- Email: [email protected]
Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response Qingping Lyu, Xiaojun Pang, Zibin Zhang, Yuyu Wei, Jinxu Hong, Huai Chen* Department of Neurosurgery, Hangzhou Red Cross Hospital/Zhejiang Chinese Medicine and Western Medicine Integrated Hospital, 208 Huancheng Road East, HangZhou 310003, Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2019 Accepted 13 November 2019 Available online xxx
Ischemic stroke is a leading cause of death among human in the world, and a critical cause for long-term disability. Accumulating studies have indicated that inflammatory response regulated by microglia contributes a lot to neuronal death, but the molecular mechanism still remains unclear. V-set and immunoglobulin domain-containing 4 (Vsig4), a complement receptor of the immunoglobulin superfamily (CRIg) that specifically expresses in resting tissue-resident macrophages, plays a critical role in regulating various inflammatory diseases via multiple signaling pathways. However, the effects of Vsig4 on ischemic stroke have not been investigated. In this study, we identified that Vsig4 expression was decreased after cerebral ischemic injury induced by middle cerebral artery occlusion (MCAO). Immunofluorescence staining showed that Vsig4 was co-localized with Iba1 in microglial cells from the infarct region of MCAO-operated mice. After over-expressing Vsig4 in mice, MCAO-induced infarction area and neurological deficits score were markedly attenuated. In addition, neurological dysfunction due to MCAO surgery was improved by Vsig4 over-expression. Microglial M1 polarization was detected in mice with MCAO surgery, which was markedly inhibited by Vsig4 over-expression, as evidenced by the markedly reduced expression of CD16, CD11b, inducible nitric oxide synthase (iNOS) and interleukin 6 (IL-6); however, the expression of M2-like phenotype hallmarks such as arginase 1 (Arg1), CD206, IL-10 and Ym-1 was significantly up-regulated. Mechanistically, the anti-inflammatory role of Vsig4 was mainly through the blockage of toll-like receptor 4/nuclear factor kappa B (TLR4/NF-kB) signaling via the in vivo and in vitro experiments. Also, we found that microglial TLR4 expression in the cerebral infarct area of MCAO mice was highly suppressed by Vsig4 over-expression. In vitro, the neuron-glial mixed culture by fluorescent staining showed that oxygen glucose deprivation (OGD) treatment led to significant cell death, while being attenuated by Vsig4 over-expression in primary microglial cells. Finally, we showed that Vsig4 could interact with TLR4 and repress its expression, subsequently alleviating ischemic stroke. Collectively, our findings demonstrated that microglial Vsig4 protected against post-stroke neuroinflammation mainly through interacting with TLR4. © 2019 Published by Elsevier Inc.
Keywords: Ischemic stroke Vsig4 Neuro-inflammation Microglial M1 polarization TLR4
1. Introduction Ischemic stroke is one of the leading causes for human death across the world, and also a pivotal factor for permanent disability among adults [1]. Increasing studies have elucidated that the
* Corresponding author. Department of Neurosurgery, Zhejiang Integrated Traditional and Western Medicine Hospital, No.208, Huancheng East Road, Hangzhou, 310003, China. E-mail address: [email protected] (H. Chen).
molecular and cellular mechanisms contributing to neuron death and neuro-inflammation are complicated, which still require further exploration. Excessive inflammatory response is crucial for the progression of ischemic stroke, which could result in the elevated infarction area and neuron death [2e4]. Unfortunately, the clinical analysis suggest that therapeutic strategies to inhibit inflammation has not yet exhibited the expected outcomes during the treatment of cerebral ischemia [5]. Herein, it is urgently needed to gain deeper insights into neuro-inflammation following cerebral ischemic stroke. Toll-like receptor 4 (TLR4) is one of the important receptors associated with innate immunity and various inflammatory
https://doi.org/10.1016/j.bbrc.2019.11.077 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
2
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
reactions [6]. In the central nervous system (CNS), TLR4 is mainly expressed in glial cells, including microglia. The up-regulated protein expression of TLR4 promotes the NF-kB transcriptional activity and thereafter leads to the inflammatory responses in ischemic brain [7,8]. Restraining the activation of TLR4/NF-kB signaling pathway represents a promising therapeutic strategy against ischemic stroke progression [9]. In addition, microglial cells, known as the resident macrophages, are reported to supply the primary immune defense in CNS. During ischemic stroke progression, microglial cells can be activated to eliminate the injured cells. The activated microglia secret enormous pro-inflammatory meditators in the brain, accompanied with the reduced release of antiinflammatory factors, which subsequently enhances the progression of stroke [10,11]. Herein, it is interesting to explore more molecular mechanisms that reveal the neuro-inflammation in microglial cells that regulate ischemic damage. V-set immunoglobulin-domain-containing 4 (Vsig4) is a critical membrane protein belonging to complement receptor of the immunoglobulin superfamily (CRIg). Accordingly, the expression of Vsig4 is significantly restricted to tissue macrophages, such as the peritoneal macrophages and liver-residential Kupffer cells [12,13]. Recently, Vsig4 was suggested to reduce IL-2 expression and T-cell proliferation through bonding T-cell ligand or receptor that has not been well understood [14]. Vsig4 regulates inflammation in macrophages that are involved in diabetes resistance [15]. In macrophages, Vsig4 impeded IL-1b secretion by repressing the NLRP3 inflammasome [16]. High fat diet-induced hepatic steatosis was also attributed to the down-regulation of Vsig4 largely via the excessive release of pro-inflammatory meditators [17]. More recently, promoting Vsig4 expression could attenuate lipid accumulation and fibrosis in liver of high fat diet-challenged mice, which was partly dependent on the suppression of proinflammatory factors in macrophages [18]. Despite Vsig4 shows protective effects against the progression of autoimmune disease or hepatitis, its effects on cerebral ischemic stroke are still unclear. Herein, in the study, we explored if microglial Vsig4 meditated neuro-inflammation following ischemic damage in vivo and in vitro, and whether the involvement was associated with TLR4 signaling.
1.5 h of occlusion, the filament was withdrawn to induce reperfusion. During MCAO surgery, body temperature was maintained at 37 ± 0.5 C using a heating pad. The Sham group mice were subjected to the same operation except that the filament was not inserted into internal carotid artery (ICA). 2.2. Cell culture Primary microglia cells were isolated and cultured as previously described [20]. Microglia cells were finally collected through shaking and planted in plates for further analysis. The primary cortical neurons were isolated from C57BL/6J mouse brain and incubated in neurobasal medium (Gibco, USA) supplemented with 2% B27 (Gibco) and 1% GlutaMax (Gibco). For microglia and neuron mix culture, the Transwell® plates (0.4-mm pore size, Corning, USA) were used. Primary neurons were planted in the lower chamber of the Transwell plates and incubated with microglia, which were then subjected to OGD/R. To perform OGD, the cell cultures were exposed to transient OGD for 1 h and returned to normal culture conditions for different time. As for OGD, the culture medium was replaced with serum-free and glucose-free Locke’s buffer. Then, the cultures were incubated in an experimental hypoxia chamber with a saturated atmosphere of 95% N2 and 5% CO2. Cells in control group were cultured with normal levels of glucose and incubated for the same time in a humidified atmosphere of 95% air and 5% CO2. Primary microglia cells were infected with AAV-Vsig4 or AAV-NC diluted in medium at a multiplicity of infection of 50 for 24 h. 2.3. Infarct volume determination Mice were euthanized after neurobehavioral test through rapid decapitation. The brain samples were rapidly removed and stained with 2,3,5-triphenyltetrazolium chloride (TTC; Sigma Aldrich, St. Louis, USA) as previously demonstrated [3]. The infarction area was calculated by an examiner blind to each treatment. Magnetic resonance imaging (MRI) was also used to evaluate the infarction area in mice at 3 d after ischemia by the use of a 7T MRI Bruker scanner (Germany).
