- Email: [email protected]
Mesenchymal stem cells enhance microglia M2 polarization and attenuate neuroinflammation through TSG-6
Brain Research 1724 (2019) 146422
Contents lists available at ScienceDirect
Brain Research journal homepage: www.elsevier.com/locate/brainres
Research report
Mesenchymal stem cells enhance microglia M2 polarization and attenuate neuroinflammation through TSG-6
T
⁎
Yi Liua,b, ,1, Rong Zengc,1, Yezhong Wangb, Wenhui Huangb, Bin Hub, Guohui Zhud, Run Zhangd, Feng Lid, Jianbang Hand, Yongshi Lie a
Department of Neurosurgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China Department of Neurosurgery and Neurosurgical Disease Research Centre, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China c Department of Radiotherapy, Oncology Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China d Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China e Department of Neurosurgery, GuangDong 999 Brain Hospital, Guangzhou, China b
H I GH L IG H T S
suppress the inflammatory response in the LPS-induced neuroinflammation mouse model • MSCs prevented microglial M1 activation and promoted M2 polarization. • MSCs • TSG-6 play a key role in the MSC-mediated microglial polarization.
A R T I C LE I N FO
A B S T R A C T
Keywords: Mesenchymal stem cells TSG-6 Microglia polarization Neuroinflammation
Microglia are the primary cells that exert immune function in the central nervous system (CNS), and they play an important role in the pathogenesis and progression of neuroinflammation-related diseases. Mesenchymal stem cells (MSCs) have been demonstrated to promote functional recovery in many neurological diseases. The mechanisms underlying this may be that MSCs can reduce inflammatory responses through various soluble factors. Among these factors, tumor necrosis factor-α-induced gene/protein 6 (TSG-6) is a key factor influencing MSCs immunomodulatory properties; however, the precise mechanisms underlying the anti-inflammatory effects are not fully understood. Here, we aim to investigate the potential effects of MSCs on neuroinflammation and to reveal the underlying mechanisms. First, we confirmed that administration of MSCs could inhibit the lipopolysaccharide (LPS)-induced neuroinflammatory responses in a mouse model. Then, we found that MSCs promoted M2 polarization and inhibited M1 polarization both in vivo and in vitro. Moreover, we demonstrated that the effect of MSCs on microglial polarization was dependent on TSG-6. This study demonstrated that MSCs promoted M2 polarization of microglia via TSG-6, thus conferring anti-neuroinflammatory effects.
1. Introduction Neuroinflammation is an important pathological process involved in traumatic brain injury (TBI) (Borlongan et al., 2015), stroke (Xia et al., 2015), intracerebral hemorrhage (ICH) and various neurodegenerative diseases (Alam et al., 2016; Glass et al., 2010; Machado et al., 2016). Under normal conditions, neuroinflammation maintains homeostasis and promotes tissue repair. However, unregulated neuroinflammation can be detrimental to the brain. Therefore, controlling harmful inflammatory responses is a promising therapeutic approach for
neurological diseases (DiSabato et al., 2016). As an important type of immune cell in the CNS, microglia play a crucial role in initiating and maintaining neuroinflammatory responses (Franco and Fernández-Suárez, 2015). Similar to macrophages, microglial cells show plasticity and can polarize toward the classical M1 or alternative M2 phenotype (Boche et al., 2013). M1-polarized microglia are characterized by increased proinflammatory cytokine levels, including TNF-a, IL-1β, and IL-6, and increased iNOS and CD16 levels, whereas M2-polarized microglia have high expression of M2 phenotype markers, such as Arg-1, CD206, Ym-1/2, and TGFβ. Functionally, M1
⁎
Corresponding author at: Department of Neurosurgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China E-mail address: [email protected] (Y. Liu). 1 Equal contributors. https://doi.org/10.1016/j.brainres.2019.146422 Received 15 June 2019; Received in revised form 24 August 2019; Accepted 27 August 2019 Available online 28 August 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.
Brain Research 1724 (2019) 146422
Y. Liu, et al.
Fig. 1. MSCs suppress the inflammatory response in the LPS-Induced Neuroinflammation Mouse Model. Neuroinflammation was induced by intraperitoneal injection of LPS. Twenty-four hours after LPS stimulation, MSCs were infused ICV, and control mice were infused with PBS. The mRNA expression level was determined by qRT-PCR. A: MSCs inhibited LPS-induced mRNA expression of TNF-a, IL-6, and IL-1β; B: MSCs prevented the LPS-induced downregulation of TGF-β and IL-10. Data are expressed as the means ± SD of three independent experiments. n = 3 #p < 0.01, versus control group; *p < 0.05, **p < 0.01, versus LPStreated group.
The cells were harvested, resuspended in PBS, and stained with phycoerythrin- or FITC-conjugated mouse monoclonal antibodies against CD44, CD90, CD34, CD31, and CD45 (eBioscience, San Diego, CA, USA) on ice for 20 min. Mouse IgG1 isotype-control antibodies were used in parallel as negative controls. The stained cells were then washed twice and resuspended in cold buffer and analyzed with flow cytometry (FACS Calibur; BD Biosciences). The results are expressed as the percentage of positively stained cells relative to the total cell number. MSCs from only passages 3–8 were used in the experiments.
microglia exacerbate neuronal injury and impede cellular repair after CNS disruption. In contrast, M2 microglia confer neuroprotection and promote recovery and remodeling (Hu et al., 2015). Therefore, inhibiting M1 microglia polarization and promoting M2 microglia polarization may be a viable strategy for treating neuroinflammation-related diseases (Jiao et al., 2018; Li et al., 2018). Recent studies have shown that MSCs administration could promote functional recovery in many neurological diseases (Jaimes et al., 2017; Park et al., 2016). The mechanism underlying this effect may be a reduction in inflammation responses through various soluble factors, including neurotrophic growth factors, chemokines, cytokines, and extracellular matrix proteins (Teixeira et al., 2017). In our previous studies, we demonstrated that MSCs can reduce inflammatory factor production in microglia through TSG-6 (Liu et al., 2014). Whether MSCs can enhance microglial M2 polarization through TSG-6 and thereby exert neuroprotective effects has not been investigated. In this study, we investigated the mechanisms underlying beneficial effects of MSCs on inflammation in the CNS. Our results demonstrated that MSCs administration inhibited LPS-induced neuroinflammatory responses in a mouse model by mediating the microglial activation status. Furthermore, we speculated that the polarization effects of MSCs on microglial status were mediated by TSG-6 expression. We confirmed this hypothesis by demonstrating that transfection of MSCs with TSG-6 siRNA led to a decreased effect on microglial polarization. MSCs can change macrophage/microglia polarization and inhibit a variety of inflammatory diseases (Di et al., 2017; Song et al., 2019; Watanabe et al., 2019), but whether MSCs can regulate microglia polarization through TSG-6 in inflammatory CNS diseases has not been reported. Based on our in vitro and in vivo research, we propose that MSCs can promote M2 polarization of microglia in neuroinflammatory responses and that TSG-6 plays an important role in microglia polarization induced by MSCs.
2.2. MSCs transfection with small interfering RNA (siRNA) A total of 2 × 105 MSCs were plated in 6-well dishes and cultured for 24 h; then, the cells were transfected with TSG-6 siRNA or control siRNA (sc-39820 and sc-37007, respectively; Santa Cruz Biotechnology Inc., Paso Robles, USA) using Lipofectamine 3000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). To confirm the silencing effect of the siRNA, RNA was extracted from aliquots of the cells after 48 h, and TSG-6 expression was analyzed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). TSG6 secreted into the supernatant was detected by ELISA. 2.3. Animal experiments Male C57BL/6 mice (25–30 g, approximately 8 weeks old) were obtained from The Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were maintained in specific pathogen-free conditions with a 12 h light/12 h dark cycle and standard temperature and humidity conditions. In addition, the mice were provided a regular diet daily and clean water ad libitum. All animal experiments were performed according to guidelines for the care and use of laboratory animals (Ministry of Science and Technology of China, 2006) and approved by the Animal Care and Use Committee of Sun Yat-sen University. Neuroinflammation was induced by intraperitoneal injection of LPS (1 mg/kg). Twenty-four hours after LPS stimulation, a hole was drilled in the right scalp of each anesthetized mouse at coordinates 0 mm caudal to the bregma, 1 mm lateral to the midline, and 3 mm beneath the dura mater. MSCs were prepared as previously described, and the cell concentration was adjusted to 150,000 cells/5 μl PBS. MSCs were infused intracerebroventricularly (ICV) over 5 min, and the needle was left in place for another 5 min. Control mice were infused with 5 μl PBS alone. MSCs transfected with TSG-6 siRNA or control siRNA control
2. Materials and methods 2.1. MSCs isolation, expansion and characterization MSCs were prepared from mouse bone marrow (BM) cells as previously described (Rahmat et al., 2013) with minor modifications. MSCs were isolated from the BM of male C57BL/6 mouse tibias and femurs. MSCs were confirmed by flow cytometry analysis after three passages. 2
Brain Research 1724 (2019) 146422
Y. Liu, et al.
Fig. 2. MSCs promoted microglia M2 polarization in the LPS-Induced Neuroinflammation Mouse Model. The percentages of M1/M2 microglia surface markers were determined by immunofluorescence staining. A: Representative immunofluorescence staining of cortex costained for Iba-1 (red) and CD16/32 (green) or costained for Iba-1 (red) and CD206 (green), Scale bar = 50 μm. B: The M1 marker genes CD16 and iNOS were determined by qRT-PCR assay. C: qRT-PCR for mRNA expression of the M2 markers CD206 and Arg1. Data were expressed as the means ± SD of three independent experiments. n = 3. # p < 0.01, versus control group; **p < 0.01, versus LPS-treated group.
