Dectin-1/Syk signaling triggers neuroinflammation after ischemic stroke in mice
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.
Adult male C57BL/6 J 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 via 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 the intervention.
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, decreased the brain infarct volume, and improved neurological functions on day 3 after ischemic stroke. In addition, the in vitro data revealed that Dectin-1, Syk, and p-Syk expression was increased following the 3-h OGD and 0, 3, and 6 h of reperfusion in BV2 microglial cells. LAM and PIC also decreased TNF-α and iNOS expression 3 h after OGD/R induction.
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.
KeywordsDectin-1 Syk inflammation ischemic stroke
Dendritic cell-associated C-type lectin-1
Spleen tyrosine kinase
Phosphorylated spleen tyrosine kinase
Tumor necrosis factor-α
Inducible nitric oxide synthase
Immune-receptor tyrosine-based activation motif
C-type lectin receptor
Src homology-2 domains
C-type lectin-like receptor 2
C-type lectin domain-containing 9A
Modified neurological severity score
Dulbecco’s modified Eagle’s medium
Protein kinase B
Phosphoinositide-dependent protein kinase-1
Nuclear factor kappa B
Reactive oxygen species
NLR family pyrin domain-containing 3
Damage-associated molecular patterns
In recent decades, ischemic stroke has become one of the most common causes of disability and mortality worldwide. Although the pathophysiology of cerebral ischemic 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 , and inhibition of inflammatory responses has been demonstrated to improve the outcome following a stroke [4, 5]. However, the detailed mechanisms by which the inflammatory response is triggered after a 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 (DAMPs) and triggers inflammatory signals by recruiting its downstream molecular tyrosine kinase (spleen tyrosine kinase; Syk) [6, 7, 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, 10, 11, 12]. Previous studies have reported that immune receptors that initiate the inflammatory response play an important role in stroke progression [13, 14, 15, 16]. However, to the best of our knowledge, whether the immune receptor Dectin-1 is involved in the inflammatory response following a stroke has not yet been investigated.
Syk is a nonreceptor protein tyrosine kinase that is observed 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 . 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 . Syk has been reported to play an indispensable role in acute and chronic inflammation, and inhibition of Syk impedes brain tissue damage following an ischemic stroke [23, 24, 25, 26]. However, to the best of our knowledge, the specific role of Dectin-1 and its downstream target Syk in ischemic stroke has not yet been investigated.
Materials and methods
Cerebral focal ischemia
Adult male C57BL/6 J 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 . 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; Merck KGaA) following 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. In order 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 s each and then treated with FD Cresyl Violet SolutionTM (FD Neoro Technologies) for 2 min. After washing in distilled water, the sections were dehydrated through an alcohol series, cleared in xylene, and coverslipped with neutral resin. In the present study, the stained sections were scanned and the infarct areas measured using the ImageJ software. The total infarct volume was obtained by indirect methods as previously described .
Drug administration in vivo and in vitro
The Dectin-1 receptor antagonist LAM (Sigma-Aldrich; Merck KGaA) was diluted to 10 mg/ml in vehicle (saline) according to the manufacturer’s protocol. 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 the stroke. For the in vitro experiment, the optimal dose of LAM was also assessed (the Additional file 1). Then, BV2 cells were preincubated with LAM for 1 h before induction with oxygen-glucose deprivation/reoxygenation (OGD/R) or lipopolysaccharide (LPS; 1,000 ng/ml).