2. Materials and methods
2.4. Western blot
2.1. Animals and treatments
The protein of cerebral tissues or cells was extracted using RIPA buffer (Beyotime, Nanjing, China) according to the manufacturer’s protocols. Equal amounts of brain tissue protein were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, USA). Then, the membranes were incubated with primary antibodies against Vsig4 (1:500; Abcam, USA), TLR4 (1:1000; Abcam), NF-kB (1:1000; Cell Signaling Technology, USA), peNFekB (1:1000; Cell Signaling Technology) and GAPDH (1:1000; Abcam) overnight at 4 C, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime) and visualized using ECL chemiluminescence kit (Millipore). GAPDH was used as a loading control.
Vsig4 was at first subcloned into a pAAV-CMV bGlobin-MCSeGFP-3Flag vector (AAV-Vsig4), and the empty control vector was pAAV-CMV bGlobin-MCS-eGFP-3Flag (AAV-NC). The AAV-Vsig4 and AAV-NC were packaged by GeneChem Technology (Shanghai, China). The packaged AAVs were then concentrated in PBS at the following titers for the subsequent analysis: AAV-Vsig4, 2 1012 genome copies per milliliter, and AAV-NC, 1 1012 genome copies per milliliter. Adult male C57BL/6J mice (weighed 20e25 g) were purchased from the Animal Research Center of Zhejiang University (Zhejiang) and fed in SPF condition with a 12- to 12-h light/dark cycle and free access to water and food. All animal studies were approved by the Animal Care Committee of Zhejiang Integrated Traditional and Western Medicine Hospital (Zhejiang, China) according to the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications, revised 1996). At least six mice were involved for analysis of each data point. 3 weeks before MCAO operation, the concentrated viral solution (4 ml) containing AAV-NC or AAV-Vsig4 was stereotaxically delivered into the ipsilateral lateral ventricle in the MCAO/AAV-NC and MCAO/ AAV-Vsig4 groups, respectively. Then, the mice cerebral ischemia/ reperfusion (I/R) models were induced through MCAO surgery by the intraluminal filament according to previous studies [19]. After
2.5. Quantitative real-time polymerase chain reaction (RT-qPCR) Total RNA was isolated from peri-infarct tissues or cells with TRIzol regent (Invitrogen, USA), and was then reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The RT-qPCR was conducted using the UltraSYBR Mixture (CWBio, China), specific primers (Supplementary Table S1) and cDNA on the Mx3000P Real-Time PCR System (Agilent Technologies, USA). The GAPDH mRNA expression was defined as internal control.
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
2.6. Immunostaining
(Abcam), respectively, following the manufacturers’ protocols.
Animals were anesthetized and intracardially perfused with 0.9% sodium chloride followed by 4% paraformaldehyde. After fixing for 12 h, brain samples were dehydrated in gradient sucrose solutions of 10, 20, and 30% at 4 C. Then, the brains were embedded in optimal cutting temperature compound and cut into 10-mm sections for further analysis. Tissue sections were blocked in 0.3% Triton, 3% goat serum (Solarbio, Beijing, China), and 1% bovine serum albumin (BSA, Sigma-Aldrich). The sections were then incubated with antibody against Vsig4 (1:200; Abcam), Iba1 (1:200; Santa Cruz Biotechnology, USA), NeuN (1:200; Abcam) and/ or TLR4 (1:250; Santa Cruz Biotechnology) overnight at 4 C. Next, sections were incubated with appropriate secondary antibodies (Abcam) and DAPI (Sigma-Aldrich). The positive expression of each signal was analyzed using Image J software (NIH, USA). TUNEL staining in tissue sections and cells was conducted using the In Situ Cell Death Detection Kit (Roche, USA) and TUNEL Assay Kit
2.7. Neurological score assessment
3
Neurological score was performed 3 d after reperfusion following MCAO by two investigators blind to the experiment treatments. Neurological impairment after ischemic insult was calculated using a 5-point scale: 0 (no significant deficits), 1 (failure to fully extend left forepaw), 2 (circling to the left), 3 (fail to the left), 4 (falling to walk spontaneously combined with depressed levels of consciousness). 2.8. Behavioral analysis A rotarod test was conducted using the Rotamex 5 apparatus (Columbus Instruments, USA) as previously suggested [21]. Each mouse underwent at least 3 trials daily with an inter-trial interval of 20 min.
Fig. 1. Vsig4 expression was down-regulated post-stroke. (a) RT-qPCR and (b) western blot analysis of Vsig4 at 2 h, 6 h, 24 h, 3 d, 7 d and 28 d after ischemia stroke. (c) Co-staining for Iba1 (green) and Vsig4 (red) at 3 d after reperfusion in the peri-infarct area. Relative quantification of Vsig4-positive cells was exhibited. (d) RT-qPCR and (e) western blot analysis of Vsig4 in primary microglia after treatment with OGDR for the shown time (2 h, 6 h, 24 h, 36 h and 48 h). The data were expressed as mean ± SEM. þP < 0.05 and þþP < 0.01 vs. Sham or Con group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
4
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
2.9. Co-immunoprecipitation The lysates from cerebral tissues or primary microglia were extracted and prepared using RIPA lysis buffer (Beyotime). Protein extracts were then incubated with antibody against Vsig4 or the control IgG at 4 C overnight. Then the immune complexes were linked to protein A/G-agarose beads (Thermo Fisher Scientific) for 4 h. The eluted proteins were harvested for immunoblotting analysis. 2.10. Statistical analysis All results are expressed as mean ± SEM. Differences between groups were compared through the two-tailed Student’s t tests and one-way ANOVA followed by Tukey’s post hoc test. Value of
p < 0.05 was considered statistically significant. GraphPad Prism (Version 7.0) was used for data analysis. 3. Results 3.1. Vsig4 expression was down-regulated after stroke At first, the expression change of Vsig4 in mice post stroke was investigated. As displayed in Fig. 1a and b, we found that Vsig4 expression from mRNA and protein levels was marginally downregulated at 24 h after MCAO operation, especially reduced at 3 d after reperfusion, and gradually up-regulated. Co-immunofluorescent staining suggested that Vsig4 expression was obviously decreased in microglial cells that were marked by Iba1 [22] (Fig. 1c). Consistently, OGDR treatment restrained microglial Vsig4
Fig. 2. Vsig4 attenuates cerebral I/R injury in mice at 3 d after reperfusion. (a) TTC staining of brain sections from each group of mice. (b) Quantification of infarct area, and (c) neurological scores. (d) Photomicrographs through MRI scanning indicating the infarction area marked by the red line. (e) Representative images of NeuN counterstained with DAPI in the infarct region. (f) Quantification of NeuN-positive levels was exhibited. (g) TUNEL staining for brain sections. (h) Quantification of TUNEL-positive cells was displayed. (i) Results for rotarod test after cerebral ischemia. The data were expressed as mean ± SEM. þP < 0.05, þþP < 0.01 and þþþP < 0.001; ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 3. Vsig4 inhibits M1 like phenotype and promotes M2 like phenotype in mice 3 d post stroke. RT-qPCR analysis for (a) M1 like phenotype marks (CD16, CD11b, iNOS and IL6), and (b) M1 like phenotype marks (Arg-1, CD206, IL-10 and Ym-1). (c) Western blot analysis for peNFekB in the cerebral tissues of each group of mice. (d) Co-staining for Iba1 (green) and TLR4 (red) at 3 d after reperfusion in the peri-infarct area. Relative quantification of (e) Iba1-and (f) TLR4-positive cells was displayed. (g) Western blot analysis for TLR4 in the cerebral tissues of mice. The data were expressed as mean ± SEM. þP < 0.05, þþP < 0.01 and þþþP < 0.001; ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
6
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 4. Vsig4-inhibited inflammation in microglia is through interacting with TLR4 and inhibited its expression. (a) Representative results for immunofluorescence staining of NeuN (green) and PI (red) in neuron-glial mixed culture after OGD/R treatment for 24 h. Quantification of NeuN- and PI-positive cells was shown. (beg) Primary microglia cells were infected with AAV-Vsig4, followed by OGD/R treatment for another 24 h. Then, all cells were harvested for subsequent analysis. (b) RT-qPCR analysis for inflammation-associated
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
