectin–1 or Syk siRNA group; iii) OGD/R + control siRNA group; iv) OGD/R + Dectin–1 or Syk siRNA group.

Western blotting. Total proteins from the ischemic cortical area brain tissues or BV2 microglial cells were collected in equal amounts of cell lysate, and the separated proteins in the supernatant were subsequently transferred. For immunoblotting, the following primary antibodies were used: anti-Dectin–1 (1:1,000; Abcam, Cambridge, UK), anti-Syk (1:1,000;Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-Syk (1:1,000; Cell Signaling Technology, Inc.), anti-TNF-α (1:1,000; Cell Signaling Technology, Inc.) and anti-iNOS (1:1,000; Cell Signaling Technology, Inc.). Western blotting was performed at 6 h and 3, 5 and 7 days after ischemic stroke for the mouse tissues and at 0, 3, 6 and 12 h after OGD/R treatment and 3, 6, 12 and 24 h after LPS treatment for the BV2 microglial cells. Then, the optimal time points of 3 days for the in vivo experiments and 3 h for OGD/R treatment and 24 h for LPS treatment in vitro were selected for the subsequent experiments.

Immunofluorescence analysis. Brain tissue was removed and then immersed in 4% paraformaldehyde for 5 hours and 20% sucrose for 3 d at 4°C to process the samples for immunofluorescence. Following standard histological procedures, the sections were preincubated with 5% goat serum at room temperature for 60 min. Subsequently, the sections were incubated overnight at 4°C in a mixture containing a rabbit polyclonal anti-Iba1 antibody (1:500; Abcam) and a rat polyclonal anti-CD68 antibody (1:500; Abcam). After washing 3 times in PBS for 10 min per wash, the sections were incubated with a fluorescent secondary immunoglobulin G antibody (polyclonal anti-rabbit Alexa Fluor 594-conjugated red antibody; 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA) for 2 h at room temperature. After washing 3 times in PBS for 10 min per wash, the sections were stained with DAPI, sealed and mounted, and imaged using a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The numbers of target cells were counted using the ImageJ software.

Statistical analysis. The data are presented as the mean ± standard deviation from 3 independent experiments. Comparisons between groups were performed using one-way ANOVA followed by the Student-Newman-Keuls test, and changes in the behavioral responses to drug stimuli over time among groups were tested using two-way ANOVA with repeated measures followed by the Bonferroni post hoc test. The statistical significance of differences was analyzed with the SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Images were created with the GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). p<0.05 was considered a statistically significant difference.

Dectin–1 was significantly increased in the ischemic brain tissue after stroke and the BV2 microglial cells after OGD/R-induced injury in vitro.

To investigate the potential role of Dectin–1 in the progression of ischemic stroke, first we examined the Dectin–1 protein levels in ischemic brain tissue and BV2 cells in the OGD/R model. Fig. 2A-B showed that the Dectin–1 expression level was significantly higher in the ischemic group than in the sham group; the expression level increased significantly at day 1, peaked at day 3, and decreased at day 5 after ischemic stroke (n = 3/group, p<0.05). Fig. 2C-D showed that the Dectin–1 expression level was higher in the BV2 cells after 3-hour OGD followed by 0, 3, and 6 hours of reperfusion in the OGD/R group than in the control group cells (n = 3/group, p < 0.05). These results demonstrate increased Dectin–1 expression in response to stroke.

Syk and p-Syk were significantly upregulated in the ischemic brain tissue after stroke and BV2 microglial cells with OGD/R-induced injury in vitro.

To investigate whether Syk signaling was involved in the progression of ischemic stroke, the Syk and p-Syk protein levels were tested in ischemic brain tissue and BV2 cells in the OGD/R model. Fig. 3A-B showed that the Syk expression was significantly increased at days 1, 3, 5, and 7 after stroke in the ischemic group compared to that of the sham group (n = 3/group, p<0.05). Additionally, p-Syk expression was significantly enhanced at days 3, 5, and 7 after stroke. Fig. 3C-D showed that the Syk and p-Syk expression levels were higher in the BV2 cells subjected to a 3-hour OGD followed by a 0, 3, or 6-hour reperfusion in the OGD/R group compared to those of the control group cells (n = 3/group, p < 0.05). Therefore, the Syk and p-Syk expression levels are enhanced after stroke, and day 3 after stroke in the in vivo study and a 3-hour OGD followed by a 3-hour reperfusion in the in vitro study were chosen for the subsequent experiments.

