Mesenchymal stem cells alleviate Japanese encephalitis virus-induced neuroinflammation and mortality
- Peiyu Bian†1,
- Chuantao Ye†1,
- Xuyang Zheng†1,
- Jing Yang2,
- Wei Ye2,
- Yuan Wang2,
- Yun Zhou1,
- Hongwei Ma2,
- Peijun Han2,
- Hai Zhang3,
- Ying Zhang1,
- Fanglin Zhang2,
- Yingfeng Lei2Email author and
- Zhansheng Jia1Email author
© The Author(s). 2017
Received: 15 September 2016
Accepted: 21 January 2017
Published: 16 February 2017
Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. Japanese encephalitis (JE) caused by JEV is characterized by extensive inflammatory cytokine secretion, microglia activation, blood-brain barrier (BBB) breakdown, and neuronal death, all of which contribute to the vicious cycle of inflammatory damage. There are currently no effective treatments for JE. Mesenchymal stem cells (MSCs) have been demonstrated to have a therapeutic effect on many central nervous system (CNS) diseases by regulating inflammation and other mechanisms.
In vivo, 8- to 10-week-old mice were infected intraperitoneally with JEV and syngeneic bone marrow MSCs were administered through the caudal vein at 1 and 3 days post-infection. The mortality, body weight, and behavior were monitored daily. Brains from each group were harvested at the indicated times for hematoxylin and eosin staining, immunohistochemical observation, flow cytometric analysis, TUNEL staining, Western blot, quantitative real-time polymerase chain reaction, and BBB permeability assays. In vitro, co-culture and mixed culture experiments of MSCs with either microglia or neurons were performed, and then the activation state of microglia and survival rate of neurons were tested 48 h post-infection.
MSC treatment reduced JEV-induced mortality and improved the recovery from JE in our mouse model. The inflammatory response, microglia activation, neuronal damage, BBB destruction, and viral load (VL) were significantly decreased in the MSC-treated group. In co-culture experiments, MSCs reprogrammed M1-to-M2 switching in microglia and improved neuron survival. Additionally, the VL was decreased in Neuro2a cells in the presence of MSCs accompanied by increased expression of interferon-α/β.
MSC treatment alleviated JEV-induced inflammation and mortality in mice.
KeywordsMesenchymal stem cells Immunomodulation Japanese encephalitis virus Inflammation
Viral encephalitis caused by multiple emerging and re-emerging viruses is characterized by overwhelming inflammation in the brain . There are currently no effective methods to eliminate the virus or to cure the neuroinflammation, leading to thousands of people succumbing to the devastating illness and the survivors often suffering from permanent neurological deficits. Japanese encephalitis (JE) caused by the JE virus (JEV) is the most prevalent type of viral encephalitis .
JEV is a single-stranded, positive-sense RNA (~11 kb, monopartite, linear) virus belonging to the genus Flavivirus in the family Flaviviridae . Globally, more than 67,900 cases of JEV infection are reported annually, among which approximately 30% are fatal and 50% suffer from permanent neuropsychiatric sequelae [4, 5]. Children are more susceptible to JEV, but there is an increasing occurrence in the middle-aged population . Although the development of a JE vaccine has markedly reduced the incidence, its protection is not always effective . JE is characterized by extensive neuroinflammation in the central nervous system (CNS) with robust and uncontrolled production of pro-inflammatory cytokines (e.g., tumor necrosis factor (TNF)-α and interferon (IFN)-γ) and chemokines (e.g., MCP-1/CCL2) [8–11]. Increased activation of microglia following JEV infection also contributes to the inflammatory response. During JE, neurons can be damaged by JEV directly or indirectly by the cytokine storm through the bystander effect . Meanwhile, breakdown of the blood-brain barrier (BBB) integrity also accelerates the progression of JE . There are currently no effective anti-JEV or other satisfactory therapeutic methods but life-sustaining treatment .