expression mainly through a time-dependent manner, particularly after 24 h (Fig. 1d and e). Therefore, the expression of microglial Vsig4 was decreased under ischemic-hypoxic condition. 3.2. Vsig4 attenuates cerebral I/R injury in mice at 3 d after reperfusion To further reveal the biological function of Vsig4 in ischemic stroke, an adenovirus-packaged Vsig4 expression vector was used through direct injection into the mouse brain. TTC results indicated that MCAO surgery resulted in larger infarction area, and overexpressing Vsig4 markedly reduced the infarction volume (Fig. 2a and b). We also found that MCAO-induced neurological deficit score was significantly alleviated by the over-expression of Vsig4 (Fig. 2c). MRI assay confirmed the results that promoting Vsig4 expression could decrease the infarct size of mice with ischemic stroke (Fig. 2d). Immunofluorescent results showed that Vsig4 increase greatly rescued the neuron survival, as evidenced by the obviously increased number of NeuN-positive cells (Fig. 2e and f). In parallel, MCAO-induced cell death in cerebral infarct area was markedly alleviated in mice infected with AAV-Vsig4, as detected by the down-regulated number of TUNEL-positive cells (Fig. 2g and h). The rotarod test suggested that the latency to fall off the rotarod was much longer in the AAV-Vsig4-injected group compared to the MCAO group (Fig. 2i). Thus, promoting Vsig4 expression inhibited infarction area and protected neurological function in mice after MCAO surgery. 3.3. Vsig4 inhibits M1 like phenotype but promotes M2 like phenotype in mice 3 d post stroke Inflammation plays a key role in regulating the pathogenesis of ischemic stroke, which is closely associated with the release of proinflammatory cytokines or factors by M1 microglia; conversely, the M2 like phenotype microglia that produce anti-inflammatory cytokines are repressed [23]. We then found that the mRNA levels of pro-inflammatory factors including CD16, CD11b, iNOS and IL-6 were markedly elevated in the cerebral infarct area, while being down-regulated in AAV-Vsig4 mice (Fig. 3a). In contrast, antiinflammatory meditators such as Arg-1, CD206, IL-10 and Ym-1 reduced by MCAO were significantly restored by over-expressing Vsig4 (Fig. 3b). TLR4/NF-kB signaling pathway is involved in inducing M1 microglia to promote inflammatory response [7e9]. Western blot and/or immunofluorescent analysis suggested that MCAO operation led to significant increases of NF-kB and TLR4, and these effects were clearly abrogated in mice injected with AAVVsig4. Also, immunofluorescent staining demonstrated that TLR4 was markedly expressed in microglia of the infarction area from MCAO-treated mice, as evidenced by the obvious co-localization with Iba1 (Fig. 3ceg). Collectively, Vsig4-inhibited the secretion of pro-inflammatory factors might be associated with the blockage of TLR4/NF-kB signaling. 3.4. Vsig4-associated inflammation inhibition in microglia is rely on interacting with TLR4 pathway To further validate the effects of Vsig4 on ischemic stroke, as well as the underlying molecular mechanism, in vitro experiments using primary microglia were then performed. As shown in Fig. 4a,
7
the neuron-glial mixed culture by fluorescent staining suggested that OGD/R treatment led to significant decrease in the number of NeuN-positive cells, while resulted in obvious increase in TUNELpositive cells, and notably these results were reversed by overexpressing Vsig4 in microglial cells. By RT-qPCR analysis, the primary microglial cells after OGD/R treatment showed markedly increased mRNA levels of CD16, CD11b, iNOS and IL-6, while the decreased expression of Arg1, CD206, IL-10 and Ym-1. Importantly, these results were reversed in microglia with Vsig4 over-expression (Fig. 4b). What’s more, OGD/R-reduced expression of Vsig4, and -elevated expression of peNFekB and TLR4 were greatly resumed by AAV-Vsig4 in primary microglial cells (Fig. 4cef). As showed in Fig. 4g, immunofluorescent staining confirmed the expression change of Vsig4 and TLR4 in OGD/R-stimulated microglial cells with or without Vsig4 over-expression as detected by western blotting. Finally, we explored if an association existed between Vsig4 and TLR4 in microglia. Using co-immunoprecipitation analysis, Vsig4 appeared to interact with TLR4 both in vivo and in vitro (Fig. 4h). Therefore, Vsig4 could interact with microglial TLR4, and then modulate the progression of ischemic stroke via regulating inflammatory response. 4. Discussion Ischemic cerebral stroke results in permanent injury to the CNS, and this process includes a critical step if post-ischemic inflammatory response, which then influence the physical function in individuals [1e4]. Increasing studies have illustrated that excessive inflammatory response could be detected in ischemic models both in vivo and in vitro, as indicated by the elevated microglial M1 polarization. M1 like phenotype microglial cells show significantly cytotoxicity through releasing a large number of pro-inflammatory factors, such as iNOS, CD11b, IL-1b and IL-6. In contrast, the release of anti-inflammatory meditators (Arg1, CD206, Ym-1, IL-10, etc) by M2-like phenotype microglia is blunted [23,24]. Vsig4, as a B7associated protein that is specifically expressed in macrophages, regulates suppressive signals to inhibit NLRP3 inflammasome and subsequent Il-1b transcription, and this process was related to the inhibition of NF-kB [16]. In our study, we found that Vsig4 expression was markedly decreased during ischemic stroke progression in MCAO-operated mice, and in OGD/R-treated primary microglial cells. Meanwhile, Vsig4 was found to co-localize with Iba-1, suggesting its expression in microglial cells in the infarct region of MCAO-mice. Moreover, the MCAO surgery-induced neuronal death, infarct area enlargement and functional impairment in mice were significantly alleviated by Vsig4 overexpression. Promoting Vsig4 expression greatly inhibited the microglial M1 polarization, while promoted M2-like phenotype. This anti-inflammatory effect regulated by Vsig4 was largely associated with the blockage of TLR4/NF-kB signaling. Notably, the Coimmunoprecipitation analysis illustrated that Vsig4 could interact with TLR4, thereafter modulating the progression of cerebral ischemic stroke (Fig. 4i). Thus, these results provided evidence that targeting and improving Vsig4 expression could ameliorate ischemic stroke, and it could be served as a novel and effective therapeutic signal to develop useful treatment for stroke or associated disease. The innate immune system is an essential factor involved in the pathophysiology of cerebral ischemic stroke. It is generally
genes as exhibited. (cef) Western blot results for Vsig4, TLR4 and peNFekB. (g) Immunofluorescence staining of Vsig4 (red) and TLR4 (green) in primary microglia. (h) The lysates from cerebral tissue and microglia were immunoprecipitated with anti-Vsig4. Then, immunoprecipitates were analyzed using western blot with anti-Vsig4 and anti-TLR4. (i) Proposed schematic mechanism demonstrating microglial Vsig4 meditated ischemic neuro-inflammation. The data were expressed as mean ± SEM. þP < 0.05, þþP < 0.01 and þþþ P < 0.001; ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
8
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
accepted that the pro-inflammatory response is originally regulated by microglia-triggered CNS injury in ischemic stroke [25]. Microglia cells are extremely activated and then release excessive pro-inflammatory meditators, such as CD11b, iNOS, CD16 and IL-6, immediately following ischemic stroke, which result in neuronal apoptosis and accelerate brain injury consequently [23,24]. M1 microglia is a toxic cellular state linked to an elevation of proinflammatory meditators. By contrary, M2-like phenotype microglia promote secretion anti-inflammatory factors (Arg1, CD206, Ym-1, IL-10, IL-4, etc) [10,11,26]. Increasing evidences suggest that ischemic animal models with M1-polarized microglia intensify neuron death whereas the M2 microglia could protect neurons and improve post-injury tissue repairmen [27]. As reported, TLR4 is primarily expressed in microglial cells and regulates microglial activation partly via promoting the activation of NF-kB signaling and subsequent release of inflammatory cytokines or chemokine [7e9]. Suppressing TLR4 showed beneficial effects on tissue homeostasis and ameliorates cerebral ischemic damage [28]. In our study, we confirmed that MCAO-induced mice with ischemic stroke showed M1-like phenotype microglia, accompanied with significantly up-regulated expression of pro-inflammatory regulators and down-regulated anti-inflammatory signals. Notably, these effects were reversed by Vsig4 over-expression. Recent reports have provided insights that inflammatory activation by microglial cells can elicit apoptotic cell death both acute under in vitro and in vivo ischemic conditions [29,30]. Because we found an up-regulated expression and activation level of in inflammatory molecules in microglial cells, we then calculated the functional role of inflammation regulated by Vsig4 in neuronal cell death under ischemic conditions. Mix culture of primary microglial cells and neurons showed that OGD/R-induced neuron death as proved by the downregulated NeuN-positive levels was significantly rescued by Vsig4 over-expression in primary microglial cells. Recent studies have provided evidence that Vsig4 could suppress inflammatory response in macrophages associated with the inhibition of NF-kB signaling; however, Vsig4 insufficiency aggravated inflammatory damage in in vitro and in vivo [16]. Additionally, in response to inflammatory stimuli, Vsig4 reduced macrophage activation and inhibited M1 polarization [17]. More recently, the deficiency of Vsig4 accelerated the production of pro-inflammatory cytokines, which was involved in high fat diet-induced insulin resistance, lipid deposition and fibrosis in hepatic tissues [18]. Therefore, we supposed that Vsig4-alleviated cerebral stroke was largely dependent on its suppression to microglial inflammatory response. More importantly, we found that Vsig4 could interact with TLR4, thus inhibiting its expression and the subsequent inflammatory response. However, there is still possibility that microglial Vsig4 might interact with other molecules in ischemiaassociated cerebral immune response. As for this, further analysis is still necessary to reveal more details regulated by Vsig4 during cerebral ischemic damage. Together, these results showed that Vsig4 expression was reduced in infarction region of mice with cerebral ischemic stroke. We also found that promoting Vsig4 expression inhibited M1 polarization in microglia by blocking NF-kB activation via interacting with TLR4 and repressing TLR4 expression, subsequently reducing the release of pro-inflammatory response and neuronal death (Fig. 4i). Together, Vsig4 is considered as a promising and effective therapeutic target for ischemic stroke or related diseases.