administration, and whole brains were removed for additional experiments.
were injected according to the same procedures. Four mice were used in the control group, and six mice were used in the LPS, LPS+MSC, LPS +siTSG6-MSC and LPS + siCtrl-MSC treatment groups. No animals died after transplantation. The animals were sacrificed 24 h after MSCs 3
Brain Research 1724 (2019) 146422
Y. Liu, et al.
Fig. 3. MSCs promoted microglia M2 polarization in BV2 microglial cells. MSCs produced more TSG-6 after LPS treatment and decreased LPS-induced M1 marker gene expression and promoted M2 marker gene expression in BV2 microglial cells. A: After treatment with LPS the mRNA levels of TSG-6 in MSCs were significantly increased. B: The secretion levels of TSG-6 in MSCs were detected by ELISA. C: qRT-PCR for mRNA expression of M1 markers (CD16, iNOS) in BV2 microglial cells. D: qRT-PCR for mRNA expression of M2 markers (CD206, Arg1) in BV2 microglial cells. E: MSCs significantly decreased iNOS expression, whereas they increased CD206 expression in BV2 cells, evidenced by Western blot assay. Data were expressed as the means ± SD of three independent experiments. n = 3, #p < 0.01, versus control group; **p < 0.01, versus LPS-treated group.
2.4. BV2 cell culture
2.6. Enzyme-linked immunosorbent assay (ELISA)
The BV2 murine microglial cell line was obtained from Xiehe Medical University (Beijing, China). The cells were cultured in a humidified incubator at 37 °C with 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS, 100 μg/ml streptomycin, 100 U/ml penicillin (HyClone, Logan, UT, USA), and 2 mmol/l glutamine (Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO2. A 6-well transwell system (0.4-μm pore size membrane; Corning, Cambridge, MA, USA) was used to assess the effect of MSCs on BV2 cells. A total of 5 × 105 BV2 cells were placed in the lower chamber and stimulated with 100 ng/mL LPS. After stimulation, 1.0 × 105 MSCs were placed in the upper chamber. BV2 cells without LPS stimulation were used as controls. After 24 h, the microglia were washed and harvested for further studies.
TSG-6 levels in the supernatants were measured with a TSG-6 ELISA kit (MyBiosource, San Diego, CA, USA) and a multidetection microplate reader according to the manufacturer's instructions. 2.7. Immunofluorescence and laser-scanning confocal microscopy Immunofluorescence staining and laser-scanning confocal microscopy were performed as previously described. Brain sections (14-μm thickness) were fixed and washed. The slides were blocked for 60 min and incubated at 4 ℃ overnight with primary antibodies, including rabbit anti-Iba1 (1:800; Abcam, Cambridge, UK), rat anti-CD16/32 (1:800; Abcam, Cambridge, UK), and goat anti-CD206 (Santa Cruz, TX, USA). After that, the sections were incubated with an appropriate fluorescent secondary antibody (1:500) and stained with DAPI (Beyotime, China). The sections were imaged with a confocal microscope (TCS SP5; Lecia, Solms, Germany). Cell numbers were calculated by counting the cells in random microscopic fields in a blinded fashion. The data are expressed as the percentage of CD16/32+ or CD206+ cells to Iba-1+ cells.
2.5. RNA extraction, cDNA synthesis, and qRT-PCR Total RNA was extracted from the brain tissues and BV2 cells using a PrimeScript RT reagent kit (Takara, Japan) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized and used for qRT-PCR using a SYBR Green Kit (Takara, Japan), genespecific primers, and a 7500 Sequence Detection system (Applied Biosystems, USA). GAPDH was used as an endogenous control. The primers that were used are listed in the Supplementary Table.
2.8. Western blotting analysis Western blotting was performed to assess the expression of iNOS 4
Brain Research 1724 (2019) 146422
Y. Liu, et al.
(caption on next page)
5
Brain Research 1724 (2019) 146422
Y. Liu, et al.
Fig. 4. MSCs with TSG-6 knockdown had less impact on reducing the polarization of M1 microglia and promoted M2 polarization. The mRNA and secretion levels of TSG-6 were significantly lower in siTSG6-MSCs than in MSCs or control siRNA-transfected MSCs (siCtrl-MSCs) A: Relative expression level of TSG-6 mRNA was determined by qRT-PCR. B: The secretion levels of TSG-6 were detected by ELISA. siTSG6-MSCs had a decreased effect on altering the expression of pro- and antiinflammatory in mouse model. C, D: The mRNA expression levels of pro-inflammatory TNF-a, IL-6, IL-1β and anti-inflammatory TGF-β, IL-10 were determined by qRT-PCR. E, F: qRT-PCR for mRNA expression of the M1 markers CD16 and iNOS, and the M2 markers CD206 and Arg1. G, H: Representative immunofluorescence staining of cortex costained for Iba-1 (red) and CD16/32 (green), or costained for Iba-1 (red) and CD206 (green). Scale bars = 50 μm. I: qRT-PCR for mRNA expression of M1 markers (CD16, iNOS) in BV2 microglial cells. J: The iNOS protein levels in BV2 microglial cells were determined by Western blotting. K: qRT-PCR for mRNA expression of M2 markers (CD206, Arg1) in BV2 microglial cells. L: The CD206 protein levels in BV2 microglial cells were determined by Western blotting. Data were expressed as the means ± SD of three independent experiments. n = 3, **p < 0.01.
with Iba-1 was decreased after LPS stimulation compared with that in the control group. However, MSCs administration attenuated M1 marker expression and promoted M2 marker expression (Fig. 2A). Consistent with the immunofluorescence results, the mRNA expression of M1 genes (iNOS, CD16) was markedly increased after LPS stimulation, whereas MSCs treatment prevented this effect and increased the levels of M2 genes (Arg-1 and CD206) after LPS stimulation in mice (Fig. 2B, C). These results suggest that MSCs administration inhibited microglia polarization to the M1 phenotype and instead promoted a shift to the M2 phenotype in the LPS-induced neuroinflammation mouse model.
and CD206 in BV2 cells. Total proteins were extracted using RIPA lysis buffer (sc-24948; Santa Cruz Biotechnology, CA, USA). Protein concentrations were estimated using a BCA protein assay kit (Thermo Scientific, Pierce, Rockford, IL, USA), and 20 μg protein was loaded per lane. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked using 5% nonfat dry milk in TBST and then incubated with rabbit anti-iNOS (Cell Signaling Technology, Danvers, MA, USA) or goat anti-CD206 (Santa Cruz, TX, USA) primary antibody at a 1:1000 dilution at 4°C overnight. Diluted (1:5000) horseradish peroxidase (HRP)-conjugated goat anti-rabbit or donkey anti-goat (Beyotime, Hangzhou, China) secondary antibodies were applied for 1 h at room temperature. The immunoblots were visualized using enhanced chemiluminescence (ECL; Thermo Scientific), and the cellular expression levels were normalized to GAPDH (Cell Signaling Technology).
3.4. MSCs prevented LPS-induced M1 microglial activation and promoted microglial polarization toward the M2 phenotype in microglial cells To elucidate the potential mechanisms responsible for the effects of MSCs on microglial polarization, we measured TSG-6 gene expression and secretion levels after LPS stimulation. Our data showed that the mRNA and secretion levels of TSG-6 in MSCs after treatment with LPS were significantly increased in vitro (Fig. 3A, B). Then, in a co-culture experiment, we examined M1 or M2 polarization in BV2 cells. The qRTPCR results showed that expression of the M1 marker genes CD16 and iNOS was increased after LPS stimulation. However, MSCs treatment reduced LPS-induced M1 marker gene expression (Fig. 3C). Furthermore, significant increases in the M2 marker genes Arg-1 and CD206 were observed in the MSCs-treated groups compared with those in the LPS group (Fig. 3D). In addition, the Western blot results showed that MSCs decreased LPS-induced iNOS expression and promoted the protein expression of the M2 marker CD206 (Fig. 3E). In conclusion, MSCs prevented LPS-induced M1 microglial polarization and promoted the M2 phenotype in vitro.