Blockage of Dectin–1 rescued the brain infarct volume and neurological impairment after stroke.

To further assess the potential role of Dectin–1 in ischemic stroke, we examined the brain infarct volume and neurological impairment using neurological functional tests in ischemic mice with or without LAM (Dectin–1 antagonist) treatment. Fig. 4A-B showed that the infarct volume was reduced in the ischemic mice with LAM treatment compared to that of the ischemic mice without treatment (n = 3/group, p<0.05). Fig. 4C-E showed that neurological function was significantly improved in the ischemic mice with LAM treatment compared to that of the ischemic mice without treatment (n = 9/group, p<0.05). These results suggest that Dectin–1 may have a deleterious effect on the pathophysiological process of stroke.

Blockade of Decin–1 inhibited Dectin–1/Syk signaling.

To further assess the potential role of Dectin–1 in ischemic stroke, Dectin–1/Syk signaling was examined by western blotting. As shown in Fig. 5A-B, Dectin–1 and p-Syk expression in the ischemic brain tissue was significantly enhanced in the ischemia and ischemia + saline groups compared to that of the sham group (n = 3/group, p<0.05). However, LAM (dectin–1 antagonist) treatment significantly reduced Dectin–1 and p-Syk expression in the ischemia group compared to that of the ischemia and the ischemia + saline groups (n = 3/group, p<0.05). Dectin–1 and p-Syk expression was significantly increased in in the BV2 cells following OGD/R exposure. LAM pretreatment obviously reduced the expression of these proteins in the BV2 cells following OGD/R exposure (Fig. 5C-D, n = 3/group, p<0.05). Taken together, these results provide evidence for the potential involvement of Dectin–1/Syk signaling in ischemic stroke.

Blockade of Decin–1 diminished microglial activation and reduced the expression levels of inflammatory cytokines.

To further investigate the potential role of Dectin–1 in ischemic stroke, microglial activation was assessed by Iba–1 and CD68 staining, and the expression of inflammatory cytokines was examined by western blotting. Three days after stroke, Iba–1 and CD68 positive cells were detected in the ischemic brain tissue, although the number of these cells was largely decreased after LAM (dectin–1 antagonist) treatment (Fig. 6A-B). Fig. 6C-D showed that TNF-α and iNOS expression was significantly enhanced in the ischemia and ischemia + saline groups compared to that of the sham group (n = 3/group, p<0.05). LAM treatment significantly reduced TNF-α and iNOS expression in the ischemia group compared to that of the ischemia and ischemia + saline groups (n = 3/group, p<0.05). Fig. 6E-F showed similar results for BV2 cells exposed to OGD/R in the in vitro experiments. Taken together, these results suggest that Dectin–1 is involved in the inflammatory response after stroke.

Blockade of Syk partly reduced the brain infarct volume and improved the neurological outcomes after stroke.

To further explore whether Dectin–1/Syk signaling played a crucial role in the progression of ischemic stroke, PIC (Syk inhibitor) was applied, and the brain infarct volume and neurological functional tests were evaluated. Fig. 7A-B indicated that the infarct volume was reduced in ischemic mice with PIC treatment compared to that of ischemic mice without treatment (n = 3/group, p<0.05). Fig. 7C-E showed that functional impairments after stroke were significantly attenuated at days 3, 5 and 7 after PIC treatment in the ischemic stroke model mice compared to those of the mice in the ischemia and ischemia + DMSO groups (n = 9/group，p<0.05).

PIC treatment attenuated p-Syk expression in ischemic brain tissue and in BV2 cells with OGD/R exposure.