Acknowledgement This work was supported by Chinese Medicine Research program of Zhejiang province (No. 2019ZA-089).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.077.
References [1] W.N. Kernan, et al., Pioglitazone after ischemic stroke or transient ischemic attack, N. Engl. J. Med. 374 (14) (2016) 1321e1331. [2] X. Wang, et al., The evolving role of neuro-immune interaction in brain repair after cerebral ischemic stroke, CNS Neurosci. Ther. 24 (12) (2018) 1100e1114. [3] H. Yan, et al., Long noncoding RNA MEG3 activation of p53 mediates ischemic neuronal death in stroke, Neuroscience 337 (2016) 191e199. [4] S. Yao, et al., miR-455 inhibits neuronal cell death by targeting TRAF3 in cerebral ischemic stroke, Neuropsychiatric Dis. Treat. 12 (2016) 3083. [5] M.D. Ginsberg, Neuroprotection for ischemic stroke: past, present and future, Neuropharmacology 55 (3) (2008) 363e389. [6] M. Fukata, et al., Innate immune signaling by Toll-like receptor-4 (TLR4) shapes the inflammatory microenvironment in colitis-associated tumors, Inflamm. Bowel Dis. 15 (7) (2009) 997e1006. [7] H. Zhu, et al., Curcumin attenuates acute inflammatory injury by inhibiting the TLR4/MyD88/NF-kB signaling pathway in experimental traumatic brain injury, J. Neuroinflammation 11 (1) (2014) 59. [8] B. Han, et al., Electroacupuncture modulated the inflammatory reaction in MCAO rats via inhibiting the TLR4/NF-kB signaling pathway in microglia, Int. J. Clin. Exp. Pathol. 8 (9) (2015) 11199. [9] Y.W. Li, et al., Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NF-kB p65 signal pathway, Gene 542 (1) (2014) 46e51. [10] C. Schmidt, et al., Phosphoinositide 3-kinase g restrains neurotoxic effects of microglia after focal brain ischemia, Mol. Neurobiol. 53 (8) (2016) 5468e5479. [11] Y. Liu, et al., CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway, J. Cereb. Blood Flow Metab. 35 (10) (2015) 1623e1631. [12] F. Zheng, et al., Monitoring liver macrophages using nanobodies targeting Vsig4: concanavalin A induced acute hepatitis as paradigm, Immunobiology 220 (2) (2015) 200e209. [13] S.J.L. van der Tuin, et al., Lipopolysaccharide lowers cholesteryl ester transfer protein by activating F4/80þ Clec4fþ Vsig4þ Ly6C Kupffer cell subsets, J. Am. Heart Assoc. 7 (6) (2018) e008105. [14] L. Vogt, et al., VSIG4, a B7 family-related protein, is a negative regulator of T cell activation, J. Clin. Investig. 116 (10) (2006) 2817e2826. [15] W. Fu, et al., Early window of diabetes determinism in NOD mice, dependent on the complement receptor CRIg, identified by noninvasive imaging, Nat. Immunol. 13 (4) (2012) 361. [16] X. Huang, et al., VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1b in macrophages, Sci. Adv. (1) (2019) 5, eaau7426. [17] J. Li, et al., VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism, Nat. Commun. 8 (1) (2017) 1322. [18] Y. Li, et al., Expression of Vsig4 attenuates macrophage-mediated hepatic inflammation and fibrosis in high fat diet (HFD)-induced mice, Biochem. Biophys. Res. Commun. 516 (3) (2019) 858e865. [19] X. Xu, et al., MicroRNA-1906, a novel regulator of toll-like receptor 4, ameliorates ischemic injury after experimental stroke in mice, J. Neurosci. 37 (43) (2017) 10498e10515. [20] L.K. Parvathenani, et al., P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer’s disease, J. Biol. Chem. 278 (15) (2003) 13309e13317. [21] X. Tang, et al., Genetic deletion of Krüppel-like factor 11 aggravates ischemic brain injury, Mol. Neurobiol. 55 (4) (2018) 2911e2921. [22] J. Tischer, et al., Inhomogeneous distribution of Iba-1 characterizes microglial pathology in Alzheimer’s disease, Glia 64 (9) (2016) 1562e1572. [23] Y. Tang, et al., Differential roles of M1 and M2 microglia in neurodegenerative diseases, Mol. Neurobiol. 53 (2) (2016) 1181e1194. [24] J. Pan, et al., Malibatol A regulates microglia M1/M2 polarization in experimental stroke in a PPARg-dependent manner, J. Neuroinflammation 12 (1) (2015) 51. [25] D. Amantea, et al., Rational modulation of the innate immune system for neuroprotection in ischemic stroke, Front. Neurosci. 9 (2015) 147. [26] C.Y. Xia, et al., Selective modulation of microglia polarization to M2 phenotype for stroke treatment, Int. Immunopharmacol. 25 (2) (2015) 377e382. [27] H.L. Meng, et al., Neuronal soluble fas ligand drives M1-microglia polarization after cerebral ischemia, CNS Neurosci. Ther. 22 (9) (2016) 771e781. [28] S.L. Wang, et al., Dexmedetomidine preconditioning plays a neuroprotective role and suppresses TLR4/NF-kB pathways model of cerebral ischemia reperfusion, Biomed. Pharmacother. 93 (2017) 1337e1342. [29] M. Zhang, et al., The cystathionine b-synthase/hydrogen sulfide pathway contributes to microglia-mediated neuroinflammation following cerebral ischemia. Brain, behavior, and, Immunity 66 (2017) 332e346. [30] X. Liu, et al., Salidroside provides neuroprotection by modulating microglial polarization after cerebral ischemia, J. Neuroinflammation 15 (1) (2018) 39.