2.9. Statistical analysis All experiments were completed at least three times, and the data are expressed as the mean ± SD. Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) or two-tailed Student’s t-test was used to determine the significance between groups. Significance was indicated at p < 0.05. 3. Results 3.1. Characterization of MSCs After three passages, MSCs were confirmed by flow cytometry analysis, and the cells were positive for CD44 (93.90%) and CD90 (98.57%) but not CD34 (0.58%), CD31 (0.13%) or CD45 (0.36%) (Supplementary Figure).
3.5. MSCs with TSG-6 knockdown had little effect on reducing M1 microglia polarization and promoted M2 polarization
3.2. MSCs suppress the inflammatory response in an LPS-induced neuroinflammation mouse model
To obtain further insight into the role of TSG-6 in modulating microglia activities, we knocked down TSG-6 in MSCs with siRNAs. The mRNA and secretion levels of TSG-6 were significantly lower in siTSG6MSCs than in MSCs or control siRNA-transfected MSCs (siCtrl-MSCs) (Fig. 4 A, B). After successful transfection, we repeated part of the above in vivo and in vitro experiments. The expression levels of the inflammatory cytokines TNF-a, IL-6, and IL-1β were significantly increased, and the expression levels of anti-inflammatory TGF-β1 and IL10 were significantly decreased in neuroinflammation mouse model in siTSG6-MSCs group compared with MSCs or siCtrl-MSCs groups (Fig. 4C and D). Next, we assessed microglial polarization in neuroinflammation mouse model. siTSG6-MSCs had little effect on reducing expression levels of the M1 marker genes CD16 and iNOS or the percentage of CD16/32 coexpression with Iba-1 compared to nontransfected MSCs or siCtrl-MSCs (Fig. 4E,G). Moreover, expression levels of the M2 microglia marker genes CD206 and Arg1 and the level of CD206 coexpression with Iba-1 in the cortex were significantly lower in siTSG6-MSCs than in nontransfected MSCs or siCtrl-MSCs (Fig. 4F, H). In the in vitro studies, the expression levels of the M1 marker genes CD16 and iNOS and protein levels of iNOS were significantly higher in LPS-stimulated BV2 cells cocultured with siTSG6-MSCs than in
To investigate the effect of MSCs on neuroinflammation in a mouse model in vivo, we analyzed the mRNA expression of TNF-a, IL-6, IL-1β, TGF-β1 and IL-10 with qRT-PCR. As shown in Fig. 1A, the mRNA expression levels of the proinflammatory cytokines TNF-a, IL-6 and IL-1β were significantly higher after LPS stimulation than after PBS treatment. Additionally, the expression of the anti-inflammatory cytokines TGF-β1 and IL-10 decreased after LPS stimulation compared with that in the control group (Fig. 1B). MSCs administration attenuated LPSinduced proinflammatory gene expression and promoted anti-inflammatory gene expression. 3.3. MSCs promoted microglial M2 polarization in vivo CD16 and CD32 are cell surface markers for the M1 microglial phenotype, whereas CD206 is a cell surface marker of the M2 phenotype. First, we analyzed M1/M2 surface markers by immunofluorescence. The results revealed that CD16/32 coexpression with Iba1 in the cortex was significantly increased, and CD206 coexpression 6
Brain Research 1724 (2019) 146422
Y. Liu, et al.
In addition, recent studies have shown that TSG-6 released from MSCs ameliorates inflammation by inducing M2 polarization of macrophages/microglia in inflammatory bowel disease, DSS-induced colitis, and diabetic corneal epithelial wound healing animal models (Di et al., 2017; Song et al., 2018, 2017). However, whether MSCs can regulate microglia polarization through TSG-6, thus affecting the neuroinflammatory response, has rarely been studied. Based on the above findings, we speculated that TSG-6 secreted from MSCs plays a key role in inducing microglial M2 polarization. Therefore, we transfected MSCs with siRNAs to examine whether these cells could induce a microglia phenotype switch in vivo and in vitro. The in vivo experimental results showed that M2 marker expression was induced in the siCtrl-MSCs- and naive MSCs-treated groups of the neuroinflammatory mouse model. However, the expression of these markers was much lower in siTSG6-MSCs-treated animals. The in vitro experimental results also confirmed that the expression of M2 markers was lower in LPS-stimulated BV2 cells cultured with siTSG6-MSCs than in those cultured with MSCs and siCtrl-MSCs. Taken together, these findings suggest that TSG-6 plays a crucial role in regulating the M1 to M2 phenotypic switch. The main limitation of our research is that several soluble factors, such as PGE2 and IDO, have been reported to be involved in MSCsmediated immunoregulation. Therefore, in addition to TSG-6, it is apparent that MSCs can produce a variety of anti-inflammatory factors, and the results presented here do not rule out the effects of other antiinflammatory factors. Another limitation of our experiment is the lack of mechanistic research on TSG-6 and phenotypic alterations in microglia. Further research must be performed to determine the specific mechanisms of this action. In conclusion, we demonstrated that MSCs ameliorated LPS-induced neuroinflammation in mice by inducing microglia to switch to the M2 phenotype through TSG-6. These findings suggest that MSCs administration could be a promising treatment option for neuroinflammatory disorders.
nontransfected MSCs or siCtrl-MSCs (Fig. 4I and J). In addition, expression levels of the M2 microglia marker genes CD206 and Arg1 and protein levels of CD206 (Fig. 4 K, L) were significantly reduced in LPSstimulated BV2 cells cocultured with siTSG6-MSCs. However, siCtrlMSCs had an effect similar to nontransfected MSCs on the gene and protein expression of LPS-stimulated BV2 microglia cells. These data suggest that TSG-6 expression in MSCs plays a vital role in mediating microglial polarization both in vivo and in vitro. 4. Discussion Neuroinflammation is an important pathological process in many CNS diseases. Therapeutic targeting of inflammation represents an exciting approach for neuroprotective strategies. Microglia are the major cellular elements with immune function in the CNS, and they play an important role in the pathogenesis and progression of neuroinflammation-related diseases. It is well recognized that microglial cells can polarize into different functional phenotypes: the proinflammatory M1 phenotype and the anti-inflammatory M2 phenotype. M1 activation leads to the release of proinflammatory cytokines, while M2 activation enhances the expression of genes involved in inflammation resolution, immunomodulation, homeostasis, scavenging, angiogenesis, and wound healing (Saijo and Glass, 2011). The pro- and anti-inflammatory responses need to be balanced to prevent the potential detrimental effects of prolonged, unregulated inflammation. Accumulating evidence regarding inflammation in TBI (Borlongan et al., 2015), stroke and neurodegenerative diseases points to an uncontrolled and prolonged M1-activated state that contributes to additional neuronal damage. However, merely blocking inflammation by suppressing M1 activation will likely not induce overall beneficial effects. Therefore, inhibiting the microglia M1 phenotype while stimulating the M2 phenotype may be a potential therapeutic strategy for treating neuroinflammatory disorders. Previous studies have indicated that MSC treatment might be able to suppress neuroinflammation because it can switch microglia from the M1 to M2 phenotype in vitro as well as in animal models of subarachnoid hemorrhage (SAH), brain trauma, and PD (Nijboer et al., 2018; Teixeira et al., 2017). In our present study, we administered MSCs in an LPS-induced neuroinflammatory mouse model, and we found that MSCs treatment significantly reduced the expression of the proinflammatory genes TNFa, IL-1β and IL-6 and increased the mRNA expression of the anti-inflammatory genes TGF-β1 and IL-10. In addition, we found higher expression of the M2 microglia marker CD206 in the MSCs treatment group than in the control group of the LPS-induced neuroinflammatory mouse model. The in vitro experiments also confirmed that MSCs inhibited microglial M1 polarization while promoting M2 polarization. All these data supported previous findings that MSCs could modulate inflammatory responses by polarizing microglia from the M1 to M2 phenotype. Recent studies have reported that MSCs reduce inflammation through many soluble factors, such as prostaglandin E2 (PGE2), TGF-β1 (Kim et al., 2015), indoleamine 2,3-dioxygenase (IDO) (Shi et al., 2012), IL-4 (Park et al., 2016), hepatocyte growth factor (Kennelly et al., 2016), and TSG-6 (Song et al., 2017). TSG-6, also known as TNAIP6, is a 30-kD glycoprotein that is expressed at substantially higher levels by many cell types, including MSCs, in response to stimulation by several proinflammatory mediators compared to normal physiological conditions (Milner and Day, 2003). Several reports have shown that TSG-6 plays a pivotal role in the immunomodulatory effects of MSCs in different inflammatory disease models, such as corneal inflammation, wound injury, acute lung injury, peritonitis, and pancreatitis (Oh et al., 2010; Tuo et al., 2012; Wang et al., 2012). In our previous studies, we found that MSCs could decrease NF-κB and MAPK signaling activity, thus diminishing the expression levels of inflammatory cytokines in BV2 microglia through TSG-6.