To investigate whether Dectin–1/Syk signaling played a crucial role in the progression of ischemic stroke, PIC (Syk inhibitor) was applied, and Dectin–1/Syk signaling was examined by western blotting. Fig. A-B: p-Syk expression was significantly enhanced in the ischemic brain tissue in the ischemia and ischemia + DMSO groups compared to that of the sham group. PIC treatment significantly reduced p-Syk expression in the ischemic brain tissue (n = 3/group, *p<0.05 vs sham group; #p<0.05 vs ischemia group/ischemia + DMSO group). However, PIC treatment failed to reduce Dectin–1 expression in the ischemic brain tissue. C-D: Dectin–1 and p-Syk expression in BV2 cells with OGD/R exposure was significantly increased compared to that of the cells without OGD/R exposure. PIC pretreatment of BV2 cells with OGD/R exposure significantly reduced p-Syk expression (n = 3/group, *p<0.05 vs control group; #p<0.05 vs OGD/R group). However, Dectin–1 expression was not attenuated in the OGD/R-induced BV2 cells with PIC treatment compared to that of the OGD/R-induced BV2 cells without PIC pretreatment.

Reduction of microglial activation and inflammatory cytokine expression by inhibition of Syk signaling.

To further study the role of Dectin–1/Syk signaling in ischemic stroke, the Syk inhibitor PIC was used. Fig. 9A-B showed that PIC treatment significantly reduced the numbers of Iba–1 and CD68 positive cells in the ischemic brain tissue in the ischemia group compared to that of the ischemia and ischemia + DMSO groups (n = 3/group, p<0.05). Fig. 9C-D showed that TNF-α and iNOS expression was significantly reduced in the ischemic brain tissue in the ischemia + PIC group compared to that of the ischemia and ischemia + DMSO groups (n = 3/group, p<0.05). Fig. 9E-F showed similar results for the BV2 cells with OGD/R exposure in the in vitro experiments. Taken together, these results suggest the involvement of Dectin–1/Syk signaling in microglial activation and enhanced inflammatory cytokine expression after stroke.

LAM and PIC reduced TNF-α and iNOS production by inhibiting Dectin–1/Syk signaling in primary microglial cells with OGD/R-induced injury in vitro

To further study the proinflammatory effect of Dectin–1/Syk signaling after primary microglial cells were exposed to OGD/R-induced inflammation. As shown in Fig. 10A and B, the Dectin–1, p-Syk,TNF-α and iNOS expression levels were significantly increased in primary microglial cells after OGD/R induction. In addition, LAM pretreatment significantly reduced the Dectin–1 and p-Syk expression levels. At the same time, TNF-α and iNOS production was significantly reduced in primary microglial cells after LAM treatment (n = 3/group, p<0.05). Fig. 10C and D showed that p-Syk expression was significantly inhibited after PIC pretreatment, and PIC treatment also significantly reduced p-Syk, TNF-α and iNOS production in the OGD/R-induced primary microglial cells (n = 3/group, p<0.05).

Knockdown Dectin–1 or Syk inhibited production of proinflammatory cytokines TNF-α and iNOS in BV2 microglial cellls after OGD/R stimulation.

To further precisely evaluate the function of Dectin–1/Syk signaling to regulate TNF-α and iNOS, BV2 cells were stimulated with OGD/R after transfection with Dectin–1 siRNA or Syk siRNA. Fig.11A and B showed that knockdown Dectin–1 downregulated the expression of p-Syk, TNF-α and iNOS in BV2 cells in the OGD/R + Dectin–1 siRNA group compared to that of the OGD/R + control siRNA group (n = 3/group, p<0.05). Similarly, we found that the production of p-Syk, TNF-α and iNOS in BV2 cells were significantly decreased in OGD/R + Syk siRNA group compared to that of the OGD/R + Control siRNA group. We also found that the Dectin–1 expression was significantly reduced in BV2 cells in Dectin–1 siRNA group compared to Control siRNA group, and the Syk expression was reduced in BV2 cells in Syk siRNA group compared to that of the Control siRNA group (n = 3/group, p<0.05). These data suggested that knockdown the Dectin–1 or Syk ameliorate inflammation progression in BV2 microglial cells with OGD/R treatment.