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response Qingping Lyu, Xiaojun Pang, Zibin Zhang, Yuyu Wei, Jinxu Hong, Huai Chen* Department of Neurosurgery, Hangzhou Red Cross Hospital/Zhejiang Chinese Medicine and Western Medicine Integrated Hospital, 208 Huancheng Road East, HangZhou 310003, Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2019 Accepted 13 November 2019 Available online xxx
Ischemic stroke is a leading cause of death among human in the world, and a critical cause for long-term disability. Accumulating studies have indicated that inflammatory response regulated by microglia contributes a lot to neuronal death, but the molecular mechanism still remains unclear. V-set and immunoglobulin domain-containing 4 (Vsig4), a complement receptor of the immunoglobulin superfamily (CRIg) that specifically expresses in resting tissue-resident macrophages, plays a critical role in regulating various inflammatory diseases via multiple signaling pathways. However, the effects of Vsig4 on ischemic stroke have not been investigated. In this study, we identified that Vsig4 expression was decreased after cerebral ischemic injury induced by middle cerebral artery occlusion (MCAO). Immunofluorescence staining showed that Vsig4 was co-localized with Iba1 in microglial cells from the infarct region of MCAO-operated mice. After over-expressing Vsig4 in mice, MCAO-induced infarction area and neurological deficits score were markedly attenuated. In addition, neurological dysfunction due to MCAO surgery was improved by Vsig4 over-expression. Microglial M1 polarization was detected in mice with MCAO surgery, which was markedly inhibited by Vsig4 over-expression, as evidenced by the markedly reduced expression of CD16, CD11b, inducible nitric oxide synthase (iNOS) and interleukin 6 (IL-6); however, the expression of M2-like phenotype hallmarks such as arginase 1 (Arg1), CD206, IL-10 and Ym-1 was significantly up-regulated. Mechanistically, the anti-inflammatory role of Vsig4 was mainly through the blockage of toll-like receptor 4/nuclear factor kappa B (TLR4/NF-kB) signaling via the in vivo and in vitro experiments. Also, we found that microglial TLR4 expression in the cerebral infarct area of MCAO mice was highly suppressed by Vsig4 over-expression. In vitro, the neuron-glial mixed culture by fluorescent staining showed that oxygen glucose deprivation (OGD) treatment led to significant cell death, while being attenuated by Vsig4 over-expression in primary microglial cells. Finally, we showed that Vsig4 could interact with TLR4 and repress its expression, subsequently alleviating ischemic stroke. Collectively, our findings demonstrated that microglial Vsig4 protected against post-stroke neuroinflammation mainly through interacting with TLR4. © 2019 Published by Elsevier Inc.
Keywords: Ischemic stroke Vsig4 Neuro-inflammation Microglial M1 polarization TLR4
1. Introduction Ischemic stroke is one of the leading causes for human death across the world, and also a pivotal factor for permanent disability among adults [1]. Increasing studies have elucidated that the
* Corresponding author. Department of Neurosurgery, Zhejiang Integrated Traditional and Western Medicine Hospital, No.208, Huancheng East Road, Hangzhou, 310003, China. E-mail address: [email protected] (H. Chen).
molecular and cellular mechanisms contributing to neuron death and neuro-inflammation are complicated, which still require further exploration. Excessive inflammatory response is crucial for the progression of ischemic stroke, which could result in the elevated infarction area and neuron death [2e4]. Unfortunately, the clinical analysis suggest that therapeutic strategies to inhibit inflammation has not yet exhibited the expected outcomes during the treatment of cerebral ischemia [5]. Herein, it is urgently needed to gain deeper insights into neuro-inflammation following cerebral ischemic stroke. Toll-like receptor 4 (TLR4) is one of the important receptors associated with innate immunity and various inflammatory
https://doi.org/10.1016/j.bbrc.2019.11.077 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
2
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
reactions [6]. In the central nervous system (CNS), TLR4 is mainly expressed in glial cells, including microglia. The up-regulated protein expression of TLR4 promotes the NF-kB transcriptional activity and thereafter leads to the inflammatory responses in ischemic brain [7,8]. Restraining the activation of TLR4/NF-kB signaling pathway represents a promising therapeutic strategy against ischemic stroke progression [9]. In addition, microglial cells, known as the resident macrophages, are reported to supply the primary immune defense in CNS. During ischemic stroke progression, microglial cells can be activated to eliminate the injured cells. The activated microglia secret enormous pro-inflammatory meditators in the brain, accompanied with the reduced release of antiinflammatory factors, which subsequently enhances the progression of stroke [10,11]. Herein, it is interesting to explore more molecular mechanisms that reveal the neuro-inflammation in microglial cells that regulate ischemic damage. V-set immunoglobulin-domain-containing 4 (Vsig4) is a critical membrane protein belonging to complement receptor of the immunoglobulin superfamily (CRIg). Accordingly, the expression of Vsig4 is significantly restricted to tissue macrophages, such as the peritoneal macrophages and liver-residential Kupffer cells [12,13]. Recently, Vsig4 was suggested to reduce IL-2 expression and T-cell proliferation through bonding T-cell ligand or receptor that has not been well understood [14]. Vsig4 regulates inflammation in macrophages that are involved in diabetes resistance [15]. In macrophages, Vsig4 impeded IL-1b secretion by repressing the NLRP3 inflammasome [16]. High fat diet-induced hepatic steatosis was also attributed to the down-regulation of Vsig4 largely via the excessive release of pro-inflammatory meditators [17]. More recently, promoting Vsig4 expression could attenuate lipid accumulation and fibrosis in liver of high fat diet-challenged mice, which was partly dependent on the suppression of proinflammatory factors in macrophages [18]. Despite Vsig4 shows protective effects against the progression of autoimmune disease or hepatitis, its effects on cerebral ischemic stroke are still unclear. Herein, in the study, we explored if microglial Vsig4 meditated neuro-inflammation following ischemic damage in vivo and in vitro, and whether the involvement was associated with TLR4 signaling.
1.5 h of occlusion, the filament was withdrawn to induce reperfusion. During MCAO surgery, body temperature was maintained at 37 ± 0.5 C using a heating pad. The Sham group mice were subjected to the same operation except that the filament was not inserted into internal carotid artery (ICA). 2.2. Cell culture Primary microglia cells were isolated and cultured as previously described [20]. Microglia cells were finally collected through shaking and planted in plates for further analysis. The primary cortical neurons were isolated from C57BL/6J mouse brain and incubated in neurobasal medium (Gibco, USA) supplemented with 2% B27 (Gibco) and 1% GlutaMax (Gibco). For microglia and neuron mix culture, the Transwell® plates (0.4-mm pore size, Corning, USA) were used. Primary neurons were planted in the lower chamber of the Transwell plates and incubated with microglia, which were then subjected to OGD/R. To perform OGD, the cell cultures were exposed to transient OGD for 1 h and returned to normal culture conditions for different time. As for OGD, the culture medium was replaced with serum-free and glucose-free Locke’s buffer. Then, the cultures were incubated in an experimental hypoxia chamber with a saturated atmosphere of 95% N2 and 5% CO2. Cells in control group were cultured with normal levels of glucose and incubated for the same time in a humidified atmosphere of 95% air and 5% CO2. Primary microglia cells were infected with AAV-Vsig4 or AAV-NC diluted in medium at a multiplicity of infection of 50 for 24 h. 2.3. Infarct volume determination Mice were euthanized after neurobehavioral test through rapid decapitation. The brain samples were rapidly removed and stained with 2,3,5-triphenyltetrazolium chloride (TTC; Sigma Aldrich, St. Louis, USA) as previously demonstrated [3]. The infarction area was calculated by an examiner blind to each treatment. Magnetic resonance imaging (MRI) was also used to evaluate the infarction area in mice at 3 d after ischemia by the use of a 7T MRI Bruker scanner (Germany).