Acknowledgment This work was supported by a grant from the National Natural Science Foundation of China (No. 81601075). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.brainres.2019.146422. References Alam, Q., Alam, M.Z., Mushtaq, G., Damanhouri, G.A., Rasool, M., Kamal, M.A., Haque, A., 2016. Inflammatory process in alzheimer's and parkinson's diseases: central role of cytokines. Curr. Pharm. Des. 22, 541–548. Boche, D., Perry, V.H., Nicoll, J.A., 2013. Review: activation patterns of microglia and their identification in the human brain. Neuropathol. Appl. Neurobiol. 39, 3–18. Borlongan, C., Acosta, S., de la Pena, I., Tajiri, N., Kaneko, Y., Lozano, D., GonzalesPortillo, G., 2015. Neuroinflammatory responses to traumatic brain injury: etiology, clinical consequences, and therapeutic opportunities. Neuropsychiatr. Dis. Treat. 11, 97. Di, G., Du, X., Qi, X., Zhao, X., Duan, H., Li, S., Xie, L., Zhou, Q., 2017. Mesenchymal stem cells promote diabetic corneal epithelial wound healing through TSG-6-dependent stem cell activation and macrophage switch. Invest. Ophthalmol. Vis. Sci. 58, 4344–4354. DiSabato, D., Quan, N., Godbout, J.P., 2016. Neuroinflammation: The Devil is in the Details. J. Neurochemistry, n/a–n/a. Franco, R., Fernández-Suárez, D., 2015. Alternatively activated microglia and macrophages in the central nervous system. Prog. Neurobiol. 131, 65–86. Glass, C.K., Saijo, K., Winner, B., Marchetto, M.C., Gage, F.H., 2010. Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–934. Hu, X., Leak, R.K., Shi, Y., Suenaga, J., Gao, Y., Zheng, P., Chen, J., 2015. Microglial and macrophage polarization-new prospects for brain repair. Nat Rev Neurol. 11, 56–64. Jaimes, Y., Naaldijk, Y., Wenk, K., Leovsky, C., Emmrich, F., 2017. Mesenchymal stem cell-derived microvesicles modulate lipopolysaccharides-induced inflammatory responses to microglia cells. Stem Cells. 35, 812–823.
7
Brain Research 1724 (2019) 146422
Y. Liu, et al.
mouse bone marrow-derived mesenchymal stem cells and BV2 microglia after lipopolysaccharide stimulation. Stem Cell Res. Ther. 4, 12. Saijo, K., Glass, C.K., 2011. Microglial cell origin and phenotypes in health and disease. Nat. Rev. Immunol. 11, 775–787. Shi, Y., Su, J., Roberts, A.I., Shou, P., Rabson, A.B., Ren, G., 2012. How mesenchymal stem cells interact with tissue immune responses. Trends Immunol. 33, 136–143. Song, W., Li, Q., Ryu, M., Ahn, J., Bhang, D.H., Jung, Y.C., Youn, H., 2018. TSG-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing M2 macrophage switch in mice. Stem Cell Res. Ther. 9. Song, W.J., Li, Q., Ryu, M.O., Ahn, J.O., Ha, B.D., Chan, J.Y., Youn, H.Y., 2017. TSG-6 secreted by human adipose tissue-derived mesenchymal stem cells ameliorates DSSinduced colitis by inducing M2 macrophage polarization in mice. Sci. Rep. 7, 5187. Song, W.J., Li, Q., Ryu, M.O., Nam, A., An, J.H., Jung, Y.C., Ahn, J.O., Youn, H.Y., 2019. Canine adipose tissue-derived mesenchymal stem cells pre-treated with TNF-alpha enhance immunomodulatory effects in inflammatory bowel disease in mice. Res. Vet. Sci. 125, 176–184. Teixeira, F.G., Carvalho, M.M., Panchalingam, K.M., Rodrigues, A.J., Mendes-Pinheiro, B., Anjo, S., Manadas, B., Behie, L.A., Sousa, N., Salgado, A.J., 2017. Impact of the secretome of human mesenchymal stem cells on brain structure and animal behavior in a rat model of parkinson's disease. Stem Cells Transl. Med. 6, 634–646. Tuo, J., Cao, X., Shen, D., Wang, Y., Zhang, J., Oh, J.Y., Prockop, D.J., Chan, C.C., 2012. Anti-inflammatory recombinant TSG-6 stabilizes the progression of focal retinal degeneration in a murine model. J. Neuroinflammation. 9, 59. Wang, N., Li, Q., Zhang, L., Lin, H., Hu, J., Li, D., Shi, S., Cui, S., Zhou, J., Ji, J., Wan, J., Cai, G., Chen, X., 2012. Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6. PLoS ONE 7, e43768. Watanabe, Y., Tsuchiya, A., Seino, S., Kawata, Y., Kojima, Y., Ikarashi, S., Starkey, L.P., Lu, W.Y., Kikuta, J., Kawai, H., Yamagiwa, S., Forbes, S.J., Ishii, M., Terai, S., 2019. Mesenchymal stem cells and induced bone marrow-derived macrophages synergistically improve liver fibrosis in mice. Stem Cells Transl. Med. 8, 271–284. Xia, C.Y., Zhang, S., Gao, Y., Wang, Z.Z., Chen, N.H., 2015. Selective modulation of microglia polarization to M2 phenotype for stroke treatment. Int. Immunopharmacol. 25, 377–382.
Jiao, F., Wang, Y., Zhang, H., Zhang, W., Wang, L., Gong, Z., 2018. Histone deacetylase 2 inhibitor CAY10683 alleviates lipopolysaccharide induced neuroinflammation through attenuating TLR4/NF-κB signaling pathway. Neurochem. Res. Kennelly, H., Mahon, B.P., English, K., 2016. Human mesenchymal stromal cells exert HGF dependent cytoprotective effects in a human relevant pre-clinical model of COPD. Sci. Rep. 6, 38207. Kim, H.S., Yun, J.W., Shin, T.H., Lee, S.H., Lee, B.C., Yu, K.R., Seo, Y., Lee, S., Kang, T.W., Choi, S.W., Seo, K.W., Kang, K.S., 2015. Human umbilical cord blood mesenchymal stem cell-derived PGE2 and TGF-beta1 alleviate atopic dermatitis by reducing mast cell degranulation. Stem Cells. 33, 1254–1266. Li, C., Zhang, C., Zhou, H., Feng, Y., Tang, F., Hoi, M., He, C., Ma, D., Zhao, C., Lee, S., 2018. Inhibitory effects of betulinic acid on LPS-induced neuroinflammation involve M2 microglial polarization via CaMKKbeta-dependent AMPK activation. Front. Mol. Neurosci. 11, 98. Liu, Y., Zhang, R., Yan, K., Chen, F., Huang, W., Lv, B., Sun, C., Xu, L., Li, F., Jiang, X., 2014. Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6. J Neuroinflammation. 11, 135. Machado, V., Zoller, T., Attaai, A., Spittau, B., 2016. Microglia-mediated neuroinflammation and neurotrophic factor-induced protection in the MPTP mouse model of Parkinson's disease-lessons from transgenic mice. Int. J. Mol. Sci. 17. Milner, C.M., Day, A.J., 2003. TSG-6: a multifunctional protein associated with inflammation. J. Cell Sci. 116, 1863–1873. Nijboer, C.H., Kooijman, E., van Velthoven, C.T., van Tilborg, E., Tiebosch, I.A., Eijkelkamp, N., Dijkhuizen, R.M., Kesecioglu, J., Heijnen, C.J., 2018. Intranasal stem cell treatment as a novel therapy for subarachnoid hemorrhage. Stem Cells Dev. 27, 313–325. Oh, J.Y., Roddy, G.W., Choi, H., Lee, R.H., Ylostalo, J.H., Rosa, R.J., Prockop, D.J., 2010. Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury. Proc. Natl. Acad. Sci. U.S.A. 107, 16875–16880. Park, H.J., Oh, S.H., Kim, H.N., Jung, Y.J., Lee, P.H., 2016. Mesenchymal stem cells enhance alpha-synuclein clearance via M2 microglia polarization in experimental and human parkinsonian disorder. Acta Neuropathol. 132, 685–701. Rahmat, Z., Jose, S., Ramasamy, R., Vidyadaran, S., 2013. Reciprocal interactions of
8
Contents lists available at ScienceDirect
Brain Research journal homepage: www.elsevier.com/locate/brainres
Research report
Mesenchymal stem cells enhance microglia M2 polarization and attenuate neuroinflammation through TSG-6
T
⁎
Yi Liua,b, ,1, Rong Zengc,1, Yezhong Wangb, Wenhui Huangb, Bin Hub, Guohui Zhud, Run Zhangd, Feng Lid, Jianbang Hand, Yongshi Lie a
Department of Neurosurgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China Department of Neurosurgery and Neurosurgical Disease Research Centre, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China c Department of Radiotherapy, Oncology Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China d Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China e Department of Neurosurgery, GuangDong 999 Brain Hospital, Guangzhou, China b
H I GH L IG H T S
suppress the inflammatory response in the LPS-induced neuroinflammation mouse model • MSCs prevented microglial M1 activation and promoted M2 polarization. • MSCs • TSG-6 play a key role in the MSC-mediated microglial polarization.