LAM and PIC reduced TNF-α and iNOS production by inhibiting Dectin–1/Syk signaling in BV2 microglial cells with LPS-induced injury in vitro

To further investigate the proinflammatory effect of Dectin–1/Syk signaling in brain tissue after stroke, BV2 cells were exposed to LPS-induced inflammation. As shown in Fig. 12A and B, the Dectin–1 and p-Syk expression levels were significantly increased in the BV2 cells at 3, 6, 12 and 24 h after LPS induction. Twenty-four hours was chosen as the LPS induction time point in BV2 cells in the subsequent experiments. Fig. 12C and D showed that LAM pretreatment significantly reduced the Dectin–1 and p-Syk expression levels. At the same time, TNF-α and iNOS production was significantly reduced in the BV2 cells after LAM treatment. Fig. 12E and F showed that p-Syk expression was significantly inhibited after PIC pretreatment, but no significant difference in Dectin–1 expression was found between the LPS and LPS + PIC groups. PIC treatment also significantly reduced TNF-α and iNOS production in the LPS-induced BV2 cells.

The present study showed that the Dectin–1, Syk, p-Syk, TNF-α, and iNOS expression levels were signiﬁcantly increased in ischemic brain tissue after stroke. Either Dectin–1 antagonist (LAM) or Syk inhibitor (PIC) treatment significantly reduced the expression levels of these proteins in the ischemic brain tissue. At the same time, we also found that LAM or PIC treatment significantly reduced the infarct volume and improved the functional outcomes after stroke. Similar results were observed in the in vitro experiments. Taken together, the in vivo and complementary in vitro data support the hypothesis that Dectin–1/Syk signaling plays a vital role in neuroinflammation after stroke.

Microglia are the resident mononuclear phagocytic cells that are critical for inflammatory responses in the central nervous system (CNS). Microglial most common distinguishing feature is their rapid activation in response to pathological changes in the CNS such as ischemia[34]. Dectin–1 is expressed at a low level in the brain but is strongly upregulated after exposure to various stimuli, such as ischemia and injury [35, 36]. Dectin–1 has been reported to activate the NLRP3 (NLR family, pyrin domain-containing 3) inflammasome and enhance IL–1β production [37–39]. Activation of Dectin–1 can cause macrophage-mediated demyelination and axonal injury, and blockade of Dectin–1 can reduce inflammatory macrophage-mediated injury after spinal cord injury [40]. Low fat diet with caloric restriction reduces the expression of Dectin–1 and then attenuates white matter microglia activation during aging [41]. It has also shown that Dectin–1 signaling can trigger neuroinflammation and enable repair of injured central nervous system neurons[42]. In this study, we found that Dectin–1 was significantly increased in ischemic brain tissue after stroke, and in the OGD/R treated BV2 cells and primary microglia. The number of activated microglia were also enhanced after stroke. Dectin–1 antagonist (LAM) treatment significantly reduced the expression of the above proteins and attenuated the number of activated microglia. We also found that blockade of Dectin–1 attenuated the brain infarct volume and reduced neurological deficits and microglial activation after stroke. These data suggest that Dectin–1 overexpression may exert deleterious effects on the brain tissue and enhance neuroinflammation after ischemic stroke.