2. Materials and methods
2.4. Western blot
2.1. Animals and treatments
The protein of cerebral tissues or cells was extracted using RIPA buffer (Beyotime, Nanjing, China) according to the manufacturer’s protocols. Equal amounts of brain tissue protein were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, USA). Then, the membranes were incubated with primary antibodies against Vsig4 (1:500; Abcam, USA), TLR4 (1:1000; Abcam), NF-kB (1:1000; Cell Signaling Technology, USA), peNFekB (1:1000; Cell Signaling Technology) and GAPDH (1:1000; Abcam) overnight at 4 C, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime) and visualized using ECL chemiluminescence kit (Millipore). GAPDH was used as a loading control.
Vsig4 was at first subcloned into a pAAV-CMV bGlobin-MCSeGFP-3Flag vector (AAV-Vsig4), and the empty control vector was pAAV-CMV bGlobin-MCS-eGFP-3Flag (AAV-NC). The AAV-Vsig4 and AAV-NC were packaged by GeneChem Technology (Shanghai, China). The packaged AAVs were then concentrated in PBS at the following titers for the subsequent analysis: AAV-Vsig4, 2 1012 genome copies per milliliter, and AAV-NC, 1 1012 genome copies per milliliter. Adult male C57BL/6J mice (weighed 20e25 g) were purchased from the Animal Research Center of Zhejiang University (Zhejiang) and fed in SPF condition with a 12- to 12-h light/dark cycle and free access to water and food. All animal studies were approved by the Animal Care Committee of Zhejiang Integrated Traditional and Western Medicine Hospital (Zhejiang, China) according to the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications, revised 1996). At least six mice were involved for analysis of each data point. 3 weeks before MCAO operation, the concentrated viral solution (4 ml) containing AAV-NC or AAV-Vsig4 was stereotaxically delivered into the ipsilateral lateral ventricle in the MCAO/AAV-NC and MCAO/ AAV-Vsig4 groups, respectively. Then, the mice cerebral ischemia/ reperfusion (I/R) models were induced through MCAO surgery by the intraluminal filament according to previous studies [19]. After
2.5. Quantitative real-time polymerase chain reaction (RT-qPCR) Total RNA was isolated from peri-infarct tissues or cells with TRIzol regent (Invitrogen, USA), and was then reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The RT-qPCR was conducted using the UltraSYBR Mixture (CWBio, China), specific primers (Supplementary Table S1) and cDNA on the Mx3000P Real-Time PCR System (Agilent Technologies, USA). The GAPDH mRNA expression was defined as internal control.
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
2.6. Immunostaining
(Abcam), respectively, following the manufacturers’ protocols.
Animals were anesthetized and intracardially perfused with 0.9% sodium chloride followed by 4% paraformaldehyde. After fixing for 12 h, brain samples were dehydrated in gradient sucrose solutions of 10, 20, and 30% at 4 C. Then, the brains were embedded in optimal cutting temperature compound and cut into 10-mm sections for further analysis. Tissue sections were blocked in 0.3% Triton, 3% goat serum (Solarbio, Beijing, China), and 1% bovine serum albumin (BSA, Sigma-Aldrich). The sections were then incubated with antibody against Vsig4 (1:200; Abcam), Iba1 (1:200; Santa Cruz Biotechnology, USA), NeuN (1:200; Abcam) and/ or TLR4 (1:250; Santa Cruz Biotechnology) overnight at 4 C. Next, sections were incubated with appropriate secondary antibodies (Abcam) and DAPI (Sigma-Aldrich). The positive expression of each signal was analyzed using Image J software (NIH, USA). TUNEL staining in tissue sections and cells was conducted using the In Situ Cell Death Detection Kit (Roche, USA) and TUNEL Assay Kit
2.7. Neurological score assessment
3
Neurological score was performed 3 d after reperfusion following MCAO by two investigators blind to the experiment treatments. Neurological impairment after ischemic insult was calculated using a 5-point scale: 0 (no significant deficits), 1 (failure to fully extend left forepaw), 2 (circling to the left), 3 (fail to the left), 4 (falling to walk spontaneously combined with depressed levels of consciousness). 2.8. Behavioral analysis A rotarod test was conducted using the Rotamex 5 apparatus (Columbus Instruments, USA) as previously suggested [21]. Each mouse underwent at least 3 trials daily with an inter-trial interval of 20 min.
Fig. 1. Vsig4 expression was down-regulated post-stroke. (a) RT-qPCR and (b) western blot analysis of Vsig4 at 2 h, 6 h, 24 h, 3 d, 7 d and 28 d after ischemia stroke. (c) Co-staining for Iba1 (green) and Vsig4 (red) at 3 d after reperfusion in the peri-infarct area. Relative quantification of Vsig4-positive cells was exhibited. (d) RT-qPCR and (e) western blot analysis of Vsig4 in primary microglia after treatment with OGDR for the shown time (2 h, 6 h, 24 h, 36 h and 48 h). The data were expressed as mean ± SEM. þP < 0.05 and þþP < 0.01 vs. Sham or Con group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
4
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
2.9. Co-immunoprecipitation The lysates from cerebral tissues or primary microglia were extracted and prepared using RIPA lysis buffer (Beyotime). Protein extracts were then incubated with antibody against Vsig4 or the control IgG at 4 C overnight. Then the immune complexes were linked to protein A/G-agarose beads (Thermo Fisher Scientific) for 4 h. The eluted proteins were harvested for immunoblotting analysis. 2.10. Statistical analysis All results are expressed as mean ± SEM. Differences between groups were compared through the two-tailed Student’s t tests and one-way ANOVA followed by Tukey’s post hoc test. Value of
p < 0.05 was considered statistically significant. GraphPad Prism (Version 7.0) was used for data analysis. 3. Results 3.1. Vsig4 expression was down-regulated after stroke At first, the expression change of Vsig4 in mice post stroke was investigated. As displayed in Fig. 1a and b, we found that Vsig4 expression from mRNA and protein levels was marginally downregulated at 24 h after MCAO operation, especially reduced at 3 d after reperfusion, and gradually up-regulated. Co-immunofluorescent staining suggested that Vsig4 expression was obviously decreased in microglial cells that were marked by Iba1 [22] (Fig. 1c). Consistently, OGDR treatment restrained microglial Vsig4
Fig. 2. Vsig4 attenuates cerebral I/R injury in mice at 3 d after reperfusion. (a) TTC staining of brain sections from each group of mice. (b) Quantification of infarct area, and (c) neurological scores. (d) Photomicrographs through MRI scanning indicating the infarction area marked by the red line. (e) Representative images of NeuN counterstained with DAPI in the infarct region. (f) Quantification of NeuN-positive levels was exhibited. (g) TUNEL staining for brain sections. (h) Quantification of TUNEL-positive cells was displayed. (i) Results for rotarod test after cerebral ischemia. The data were expressed as mean ± SEM. þP < 0.05, þþP < 0.01 and þþþP < 0.001; ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 3. Vsig4 inhibits M1 like phenotype and promotes M2 like phenotype in mice 3 d post stroke. RT-qPCR analysis for (a) M1 like phenotype marks (CD16, CD11b, iNOS and IL6), and (b) M1 like phenotype marks (Arg-1, CD206, IL-10 and Ym-1). (c) Western blot analysis for peNFekB in the cerebral tissues of each group of mice. (d) Co-staining for Iba1 (green) and TLR4 (red) at 3 d after reperfusion in the peri-infarct area. Relative quantification of (e) Iba1-and (f) TLR4-positive cells was displayed. (g) Western blot analysis for TLR4 in the cerebral tissues of mice. The data were expressed as mean ± SEM. þP < 0.05, þþP < 0.01 and þþþP < 0.001; ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
6
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 4. Vsig4-inhibited inflammation in microglia is through interacting with TLR4 and inhibited its expression. (a) Representative results for immunofluorescence staining of NeuN (green) and PI (red) in neuron-glial mixed culture after OGD/R treatment for 24 h. Quantification of NeuN- and PI-positive cells was shown. (beg) Primary microglia cells were infected with AAV-Vsig4, followed by OGD/R treatment for another 24 h. Then, all cells were harvested for subsequent analysis. (b) RT-qPCR analysis for inflammation-associated
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