A R T I C LE I N FO
A B S T R A C T
Keywords: Mesenchymal stem cells TSG-6 Microglia polarization Neuroinflammation
Microglia are the primary cells that exert immune function in the central nervous system (CNS), and they play an important role in the pathogenesis and progression of neuroinflammation-related diseases. Mesenchymal stem cells (MSCs) have been demonstrated to promote functional recovery in many neurological diseases. The mechanisms underlying this may be that MSCs can reduce inflammatory responses through various soluble factors. Among these factors, tumor necrosis factor-α-induced gene/protein 6 (TSG-6) is a key factor influencing MSCs immunomodulatory properties; however, the precise mechanisms underlying the anti-inflammatory effects are not fully understood. Here, we aim to investigate the potential effects of MSCs on neuroinflammation and to reveal the underlying mechanisms. First, we confirmed that administration of MSCs could inhibit the lipopolysaccharide (LPS)-induced neuroinflammatory responses in a mouse model. Then, we found that MSCs promoted M2 polarization and inhibited M1 polarization both in vivo and in vitro. Moreover, we demonstrated that the effect of MSCs on microglial polarization was dependent on TSG-6. This study demonstrated that MSCs promoted M2 polarization of microglia via TSG-6, thus conferring anti-neuroinflammatory effects.
1. Introduction Neuroinflammation is an important pathological process involved in traumatic brain injury (TBI) (Borlongan et al., 2015), stroke (Xia et al., 2015), intracerebral hemorrhage (ICH) and various neurodegenerative diseases (Alam et al., 2016; Glass et al., 2010; Machado et al., 2016). Under normal conditions, neuroinflammation maintains homeostasis and promotes tissue repair. However, unregulated neuroinflammation can be detrimental to the brain. Therefore, controlling harmful inflammatory responses is a promising therapeutic approach for
neurological diseases (DiSabato et al., 2016). As an important type of immune cell in the CNS, microglia play a crucial role in initiating and maintaining neuroinflammatory responses (Franco and Fernández-Suárez, 2015). Similar to macrophages, microglial cells show plasticity and can polarize toward the classical M1 or alternative M2 phenotype (Boche et al., 2013). M1-polarized microglia are characterized by increased proinflammatory cytokine levels, including TNF-a, IL-1β, and IL-6, and increased iNOS and CD16 levels, whereas M2-polarized microglia have high expression of M2 phenotype markers, such as Arg-1, CD206, Ym-1/2, and TGFβ. Functionally, M1
⁎
Corresponding author at: Department of Neurosurgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China E-mail address: [email protected] (Y. Liu). 1 Equal contributors. https://doi.org/10.1016/j.brainres.2019.146422 Received 15 June 2019; Received in revised form 24 August 2019; Accepted 27 August 2019 Available online 28 August 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.
Brain Research 1724 (2019) 146422
Y. Liu, et al.
Fig. 1. MSCs suppress the inflammatory response in the LPS-Induced Neuroinflammation Mouse Model. Neuroinflammation was induced by intraperitoneal injection of LPS. Twenty-four hours after LPS stimulation, MSCs were infused ICV, and control mice were infused with PBS. The mRNA expression level was determined by qRT-PCR. A: MSCs inhibited LPS-induced mRNA expression of TNF-a, IL-6, and IL-1β; B: MSCs prevented the LPS-induced downregulation of TGF-β and IL-10. Data are expressed as the means ± SD of three independent experiments. n = 3 #p < 0.01, versus control group; *p < 0.05, **p < 0.01, versus LPStreated group.
The cells were harvested, resuspended in PBS, and stained with phycoerythrin- or FITC-conjugated mouse monoclonal antibodies against CD44, CD90, CD34, CD31, and CD45 (eBioscience, San Diego, CA, USA) on ice for 20 min. Mouse IgG1 isotype-control antibodies were used in parallel as negative controls. The stained cells were then washed twice and resuspended in cold buffer and analyzed with flow cytometry (FACS Calibur; BD Biosciences). The results are expressed as the percentage of positively stained cells relative to the total cell number. MSCs from only passages 3–8 were used in the experiments.
microglia exacerbate neuronal injury and impede cellular repair after CNS disruption. In contrast, M2 microglia confer neuroprotection and promote recovery and remodeling (Hu et al., 2015). Therefore, inhibiting M1 microglia polarization and promoting M2 microglia polarization may be a viable strategy for treating neuroinflammation-related diseases (Jiao et al., 2018; Li et al., 2018). Recent studies have shown that MSCs administration could promote functional recovery in many neurological diseases (Jaimes et al., 2017; Park et al., 2016). The mechanism underlying this effect may be a reduction in inflammation responses through various soluble factors, including neurotrophic growth factors, chemokines, cytokines, and extracellular matrix proteins (Teixeira et al., 2017). In our previous studies, we demonstrated that MSCs can reduce inflammatory factor production in microglia through TSG-6 (Liu et al., 2014). Whether MSCs can enhance microglial M2 polarization through TSG-6 and thereby exert neuroprotective effects has not been investigated. In this study, we investigated the mechanisms underlying beneficial effects of MSCs on inflammation in the CNS. Our results demonstrated that MSCs administration inhibited LPS-induced neuroinflammatory responses in a mouse model by mediating the microglial activation status. Furthermore, we speculated that the polarization effects of MSCs on microglial status were mediated by TSG-6 expression. We confirmed this hypothesis by demonstrating that transfection of MSCs with TSG-6 siRNA led to a decreased effect on microglial polarization. MSCs can change macrophage/microglia polarization and inhibit a variety of inflammatory diseases (Di et al., 2017; Song et al., 2019; Watanabe et al., 2019), but whether MSCs can regulate microglia polarization through TSG-6 in inflammatory CNS diseases has not been reported. Based on our in vitro and in vivo research, we propose that MSCs can promote M2 polarization of microglia in neuroinflammatory responses and that TSG-6 plays an important role in microglia polarization induced by MSCs.
2.2. MSCs transfection with small interfering RNA (siRNA) A total of 2 × 105 MSCs were plated in 6-well dishes and cultured for 24 h; then, the cells were transfected with TSG-6 siRNA or control siRNA (sc-39820 and sc-37007, respectively; Santa Cruz Biotechnology Inc., Paso Robles, USA) using Lipofectamine 3000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). To confirm the silencing effect of the siRNA, RNA was extracted from aliquots of the cells after 48 h, and TSG-6 expression was analyzed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). TSG6 secreted into the supernatant was detected by ELISA. 2.3. Animal experiments Male C57BL/6 mice (25–30 g, approximately 8 weeks old) were obtained from The Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were maintained in specific pathogen-free conditions with a 12 h light/12 h dark cycle and standard temperature and humidity conditions. In addition, the mice were provided a regular diet daily and clean water ad libitum. All animal experiments were performed according to guidelines for the care and use of laboratory animals (Ministry of Science and Technology of China, 2006) and approved by the Animal Care and Use Committee of Sun Yat-sen University. Neuroinflammation was induced by intraperitoneal injection of LPS (1 mg/kg). Twenty-four hours after LPS stimulation, a hole was drilled in the right scalp of each anesthetized mouse at coordinates 0 mm caudal to the bregma, 1 mm lateral to the midline, and 3 mm beneath the dura mater. MSCs were prepared as previously described, and the cell concentration was adjusted to 150,000 cells/5 μl PBS. MSCs were infused intracerebroventricularly (ICV) over 5 min, and the needle was left in place for another 5 min. Control mice were infused with 5 μl PBS alone. MSCs transfected with TSG-6 siRNA or control siRNA control
2. Materials and methods 2.1. MSCs isolation, expansion and characterization MSCs were prepared from mouse bone marrow (BM) cells as previously described (Rahmat et al., 2013) with minor modifications. MSCs were isolated from the BM of male C57BL/6 mouse tibias and femurs. MSCs were confirmed by flow cytometry analysis after three passages. 2
Brain Research 1724 (2019) 146422
Y. Liu, et al.
Fig. 2. MSCs promoted microglia M2 polarization in the LPS-Induced Neuroinflammation Mouse Model. The percentages of M1/M2 microglia surface markers were determined by immunofluorescence staining. A: Representative immunofluorescence staining of cortex costained for Iba-1 (red) and CD16/32 (green) or costained for Iba-1 (red) and CD206 (green), Scale bar = 50 μm. B: The M1 marker genes CD16 and iNOS were determined by qRT-PCR assay. C: qRT-PCR for mRNA expression of the M2 markers CD206 and Arg1. Data were expressed as the means ± SD of three independent experiments. n = 3. # p < 0.01, versus control group; **p < 0.01, versus LPS-treated group.