Similarly, the mice were treated with either the Syk inhibitor (PIC; Selleck Chemicals) or vehicle (DMSO) once daily after ischemic stroke. For the in vivo experiment, the PIC dose was 20 mg/kg/day. For the in vitro experiments, the optimal dose of PIC was also assessed (Additional file 1). Then, BV2 cells were pretreated with 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 previously described [30, 31]. Briefly, brains were removed from mice at post-natal days 1–3 and the cortices were triturated into single cells. Mixed glial were plated in 25 T culture flasks coated with poly-d-lysine and grown in DMEM/F12 with 10% fetal bovine serum, GlutaMAX (Invitrogen; ThermoFisher Scientific, Inc.) and 1% penicillin/streptomycin in a 5% CO2/37 °C incubator, changing medium after 7 days, for a total of 2 weeks. In order to harvest primary microglial cells, the flask was shaken at 150 rpm for 2 h. The fluid medium was subsequently collected and centrifuged at 1,000 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 phosphate-buffered saline (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 (ThermoFisher 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 . Briefly, BV2 cells were seeded into a 6-well plate (Corning Inc.) at a density of 1.5 × 105 cells/well 24 h prior to transfection. BV2 cells were transfected by Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) 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 the negative control. Two days after transfection, the knockdown efficiency of siRNA was determined via western blot analysis. The present study assigned the cells into the following groups: (i) control siRNA group, (ii) Dectin-1 or Syk siRNA group, (iii) OGD/R + control siRNA group, and (iv) OGD/R + Dectin-1 or Syk siRNA group.
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), anti-Syk (1:1,000; Cell Signaling Technology, Inc.), 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.
Brain tissue was removed and then immersed in 4% paraformaldehyde for 5 h and 20% sucrose for 3 days 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 three 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.) for 2 h at room temperature. After washing three 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). The numbers of target cells were counted using the ImageJ software.
The data are presented as the mean ± standard deviation from three 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.). Images were created using GraphPad Prism 6.0 software (GraphPad Software, Inc.). P < 0.05 was considered to indicate a statistically significant difference.
Dectin-1 is significantly increased in the ischemic brain tissue after stroke and the BV2 microglial cells after OGD/R-induced injury in vitro
Syk and p-Syk are significantly upregulated in the ischemic brain tissue after stroke and BV2 microglial cells with OGD/R-induced injury in vitro
Blockage of Dectin-1 rescues the brain infarct volume and neurological impairment after a stroke
Blockade of Decin-1 inhibits Dectin-1/Syk signaling
Blockade of Decin-1 diminishes microglial activation and decreases the expression levels of inflammatory cytokines
Blockade of Syk partly decreases the brain infarct volume and improves the neurological outcomes after a stroke
PIC treatment attenuates p-Syk expression in ischemic brain tissue and in BV2 cells with OGD/R exposure
Decrease of microglial activation and inflammatory cytokine expression by inhibition of Syk signaling
LAM and PIC decreases TNF-α and iNOS production by inhibiting Dectin-1/Syk signaling in primary microglial cells with OGD/R-induced injury in vitro
Knockdown of Dectin-1 or Syk inhibits production of proinflammatory cytokines TNF-α and iNOS in BV2 microglial cells following OGD/R stimulation
LAM and PIC decreases TNF-α and iNOS production by inhibiting Dectin-1/Syk signaling in BV2 microglial cells with LPS-induced injury in vitro
The present study demonstrated that the Dectin-1, Syk, p-Syk, TNF-α, and iNOS expression levels were significantly increased in ischemic brain tissue after a stroke. Either Dectin-1 antagonist (LAM) or Syk inhibitor (PIC) treatment significantly decreased the expression levels of these proteins in the ischemic brain tissue. At the same time, the present study also revealed that LAM or PIC treatment significantly decreased the infarct volume and improved the functional outcomes after a 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 a stroke.