Syk, highly expressed in the microglia, plays a vital role in the inflammatory responses after ischemic stroke [43]. The ligands produced by necrotic cells bind to the receptor of Dectin–1, resulting in recruitment and activation of Syk [44–46]. Syk inhibitor PIC reduces neuronal damage after retinal ischemia-reperfusion injury [47, 48]. Previous studies have shown that Dectin–1 can activate Syk-dependent intracellular signaling cascades [49]. Moreover, the Dectin–1/Syk signaling pathway is involved in ROS generation and NLRP3 activation in response to β-glucan particles [37, 50, 51]. Activated Syk (p-Syk) leads to subsequent activation of downstream signaling molecules, such as p85, PKB, PDK1, and NF-κB, resulting in the expression of proinflammatory genes, including TNF-α, COX–2, and iNOS [52–55]. In the present study, we found that Syk and p-Syk were significantly increased in the ischemic brain tissue after stroke and in the OGD/R model. Syk inhibitor (PIC) treatment significantly attenuated p-Syk, TNF-α and iNOS expression in the ischemic brain tissue after stroke and in the OGD/R model. Other inflammatory mediators such as JNK, p38 MAPK, and NF-kB have been linked to microglia activation[56–61] and would be assessed in the future work. We also found that the Syk inhibitor reduced microglial activation, the brain infarct volume and neurological deficits after stroke. These data suggest that Syk may also exert harmful effects on brain tissue and enhance neuroinflammation after ischemic stroke.

Our work showed that Dectin–1, Syk and p-Syk expression was signiﬁcantly increased after stroke both in vivo and in vitro. Treatment of ischemic stroke with a Dectin–1 antagonist or a Syk inhibitor signiﬁcantly reduced microglial activation, the brain infarct volume, neurological impairment, and production of the proinflammatory cytokines TNF-α and iNOS after ischemic stroke. This study may offer new ideas for effective treatment of patients who have suffered from stroke. Further studies of the pathophysiological functions of Dectin–1/Syk signaling in the activated inflammatory response may prove beneficial for clinical applications.

Dectin–1: Dendritic cell-associated C-type lectin–1 Syk: Spleen tyrosine kinase

p-Syk: Phosphorylated spleen tyrosine kinase TNF-α: Tumor necrosis factor-α

iNOS: Inducible nitric oxide synthase OGD/R: Oxygen-glucose deprivation/reoxygenation

LAM: Laminarin PIC: Piceatannol LPS: Lipopolysaccharide

DC: Dendritic cell ITAM: Immune-receptor tyrosine-based activation motif

CLR: C-type lectin receptor SH2: Src homology–2 domains

CLEC–2: C-type lectin-like receptor 2 CLEC9A: C-type lectin domain containing 9A

DMSO: Dimethyl sulfoxide mNSS: Modiﬁed neurological severity score

DMEM: Dulbecco’s modified Eagle’s medium PBS: Phosphate-buffered saline

PKB: Protein kinase B PDK1: Phosphoinositide-dependent protein kinase–1

NF-κB: Nuclear factor kappa B COX–2: Cyclooxygenase–2

ROS: Reactive oxygen species NLRP3: NLR family pyrin domain-containing 3

DAMPs: Damage-associated molecular patterns

Acknowledgements

We thank members of the laboratory of Dr. Cui for helpful discussions and suggestions.

Funding

This research was supported by the National Natural Science Foundation of China (81971134 and 81571155 to Dr. Ye and 81571210 and 81771282 to Dr. Cui), the National Natural Science Foundation of Jiangsu Province (Nos. BK20191152, BL2014031), the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (No. 17KJB320017), Jiangsu Provincial Medical Youth Talent (No. QNRC2016788), Jiangsu Commission of Heath (No. LGY2019086), Xuzhou key research and development program (Nos. KC18055, KC19131), Xuzhou Innovation Capacity Building Program (No. KC19239), Suqian Science and Technology Plan (S201714)，and the Summit of Six Top Talents Program of Jiangsu Province (No. 2017-WSN–118).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors’ contributions

Xin-chun Ye and Qi Hao contributed equally to this work. Both authors read and approved the final manuscript.

Ethics approval and consent to participate

All animal studies were conducted in compliance with protocols approved by the Jiangsu Provincial Animal Care, and all experiments conducted were in accordance with the standards and procedures of the ethics committee of Xuzhou Medical University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Dectin-1/Syk signaling triggers neuroinflammation after ischemic stroke in mice

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