administration, and whole brains were removed for additional experiments.
were injected according to the same procedures. Four mice were used in the control group, and six mice were used in the LPS, LPS+MSC, LPS +siTSG6-MSC and LPS + siCtrl-MSC treatment groups. No animals died after transplantation. The animals were sacrificed 24 h after MSCs 3
Brain Research 1724 (2019) 146422
Y. Liu, et al.
Fig. 3. MSCs promoted microglia M2 polarization in BV2 microglial cells. MSCs produced more TSG-6 after LPS treatment and decreased LPS-induced M1 marker gene expression and promoted M2 marker gene expression in BV2 microglial cells. A: After treatment with LPS the mRNA levels of TSG-6 in MSCs were significantly increased. B: The secretion levels of TSG-6 in MSCs were detected by ELISA. C: qRT-PCR for mRNA expression of M1 markers (CD16, iNOS) in BV2 microglial cells. D: qRT-PCR for mRNA expression of M2 markers (CD206, Arg1) in BV2 microglial cells. E: MSCs significantly decreased iNOS expression, whereas they increased CD206 expression in BV2 cells, evidenced by Western blot assay. Data were expressed as the means ± SD of three independent experiments. n = 3, #p < 0.01, versus control group; **p < 0.01, versus LPS-treated group.
2.4. BV2 cell culture
2.6. Enzyme-linked immunosorbent assay (ELISA)
The BV2 murine microglial cell line was obtained from Xiehe Medical University (Beijing, China). The cells were cultured in a humidified incubator at 37 °C with 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS, 100 μg/ml streptomycin, 100 U/ml penicillin (HyClone, Logan, UT, USA), and 2 mmol/l glutamine (Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO2. A 6-well transwell system (0.4-μm pore size membrane; Corning, Cambridge, MA, USA) was used to assess the effect of MSCs on BV2 cells. A total of 5 × 105 BV2 cells were placed in the lower chamber and stimulated with 100 ng/mL LPS. After stimulation, 1.0 × 105 MSCs were placed in the upper chamber. BV2 cells without LPS stimulation were used as controls. After 24 h, the microglia were washed and harvested for further studies.
TSG-6 levels in the supernatants were measured with a TSG-6 ELISA kit (MyBiosource, San Diego, CA, USA) and a multidetection microplate reader according to the manufacturer's instructions. 2.7. Immunofluorescence and laser-scanning confocal microscopy Immunofluorescence staining and laser-scanning confocal microscopy were performed as previously described. Brain sections (14-μm thickness) were fixed and washed. The slides were blocked for 60 min and incubated at 4 ℃ overnight with primary antibodies, including rabbit anti-Iba1 (1:800; Abcam, Cambridge, UK), rat anti-CD16/32 (1:800; Abcam, Cambridge, UK), and goat anti-CD206 (Santa Cruz, TX, USA). After that, the sections were incubated with an appropriate fluorescent secondary antibody (1:500) and stained with DAPI (Beyotime, China). The sections were imaged with a confocal microscope (TCS SP5; Lecia, Solms, Germany). Cell numbers were calculated by counting the cells in random microscopic fields in a blinded fashion. The data are expressed as the percentage of CD16/32+ or CD206+ cells to Iba-1+ cells.
2.5. RNA extraction, cDNA synthesis, and qRT-PCR Total RNA was extracted from the brain tissues and BV2 cells using a PrimeScript RT reagent kit (Takara, Japan) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized and used for qRT-PCR using a SYBR Green Kit (Takara, Japan), genespecific primers, and a 7500 Sequence Detection system (Applied Biosystems, USA). GAPDH was used as an endogenous control. The primers that were used are listed in the Supplementary Table.
2.8. Western blotting analysis Western blotting was performed to assess the expression of iNOS 4
Brain Research 1724 (2019) 146422
Y. Liu, et al.
(caption on next page)
5
Brain Research 1724 (2019) 146422
Y. Liu, et al.
Fig. 4. MSCs with TSG-6 knockdown had less impact on reducing the polarization of M1 microglia and promoted M2 polarization. The mRNA and secretion levels of TSG-6 were significantly lower in siTSG6-MSCs than in MSCs or control siRNA-transfected MSCs (siCtrl-MSCs) A: Relative expression level of TSG-6 mRNA was determined by qRT-PCR. B: The secretion levels of TSG-6 were detected by ELISA. siTSG6-MSCs had a decreased effect on altering the expression of pro- and antiinflammatory in mouse model. C, D: The mRNA expression levels of pro-inflammatory TNF-a, IL-6, IL-1β and anti-inflammatory TGF-β, IL-10 were determined by qRT-PCR. E, F: qRT-PCR for mRNA expression of the M1 markers CD16 and iNOS, and the M2 markers CD206 and Arg1. G, H: Representative immunofluorescence staining of cortex costained for Iba-1 (red) and CD16/32 (green), or costained for Iba-1 (red) and CD206 (green). Scale bars = 50 μm. I: qRT-PCR for mRNA expression of M1 markers (CD16, iNOS) in BV2 microglial cells. J: The iNOS protein levels in BV2 microglial cells were determined by Western blotting. K: qRT-PCR for mRNA expression of M2 markers (CD206, Arg1) in BV2 microglial cells. L: The CD206 protein levels in BV2 microglial cells were determined by Western blotting. Data were expressed as the means ± SD of three independent experiments. n = 3, **p < 0.01.
with Iba-1 was decreased after LPS stimulation compared with that in the control group. However, MSCs administration attenuated M1 marker expression and promoted M2 marker expression (Fig. 2A). Consistent with the immunofluorescence results, the mRNA expression of M1 genes (iNOS, CD16) was markedly increased after LPS stimulation, whereas MSCs treatment prevented this effect and increased the levels of M2 genes (Arg-1 and CD206) after LPS stimulation in mice (Fig. 2B, C). These results suggest that MSCs administration inhibited microglia polarization to the M1 phenotype and instead promoted a shift to the M2 phenotype in the LPS-induced neuroinflammation mouse model.
and CD206 in BV2 cells. Total proteins were extracted using RIPA lysis buffer (sc-24948; Santa Cruz Biotechnology, CA, USA). Protein concentrations were estimated using a BCA protein assay kit (Thermo Scientific, Pierce, Rockford, IL, USA), and 20 μg protein was loaded per lane. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked using 5% nonfat dry milk in TBST and then incubated with rabbit anti-iNOS (Cell Signaling Technology, Danvers, MA, USA) or goat anti-CD206 (Santa Cruz, TX, USA) primary antibody at a 1:1000 dilution at 4°C overnight. Diluted (1:5000) horseradish peroxidase (HRP)-conjugated goat anti-rabbit or donkey anti-goat (Beyotime, Hangzhou, China) secondary antibodies were applied for 1 h at room temperature. The immunoblots were visualized using enhanced chemiluminescence (ECL; Thermo Scientific), and the cellular expression levels were normalized to GAPDH (Cell Signaling Technology).
3.4. MSCs prevented LPS-induced M1 microglial activation and promoted microglial polarization toward the M2 phenotype in microglial cells To elucidate the potential mechanisms responsible for the effects of MSCs on microglial polarization, we measured TSG-6 gene expression and secretion levels after LPS stimulation. Our data showed that the mRNA and secretion levels of TSG-6 in MSCs after treatment with LPS were significantly increased in vitro (Fig. 3A, B). Then, in a co-culture experiment, we examined M1 or M2 polarization in BV2 cells. The qRTPCR results showed that expression of the M1 marker genes CD16 and iNOS was increased after LPS stimulation. However, MSCs treatment reduced LPS-induced M1 marker gene expression (Fig. 3C). Furthermore, significant increases in the M2 marker genes Arg-1 and CD206 were observed in the MSCs-treated groups compared with those in the LPS group (Fig. 3D). In addition, the Western blot results showed that MSCs decreased LPS-induced iNOS expression and promoted the protein expression of the M2 marker CD206 (Fig. 3E). In conclusion, MSCs prevented LPS-induced M1 microglial polarization and promoted the M2 phenotype in vitro.