expression mainly through a time-dependent manner, particularly after 24 h (Fig. 1d and e). Therefore, the expression of microglial Vsig4 was decreased under ischemic-hypoxic condition. 3.2. Vsig4 attenuates cerebral I/R injury in mice at 3 d after reperfusion To further reveal the biological function of Vsig4 in ischemic stroke, an adenovirus-packaged Vsig4 expression vector was used through direct injection into the mouse brain. TTC results indicated that MCAO surgery resulted in larger infarction area, and overexpressing Vsig4 markedly reduced the infarction volume (Fig. 2a and b). We also found that MCAO-induced neurological deficit score was significantly alleviated by the over-expression of Vsig4 (Fig. 2c). MRI assay confirmed the results that promoting Vsig4 expression could decrease the infarct size of mice with ischemic stroke (Fig. 2d). Immunofluorescent results showed that Vsig4 increase greatly rescued the neuron survival, as evidenced by the obviously increased number of NeuN-positive cells (Fig. 2e and f). In parallel, MCAO-induced cell death in cerebral infarct area was markedly alleviated in mice infected with AAV-Vsig4, as detected by the down-regulated number of TUNEL-positive cells (Fig. 2g and h). The rotarod test suggested that the latency to fall off the rotarod was much longer in the AAV-Vsig4-injected group compared to the MCAO group (Fig. 2i). Thus, promoting Vsig4 expression inhibited infarction area and protected neurological function in mice after MCAO surgery. 3.3. Vsig4 inhibits M1 like phenotype but promotes M2 like phenotype in mice 3 d post stroke Inflammation plays a key role in regulating the pathogenesis of ischemic stroke, which is closely associated with the release of proinflammatory cytokines or factors by M1 microglia; conversely, the M2 like phenotype microglia that produce anti-inflammatory cytokines are repressed [23]. We then found that the mRNA levels of pro-inflammatory factors including CD16, CD11b, iNOS and IL-6 were markedly elevated in the cerebral infarct area, while being down-regulated in AAV-Vsig4 mice (Fig. 3a). In contrast, antiinflammatory meditators such as Arg-1, CD206, IL-10 and Ym-1 reduced by MCAO were significantly restored by over-expressing Vsig4 (Fig. 3b). TLR4/NF-kB signaling pathway is involved in inducing M1 microglia to promote inflammatory response [7e9]. Western blot and/or immunofluorescent analysis suggested that MCAO operation led to significant increases of NF-kB and TLR4, and these effects were clearly abrogated in mice injected with AAVVsig4. Also, immunofluorescent staining demonstrated that TLR4 was markedly expressed in microglia of the infarction area from MCAO-treated mice, as evidenced by the obvious co-localization with Iba1 (Fig. 3ceg). Collectively, Vsig4-inhibited the secretion of pro-inflammatory factors might be associated with the blockage of TLR4/NF-kB signaling. 3.4. Vsig4-associated inflammation inhibition in microglia is rely on interacting with TLR4 pathway To further validate the effects of Vsig4 on ischemic stroke, as well as the underlying molecular mechanism, in vitro experiments using primary microglia were then performed. As shown in Fig. 4a,
7
the neuron-glial mixed culture by fluorescent staining suggested that OGD/R treatment led to significant decrease in the number of NeuN-positive cells, while resulted in obvious increase in TUNELpositive cells, and notably these results were reversed by overexpressing Vsig4 in microglial cells. By RT-qPCR analysis, the primary microglial cells after OGD/R treatment showed markedly increased mRNA levels of CD16, CD11b, iNOS and IL-6, while the decreased expression of Arg1, CD206, IL-10 and Ym-1. Importantly, these results were reversed in microglia with Vsig4 over-expression (Fig. 4b). What’s more, OGD/R-reduced expression of Vsig4, and -elevated expression of peNFekB and TLR4 were greatly resumed by AAV-Vsig4 in primary microglial cells (Fig. 4cef). As showed in Fig. 4g, immunofluorescent staining confirmed the expression change of Vsig4 and TLR4 in OGD/R-stimulated microglial cells with or without Vsig4 over-expression as detected by western blotting. Finally, we explored if an association existed between Vsig4 and TLR4 in microglia. Using co-immunoprecipitation analysis, Vsig4 appeared to interact with TLR4 both in vivo and in vitro (Fig. 4h). Therefore, Vsig4 could interact with microglial TLR4, and then modulate the progression of ischemic stroke via regulating inflammatory response. 4. Discussion Ischemic cerebral stroke results in permanent injury to the CNS, and this process includes a critical step if post-ischemic inflammatory response, which then influence the physical function in individuals [1e4]. Increasing studies have illustrated that excessive inflammatory response could be detected in ischemic models both in vivo and in vitro, as indicated by the elevated microglial M1 polarization. M1 like phenotype microglial cells show significantly cytotoxicity through releasing a large number of pro-inflammatory factors, such as iNOS, CD11b, IL-1b and IL-6. In contrast, the release of anti-inflammatory meditators (Arg1, CD206, Ym-1, IL-10, etc) by M2-like phenotype microglia is blunted [23,24]. Vsig4, as a B7associated protein that is specifically expressed in macrophages, regulates suppressive signals to inhibit NLRP3 inflammasome and subsequent Il-1b transcription, and this process was related to the inhibition of NF-kB [16]. In our study, we found that Vsig4 expression was markedly decreased during ischemic stroke progression in MCAO-operated mice, and in OGD/R-treated primary microglial cells. Meanwhile, Vsig4 was found to co-localize with Iba-1, suggesting its expression in microglial cells in the infarct region of MCAO-mice. Moreover, the MCAO surgery-induced neuronal death, infarct area enlargement and functional impairment in mice were significantly alleviated by Vsig4 overexpression. Promoting Vsig4 expression greatly inhibited the microglial M1 polarization, while promoted M2-like phenotype. This anti-inflammatory effect regulated by Vsig4 was largely associated with the blockage of TLR4/NF-kB signaling. Notably, the Coimmunoprecipitation analysis illustrated that Vsig4 could interact with TLR4, thereafter modulating the progression of cerebral ischemic stroke (Fig. 4i). Thus, these results provided evidence that targeting and improving Vsig4 expression could ameliorate ischemic stroke, and it could be served as a novel and effective therapeutic signal to develop useful treatment for stroke or associated disease. The innate immune system is an essential factor involved in the pathophysiology of cerebral ischemic stroke. It is generally
genes as exhibited. (cef) Western blot results for Vsig4, TLR4 and peNFekB. (g) Immunofluorescence staining of Vsig4 (red) and TLR4 (green) in primary microglia. (h) The lysates from cerebral tissue and microglia were immunoprecipitated with anti-Vsig4. Then, immunoprecipitates were analyzed using western blot with anti-Vsig4 and anti-TLR4. (i) Proposed schematic mechanism demonstrating microglial Vsig4 meditated ischemic neuro-inflammation. The data were expressed as mean ± SEM. þP < 0.05, þþP < 0.01 and þþþ P < 0.001; ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077
8
Q. Lyu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
accepted that the pro-inflammatory response is originally regulated by microglia-triggered CNS injury in ischemic stroke [25]. Microglia cells are extremely activated and then release excessive pro-inflammatory meditators, such as CD11b, iNOS, CD16 and IL-6, immediately following ischemic stroke, which result in neuronal apoptosis and accelerate brain injury consequently [23,24]. M1 microglia is a toxic cellular state linked to an elevation of proinflammatory meditators. By contrary, M2-like phenotype microglia promote secretion anti-inflammatory factors (Arg1, CD206, Ym-1, IL-10, IL-4, etc) [10,11,26]. Increasing evidences suggest that ischemic animal models with M1-polarized microglia intensify neuron death whereas the M2 microglia could protect neurons and improve post-injury tissue repairmen [27]. As reported, TLR4 is primarily expressed in microglial cells and regulates microglial activation partly via promoting the activation of NF-kB signaling and subsequent release of inflammatory cytokines or chemokine [7e9]. Suppressing TLR4 showed beneficial effects on tissue homeostasis and ameliorates cerebral ischemic damage [28]. In our study, we confirmed that MCAO-induced mice with ischemic stroke showed M1-like phenotype microglia, accompanied with significantly up-regulated expression of pro-inflammatory regulators and down-regulated anti-inflammatory signals. Notably, these effects were reversed by Vsig4 over-expression. Recent reports have provided insights that inflammatory activation by microglial cells can elicit apoptotic cell death both acute under in vitro and in vivo ischemic conditions [29,30]. Because we found an up-regulated expression and activation level of in inflammatory molecules in microglial cells, we then calculated the functional role of inflammation regulated by Vsig4 in neuronal cell death under ischemic conditions. Mix culture of primary microglial cells and neurons showed that OGD/R-induced neuron death as proved by the downregulated NeuN-positive levels was significantly rescued by Vsig4 over-expression in primary microglial cells. Recent studies have provided evidence that Vsig4 could suppress inflammatory response in macrophages associated with the inhibition of NF-kB signaling; however, Vsig4 insufficiency aggravated inflammatory damage in in vitro and in vivo [16]. Additionally, in response to inflammatory stimuli, Vsig4 reduced macrophage activation and inhibited M1 polarization [17]. More recently, the deficiency of Vsig4 accelerated the production of pro-inflammatory cytokines, which was involved in high fat diet-induced insulin resistance, lipid deposition and fibrosis in hepatic tissues [18]. Therefore, we supposed that Vsig4-alleviated cerebral stroke was largely dependent on its suppression to microglial inflammatory response. More importantly, we found that Vsig4 could interact with TLR4, thus inhibiting its expression and the subsequent inflammatory response. However, there is still possibility that microglial Vsig4 might interact with other molecules in ischemiaassociated cerebral immune response. As for this, further analysis is still necessary to reveal more details regulated by Vsig4 during cerebral ischemic damage. Together, these results showed that Vsig4 expression was reduced in infarction region of mice with cerebral ischemic stroke. We also found that promoting Vsig4 expression inhibited M1 polarization in microglia by blocking NF-kB activation via interacting with TLR4 and repressing TLR4 expression, subsequently reducing the release of pro-inflammatory response and neuronal death (Fig. 4i). Together, Vsig4 is considered as a promising and effective therapeutic target for ischemic stroke or related diseases.