Microglia are the resident mononuclear phagocytic cells that are critical for inflammatory responses in the central nervous system (CNS). The most common distinguishing feature of the microglia is their rapid activation in response to pathological changes in the CNS, such as ischemia . Dectin-1 is expressed at a low level in the brain but is strongly upregulated following exposure to various stimuli, such as ischemia and injury [34, 35]. Dectin-1 has been reported to activate the NLRP3 (NLR family, pyrin domain-containing 3) inflammasome and enhance IL-1β production [36, 37, 38]. Activation of Dectin-1 can cause macrophage-mediated demyelination and axonal injury, and blockade of Dectin-1 can decrease inflammatory macrophage-mediated injury following spinal cord injury . A low-fat diet with caloric restriction decreases the expression of Dectin-1 and then attenuates white matter microglia activation during aging . It has also been demonstrated that Dectin-1 signaling can trigger neuroinflammation and enable repair of injured central nervous system neurons . The present study demonstrated that Dectin-1 was significantly increased in ischemic brain tissue following a stroke, and in the OGD/R-treated BV2 cells and primary microglia. The number of activated microglia were also enhanced after a stroke. Dectin-1 antagonist (LAM) treatment significantly decreased the expression of the aforementioned proteins and attenuated the number of activated microglia. It was also revealed that the blockade of Dectin-1 attenuated the brain infarct volume and decreased neurological deficits and microglial activation after a stroke. These data suggest that Dectin-1 overexpression may exert deleterious effects on the brain tissue and enhance neuroinflammation following ischemic stroke.
Syk, highly expressed in the microglia, plays a vital role in the inflammatory responses after ischemic stroke . The ligands produced by necrotic cells bind to the receptor of Dectin-1, resulting in recruitment and activation of Syk [43, 44, 45]. Syk inhibitor PIC decreases neuronal damage after retinal ischemia-reperfusion injury [46, 47]. Previous studies have demonstrated that Dectin-1 can activate Syk-dependent intracellular signaling cascades . Furthermore, the Dectin-1/Syk signaling pathway is involved in ROS generation and NLRP3 activation in response to β-glucan particles [36, 49, 50]. 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 [51, 52, 53, 54]. The present study demonstrated that Syk and p-Syk were significantly increased in the ischemic brain tissue after a stroke, as well as in the OGD/R model. Syk inhibitor (PIC) treatment significantly attenuated p-Syk, TNF-α, and iNOS expression in the ischemic brain tissue after a stroke and in the OGD/R model. Other inflammatory mediators such as JNK, p38 MAPK, and NF-kB have been linked to microglia activation [55, 56, 57, 58, 59, 60] and would be assessed in future work. The present study also revealed that the Syk inhibitor decreased microglial activation, the brain infarct volume, and neurological deficits after a stroke. These data suggest that Syk may also exert harmful effects on brain tissue and enhance neuroinflammation after an ischemic stroke.
The present study demonstrated that Dectin-1, Syk, and p-Syk expression was significantly increased after a stroke both in vivo and in vitro. Treatment of ischemic stroke with a Dectin-1 antagonist or a Syk inhibitor significantly decreased microglial activation, the brain infarct volume, neurological impairment, and production of the proinflammatory cytokines TNF-α and iNOS after ischemic stroke. The present study may offer new ideas for effective treatment of patients who have suffered from a stroke. Further studies of the pathophysiological functions of Dectin-1/Syk signaling in the activated inflammatory response may prove beneficial for clinical applications.
The authors would like to thank members of Dr Cui’s laboratory for their helpful discussions and suggestions.
XY, QH, and WM contributed equally to this work. All authors read and approved the final manuscript.
The present study was supported by the National Natural Science Foundation of China (grant nos. 81971134 and 81571155 to Dr. Ye and grant nos. 81571210 and 81771282 to Dr. Cui), the National Natural Science Foundation of Jiangsu Province (grant nos. BK20191152, BK20150209 and BL2014031), the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (grant no. 17KJB320017), Jiangsu Provincial Medical Youth Talent (grant no. QNRC2016788), Jiangsu Commission of Heath (grant no. LGY2019086), Xuzhou Key Research and Development Program (grant nos. KC18055 and KC19131), Xuzhou Innovation Capacity Building Program (grant no. KC19239), Suqian Science and Technology Plan (grant no. S201714), and the Summit of Six Top Talents Program of Jiangsu Province (grant no. 2017-WSN-118).
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
The authors declare that they have no competing interests.
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