2.9. Statistical analysis All experiments were completed at least three times, and the data are expressed as the mean ± SD. Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) or two-tailed Student’s t-test was used to determine the significance between groups. Significance was indicated at p < 0.05. 3. Results 3.1. Characterization of MSCs After three passages, MSCs were confirmed by flow cytometry analysis, and the cells were positive for CD44 (93.90%) and CD90 (98.57%) but not CD34 (0.58%), CD31 (0.13%) or CD45 (0.36%) (Supplementary Figure).
3.5. MSCs with TSG-6 knockdown had little effect on reducing M1 microglia polarization and promoted M2 polarization
3.2. MSCs suppress the inflammatory response in an LPS-induced neuroinflammation mouse model
To obtain further insight into the role of TSG-6 in modulating microglia activities, we knocked down TSG-6 in MSCs with siRNAs. The mRNA and secretion levels of TSG-6 were significantly lower in siTSG6MSCs than in MSCs or control siRNA-transfected MSCs (siCtrl-MSCs) (Fig. 4 A, B). After successful transfection, we repeated part of the above in vivo and in vitro experiments. The expression levels of the inflammatory cytokines TNF-a, IL-6, and IL-1β were significantly increased, and the expression levels of anti-inflammatory TGF-β1 and IL10 were significantly decreased in neuroinflammation mouse model in siTSG6-MSCs group compared with MSCs or siCtrl-MSCs groups (Fig. 4C and D). Next, we assessed microglial polarization in neuroinflammation mouse model. siTSG6-MSCs had little effect on reducing expression levels of the M1 marker genes CD16 and iNOS or the percentage of CD16/32 coexpression with Iba-1 compared to nontransfected MSCs or siCtrl-MSCs (Fig. 4E,G). Moreover, expression levels of the M2 microglia marker genes CD206 and Arg1 and the level of CD206 coexpression with Iba-1 in the cortex were significantly lower in siTSG6-MSCs than in nontransfected MSCs or siCtrl-MSCs (Fig. 4F, H). In the in vitro studies, the expression levels of the M1 marker genes CD16 and iNOS and protein levels of iNOS were significantly higher in LPS-stimulated BV2 cells cocultured with siTSG6-MSCs than in
To investigate the effect of MSCs on neuroinflammation in a mouse model in vivo, we analyzed the mRNA expression of TNF-a, IL-6, IL-1β, TGF-β1 and IL-10 with qRT-PCR. As shown in Fig. 1A, the mRNA expression levels of the proinflammatory cytokines TNF-a, IL-6 and IL-1β were significantly higher after LPS stimulation than after PBS treatment. Additionally, the expression of the anti-inflammatory cytokines TGF-β1 and IL-10 decreased after LPS stimulation compared with that in the control group (Fig. 1B). MSCs administration attenuated LPSinduced proinflammatory gene expression and promoted anti-inflammatory gene expression. 3.3. MSCs promoted microglial M2 polarization in vivo CD16 and CD32 are cell surface markers for the M1 microglial phenotype, whereas CD206 is a cell surface marker of the M2 phenotype. First, we analyzed M1/M2 surface markers by immunofluorescence. The results revealed that CD16/32 coexpression with Iba1 in the cortex was significantly increased, and CD206 coexpression 6
Brain Research 1724 (2019) 146422
Y. Liu, et al.
In addition, recent studies have shown that TSG-6 released from MSCs ameliorates inflammation by inducing M2 polarization of macrophages/microglia in inflammatory bowel disease, DSS-induced colitis, and diabetic corneal epithelial wound healing animal models (Di et al., 2017; Song et al., 2018, 2017). However, whether MSCs can regulate microglia polarization through TSG-6, thus affecting the neuroinflammatory response, has rarely been studied. Based on the above findings, we speculated that TSG-6 secreted from MSCs plays a key role in inducing microglial M2 polarization. Therefore, we transfected MSCs with siRNAs to examine whether these cells could induce a microglia phenotype switch in vivo and in vitro. The in vivo experimental results showed that M2 marker expression was induced in the siCtrl-MSCs- and naive MSCs-treated groups of the neuroinflammatory mouse model. However, the expression of these markers was much lower in siTSG6-MSCs-treated animals. The in vitro experimental results also confirmed that the expression of M2 markers was lower in LPS-stimulated BV2 cells cultured with siTSG6-MSCs than in those cultured with MSCs and siCtrl-MSCs. Taken together, these findings suggest that TSG-6 plays a crucial role in regulating the M1 to M2 phenotypic switch. The main limitation of our research is that several soluble factors, such as PGE2 and IDO, have been reported to be involved in MSCsmediated immunoregulation. Therefore, in addition to TSG-6, it is apparent that MSCs can produce a variety of anti-inflammatory factors, and the results presented here do not rule out the effects of other antiinflammatory factors. Another limitation of our experiment is the lack of mechanistic research on TSG-6 and phenotypic alterations in microglia. Further research must be performed to determine the specific mechanisms of this action. In conclusion, we demonstrated that MSCs ameliorated LPS-induced neuroinflammation in mice by inducing microglia to switch to the M2 phenotype through TSG-6. These findings suggest that MSCs administration could be a promising treatment option for neuroinflammatory disorders.
nontransfected MSCs or siCtrl-MSCs (Fig. 4I and J). In addition, expression levels of the M2 microglia marker genes CD206 and Arg1 and protein levels of CD206 (Fig. 4 K, L) were significantly reduced in LPSstimulated BV2 cells cocultured with siTSG6-MSCs. However, siCtrlMSCs had an effect similar to nontransfected MSCs on the gene and protein expression of LPS-stimulated BV2 microglia cells. These data suggest that TSG-6 expression in MSCs plays a vital role in mediating microglial polarization both in vivo and in vitro. 4. Discussion Neuroinflammation is an important pathological process in many CNS diseases. Therapeutic targeting of inflammation represents an exciting approach for neuroprotective strategies. Microglia are the major cellular elements with immune function in the CNS, and they play an important role in the pathogenesis and progression of neuroinflammation-related diseases. It is well recognized that microglial cells can polarize into different functional phenotypes: the proinflammatory M1 phenotype and the anti-inflammatory M2 phenotype. M1 activation leads to the release of proinflammatory cytokines, while M2 activation enhances the expression of genes involved in inflammation resolution, immunomodulation, homeostasis, scavenging, angiogenesis, and wound healing (Saijo and Glass, 2011). The pro- and anti-inflammatory responses need to be balanced to prevent the potential detrimental effects of prolonged, unregulated inflammation. Accumulating evidence regarding inflammation in TBI (Borlongan et al., 2015), stroke and neurodegenerative diseases points to an uncontrolled and prolonged M1-activated state that contributes to additional neuronal damage. However, merely blocking inflammation by suppressing M1 activation will likely not induce overall beneficial effects. Therefore, inhibiting the microglia M1 phenotype while stimulating the M2 phenotype may be a potential therapeutic strategy for treating neuroinflammatory disorders. Previous studies have indicated that MSC treatment might be able to suppress neuroinflammation because it can switch microglia from the M1 to M2 phenotype in vitro as well as in animal models of subarachnoid hemorrhage (SAH), brain trauma, and PD (Nijboer et al., 2018; Teixeira et al., 2017). In our present study, we administered MSCs in an LPS-induced neuroinflammatory mouse model, and we found that MSCs treatment significantly reduced the expression of the proinflammatory genes TNFa, IL-1β and IL-6 and increased the mRNA expression of the anti-inflammatory genes TGF-β1 and IL-10. In addition, we found higher expression of the M2 microglia marker CD206 in the MSCs treatment group than in the control group of the LPS-induced neuroinflammatory mouse model. The in vitro experiments also confirmed that MSCs inhibited microglial M1 polarization while promoting M2 polarization. All these data supported previous findings that MSCs could modulate inflammatory responses by polarizing microglia from the M1 to M2 phenotype. Recent studies have reported that MSCs reduce inflammation through many soluble factors, such as prostaglandin E2 (PGE2), TGF-β1 (Kim et al., 2015), indoleamine 2,3-dioxygenase (IDO) (Shi et al., 2012), IL-4 (Park et al., 2016), hepatocyte growth factor (Kennelly et al., 2016), and TSG-6 (Song et al., 2017). TSG-6, also known as TNAIP6, is a 30-kD glycoprotein that is expressed at substantially higher levels by many cell types, including MSCs, in response to stimulation by several proinflammatory mediators compared to normal physiological conditions (Milner and Day, 2003). Several reports have shown that TSG-6 plays a pivotal role in the immunomodulatory effects of MSCs in different inflammatory disease models, such as corneal inflammation, wound injury, acute lung injury, peritonitis, and pancreatitis (Oh et al., 2010; Tuo et al., 2012; Wang et al., 2012). In our previous studies, we found that MSCs could decrease NF-κB and MAPK signaling activity, thus diminishing the expression levels of inflammatory cytokines in BV2 microglia through TSG-6.