Acknowledgement This work was supported by Chinese Medicine Research program of Zhejiang province (No. 2019ZA-089).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.077.
References [1] W.N. Kernan, et al., Pioglitazone after ischemic stroke or transient ischemic attack, N. Engl. J. Med. 374 (14) (2016) 1321e1331. [2] X. Wang, et al., The evolving role of neuro-immune interaction in brain repair after cerebral ischemic stroke, CNS Neurosci. Ther. 24 (12) (2018) 1100e1114. [3] H. Yan, et al., Long noncoding RNA MEG3 activation of p53 mediates ischemic neuronal death in stroke, Neuroscience 337 (2016) 191e199. [4] S. Yao, et al., miR-455 inhibits neuronal cell death by targeting TRAF3 in cerebral ischemic stroke, Neuropsychiatric Dis. Treat. 12 (2016) 3083. [5] M.D. Ginsberg, Neuroprotection for ischemic stroke: past, present and future, Neuropharmacology 55 (3) (2008) 363e389. [6] M. Fukata, et al., Innate immune signaling by Toll-like receptor-4 (TLR4) shapes the inflammatory microenvironment in colitis-associated tumors, Inflamm. Bowel Dis. 15 (7) (2009) 997e1006. [7] H. Zhu, et al., Curcumin attenuates acute inflammatory injury by inhibiting the TLR4/MyD88/NF-kB signaling pathway in experimental traumatic brain injury, J. Neuroinflammation 11 (1) (2014) 59. [8] B. Han, et al., Electroacupuncture modulated the inflammatory reaction in MCAO rats via inhibiting the TLR4/NF-kB signaling pathway in microglia, Int. J. Clin. Exp. Pathol. 8 (9) (2015) 11199. [9] Y.W. Li, et al., Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NF-kB p65 signal pathway, Gene 542 (1) (2014) 46e51. [10] C. Schmidt, et al., Phosphoinositide 3-kinase g restrains neurotoxic effects of microglia after focal brain ischemia, Mol. Neurobiol. 53 (8) (2016) 5468e5479. [11] Y. Liu, et al., CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway, J. Cereb. Blood Flow Metab. 35 (10) (2015) 1623e1631. [12] F. Zheng, et al., Monitoring liver macrophages using nanobodies targeting Vsig4: concanavalin A induced acute hepatitis as paradigm, Immunobiology 220 (2) (2015) 200e209. [13] S.J.L. van der Tuin, et al., Lipopolysaccharide lowers cholesteryl ester transfer protein by activating F4/80þ Clec4fþ Vsig4þ Ly6C Kupffer cell subsets, J. Am. Heart Assoc. 7 (6) (2018) e008105. [14] L. Vogt, et al., VSIG4, a B7 family-related protein, is a negative regulator of T cell activation, J. Clin. Investig. 116 (10) (2006) 2817e2826. [15] W. Fu, et al., Early window of diabetes determinism in NOD mice, dependent on the complement receptor CRIg, identified by noninvasive imaging, Nat. Immunol. 13 (4) (2012) 361. [16] X. Huang, et al., VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1b in macrophages, Sci. Adv. (1) (2019) 5, eaau7426. [17] J. Li, et al., VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism, Nat. Commun. 8 (1) (2017) 1322. [18] Y. Li, et al., Expression of Vsig4 attenuates macrophage-mediated hepatic inflammation and fibrosis in high fat diet (HFD)-induced mice, Biochem. Biophys. Res. Commun. 516 (3) (2019) 858e865. [19] X. Xu, et al., MicroRNA-1906, a novel regulator of toll-like receptor 4, ameliorates ischemic injury after experimental stroke in mice, J. Neurosci. 37 (43) (2017) 10498e10515. [20] L.K. Parvathenani, et al., P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer’s disease, J. Biol. Chem. 278 (15) (2003) 13309e13317. [21] X. Tang, et al., Genetic deletion of Krüppel-like factor 11 aggravates ischemic brain injury, Mol. Neurobiol. 55 (4) (2018) 2911e2921. [22] J. Tischer, et al., Inhomogeneous distribution of Iba-1 characterizes microglial pathology in Alzheimer’s disease, Glia 64 (9) (2016) 1562e1572. [23] Y. Tang, et al., Differential roles of M1 and M2 microglia in neurodegenerative diseases, Mol. Neurobiol. 53 (2) (2016) 1181e1194. [24] J. Pan, et al., Malibatol A regulates microglia M1/M2 polarization in experimental stroke in a PPARg-dependent manner, J. Neuroinflammation 12 (1) (2015) 51. [25] D. Amantea, et al., Rational modulation of the innate immune system for neuroprotection in ischemic stroke, Front. Neurosci. 9 (2015) 147. [26] C.Y. Xia, et al., Selective modulation of microglia polarization to M2 phenotype for stroke treatment, Int. Immunopharmacol. 25 (2) (2015) 377e382. [27] H.L. Meng, et al., Neuronal soluble fas ligand drives M1-microglia polarization after cerebral ischemia, CNS Neurosci. Ther. 22 (9) (2016) 771e781. [28] S.L. Wang, et al., Dexmedetomidine preconditioning plays a neuroprotective role and suppresses TLR4/NF-kB pathways model of cerebral ischemia reperfusion, Biomed. Pharmacother. 93 (2017) 1337e1342. [29] M. Zhang, et al., The cystathionine b-synthase/hydrogen sulfide pathway contributes to microglia-mediated neuroinflammation following cerebral ischemia. Brain, behavior, and, Immunity 66 (2017) 332e346. [30] X. Liu, et al., Salidroside provides neuroprotection by modulating microglial polarization after cerebral ischemia, J. Neuroinflammation 15 (1) (2018) 39.
Please cite this article as: Q. Lyu et al., Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.077