Acknowledgment This work was supported by a grant from the National Natural Science Foundation of China (No. 81601075). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.brainres.2019.146422. References Alam, Q., Alam, M.Z., Mushtaq, G., Damanhouri, G.A., Rasool, M., Kamal, M.A., Haque, A., 2016. Inflammatory process in alzheimer's and parkinson's diseases: central role of cytokines. Curr. Pharm. Des. 22, 541–548. Boche, D., Perry, V.H., Nicoll, J.A., 2013. Review: activation patterns of microglia and their identification in the human brain. Neuropathol. Appl. Neurobiol. 39, 3–18. Borlongan, C., Acosta, S., de la Pena, I., Tajiri, N., Kaneko, Y., Lozano, D., GonzalesPortillo, G., 2015. Neuroinflammatory responses to traumatic brain injury: etiology, clinical consequences, and therapeutic opportunities. Neuropsychiatr. Dis. Treat. 11, 97. Di, G., Du, X., Qi, X., Zhao, X., Duan, H., Li, S., Xie, L., Zhou, Q., 2017. Mesenchymal stem cells promote diabetic corneal epithelial wound healing through TSG-6-dependent stem cell activation and macrophage switch. Invest. Ophthalmol. Vis. Sci. 58, 4344–4354. DiSabato, D., Quan, N., Godbout, J.P., 2016. Neuroinflammation: The Devil is in the Details. J. Neurochemistry, n/a–n/a. Franco, R., Fernández-Suárez, D., 2015. Alternatively activated microglia and macrophages in the central nervous system. Prog. Neurobiol. 131, 65–86. Glass, C.K., Saijo, K., Winner, B., Marchetto, M.C., Gage, F.H., 2010. Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–934. Hu, X., Leak, R.K., Shi, Y., Suenaga, J., Gao, Y., Zheng, P., Chen, J., 2015. Microglial and macrophage polarization-new prospects for brain repair. Nat Rev Neurol. 11, 56–64. Jaimes, Y., Naaldijk, Y., Wenk, K., Leovsky, C., Emmrich, F., 2017. Mesenchymal stem cell-derived microvesicles modulate lipopolysaccharides-induced inflammatory responses to microglia cells. Stem Cells. 35, 812–823.
7
Brain Research 1724 (2019) 146422
Y. Liu, et al.
mouse bone marrow-derived mesenchymal stem cells and BV2 microglia after lipopolysaccharide stimulation. Stem Cell Res. Ther. 4, 12. Saijo, K., Glass, C.K., 2011. Microglial cell origin and phenotypes in health and disease. Nat. Rev. Immunol. 11, 775–787. Shi, Y., Su, J., Roberts, A.I., Shou, P., Rabson, A.B., Ren, G., 2012. How mesenchymal stem cells interact with tissue immune responses. Trends Immunol. 33, 136–143. Song, W., Li, Q., Ryu, M., Ahn, J., Bhang, D.H., Jung, Y.C., Youn, H., 2018. TSG-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing M2 macrophage switch in mice. Stem Cell Res. Ther. 9. Song, W.J., Li, Q., Ryu, M.O., Ahn, J.O., Ha, B.D., Chan, J.Y., Youn, H.Y., 2017. TSG-6 secreted by human adipose tissue-derived mesenchymal stem cells ameliorates DSSinduced colitis by inducing M2 macrophage polarization in mice. Sci. Rep. 7, 5187. Song, W.J., Li, Q., Ryu, M.O., Nam, A., An, J.H., Jung, Y.C., Ahn, J.O., Youn, H.Y., 2019. Canine adipose tissue-derived mesenchymal stem cells pre-treated with TNF-alpha enhance immunomodulatory effects in inflammatory bowel disease in mice. Res. Vet. Sci. 125, 176–184. Teixeira, F.G., Carvalho, M.M., Panchalingam, K.M., Rodrigues, A.J., Mendes-Pinheiro, B., Anjo, S., Manadas, B., Behie, L.A., Sousa, N., Salgado, A.J., 2017. Impact of the secretome of human mesenchymal stem cells on brain structure and animal behavior in a rat model of parkinson's disease. Stem Cells Transl. Med. 6, 634–646. Tuo, J., Cao, X., Shen, D., Wang, Y., Zhang, J., Oh, J.Y., Prockop, D.J., Chan, C.C., 2012. Anti-inflammatory recombinant TSG-6 stabilizes the progression of focal retinal degeneration in a murine model. J. Neuroinflammation. 9, 59. Wang, N., Li, Q., Zhang, L., Lin, H., Hu, J., Li, D., Shi, S., Cui, S., Zhou, J., Ji, J., Wan, J., Cai, G., Chen, X., 2012. Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6. PLoS ONE 7, e43768. Watanabe, Y., Tsuchiya, A., Seino, S., Kawata, Y., Kojima, Y., Ikarashi, S., Starkey, L.P., Lu, W.Y., Kikuta, J., Kawai, H., Yamagiwa, S., Forbes, S.J., Ishii, M., Terai, S., 2019. Mesenchymal stem cells and induced bone marrow-derived macrophages synergistically improve liver fibrosis in mice. Stem Cells Transl. Med. 8, 271–284. Xia, C.Y., Zhang, S., Gao, Y., Wang, Z.Z., Chen, N.H., 2015. Selective modulation of microglia polarization to M2 phenotype for stroke treatment. Int. Immunopharmacol. 25, 377–382.
Jiao, F., Wang, Y., Zhang, H., Zhang, W., Wang, L., Gong, Z., 2018. Histone deacetylase 2 inhibitor CAY10683 alleviates lipopolysaccharide induced neuroinflammation through attenuating TLR4/NF-κB signaling pathway. Neurochem. Res. Kennelly, H., Mahon, B.P., English, K., 2016. Human mesenchymal stromal cells exert HGF dependent cytoprotective effects in a human relevant pre-clinical model of COPD. Sci. Rep. 6, 38207. Kim, H.S., Yun, J.W., Shin, T.H., Lee, S.H., Lee, B.C., Yu, K.R., Seo, Y., Lee, S., Kang, T.W., Choi, S.W., Seo, K.W., Kang, K.S., 2015. Human umbilical cord blood mesenchymal stem cell-derived PGE2 and TGF-beta1 alleviate atopic dermatitis by reducing mast cell degranulation. Stem Cells. 33, 1254–1266. Li, C., Zhang, C., Zhou, H., Feng, Y., Tang, F., Hoi, M., He, C., Ma, D., Zhao, C., Lee, S., 2018. Inhibitory effects of betulinic acid on LPS-induced neuroinflammation involve M2 microglial polarization via CaMKKbeta-dependent AMPK activation. Front. Mol. Neurosci. 11, 98. Liu, Y., Zhang, R., Yan, K., Chen, F., Huang, W., Lv, B., Sun, C., Xu, L., Li, F., Jiang, X., 2014. Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6. J Neuroinflammation. 11, 135. Machado, V., Zoller, T., Attaai, A., Spittau, B., 2016. Microglia-mediated neuroinflammation and neurotrophic factor-induced protection in the MPTP mouse model of Parkinson's disease-lessons from transgenic mice. Int. J. Mol. Sci. 17. Milner, C.M., Day, A.J., 2003. TSG-6: a multifunctional protein associated with inflammation. J. Cell Sci. 116, 1863–1873. Nijboer, C.H., Kooijman, E., van Velthoven, C.T., van Tilborg, E., Tiebosch, I.A., Eijkelkamp, N., Dijkhuizen, R.M., Kesecioglu, J., Heijnen, C.J., 2018. Intranasal stem cell treatment as a novel therapy for subarachnoid hemorrhage. Stem Cells Dev. 27, 313–325. Oh, J.Y., Roddy, G.W., Choi, H., Lee, R.H., Ylostalo, J.H., Rosa, R.J., Prockop, D.J., 2010. Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury. Proc. Natl. Acad. Sci. U.S.A. 107, 16875–16880. Park, H.J., Oh, S.H., Kim, H.N., Jung, Y.J., Lee, P.H., 2016. Mesenchymal stem cells enhance alpha-synuclein clearance via M2 microglia polarization in experimental and human parkinsonian disorder. Acta Neuropathol. 132, 685–701. Rahmat, Z., Jose, S., Ramasamy, R., Vidyadaran, S., 2013. Reciprocal interactions of
8