Mesenchymal stem cells (MSCs) have been demonstrated to contribute to tissue regeneration and modulate inflammation since they were first discovered by Friedenstein in 1974 [15, 16]. MSCs have the capacity for immunoregulation, the potential to migrate to the injury site, and the ability to differentiate into multiple cell types such as adipocytes, osteocytes, chondrocytes, and neuron-like cells. All of these properties have been beneficial in treating a wide spectrum of diseases in clinical and basic studies [17–19]. It has been reported that transplantation of MSCs can upregulate the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and can improve neurological recovery in many CNS diseases [20–23]. MSCs also control local inflammation and maintain tissue homeostasis by enhancing the innate and adaptive immune responses as well as regulating the activation and function of microglia [24, 25]. In addition, MSCs regulate BBB integrity by promoting the expression of vascular endothelial growth factor and angiogenesis [26–28]. Thus, the functional features of MSCs indicate their great potential in JE treatment. However, it is unknown whether MSCs can attenuate the encephalitis caused by JEV.
This study is the first to show that MSC treatment reduced the mortality and neurological pathology in the mouse model of JE. We also demonstrated that the beneficial effect of MSCs was mediated by suppressing the overactivation of microglia, reducing neuronal death, and improving the integrity of the BBB. Furthermore, we found that MSCs decreased JEV replication through the expression of IFN-β and IFN-α.
All the mice used in this experiment were purchased from the Laboratory Animal Center of the Fourth Military Medical University (FMMU). All animal experiments were approved by the Animal Care and Use Committee of the FMMU.
Isolation and culturing of mouse bone marrow MSCs
The mouse bone marrow-derived MSCs were isolated by the adhesive screening method . Female BALB/c mice (aged 4 to 6 weeks) were euthanized by cervical dislocation and sterilized with 75% alcohol for 3 to 5 min. The femurs were removed, and both ends of each femur were cut off. The marrow cavities were then flushed with low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) repeatedly until the femurs turned white. The cell suspension was gently pipetted to separate the cells, transferred to a 15-ml centrifuge tube, and centrifuged at 1000 rpm for 5 min. The cell pellets were then resuspended in 5 ml L-DMEM, plated in 25-cm2 flasks, and cultured at 37 °C in an atmosphere with 5% CO2 and saturated humidity. After 24 h, the medium was first changed, with subsequent changes occurring every 3 days to remove the nonadherent cells. Plastic-adherent cells were cultured to 95% confluency and passaged with trypsinization. MSCs used in this study were from passages 5-8th and analyzed by flow cytometry (defining criteria: >95% positive for the stem cell surface antigens CD44 and Sca-1, and <5% negative for CD45, CD34, and I-A/I-E) and multi-differentiation evaluation (adipocytes, osteocytes, and chondrocytes, using the Mouse Mesenchymal Stem Cell Functional Identification Kit (Cyagen, China)) before the experiments (see Additional file 1: Figure S1).
Cells and virus
The JEV SA-14 strain was propagated in the mosquito cell line C6/36. The supernatant containing JEV was concentrated at 100:1 and stored in aliquots at –80 °C until further use. The virus stocks were titrated by a conventional plaque assay .
The microglia cell line N9 and the neuroblast cell line Neuro2a were purchased from ATCC and maintained in 5% FBS DMEM and 10% FBS DMEM, respectively.
MSC transplantation into JEV-infected mice
Weight-matched adult female BALB/c mice (8–10 weeks old) were randomly divided into three groups: control group (phosphate-buffered saline (PBS)); JEV-infected group (JEV); and JEV-infected and MSC-treated group (JEV + MSC). The JEV and JEV + MSC mice were intraperitoneally injected with 5 × 107 PFU of JEV in 200 μl PBS. For the cellular injections, 200 μl of either PBS alone or MSCs (5 × 105 cells per mouse) were intravenously administered to mice belonging to the JEV and JEV + MSC groups, respectively, at 1 and 3 days post-infection (dpi). The mice in each group were monitored daily to assess behavior, weight, and mortality. Behavioral scoring was performed according to the criteria previously described with minor modifications , and the mean scores of all mice in each group were calculated. After the comparative observations were repeated three times, we found an obvious difference in the manifestation of the clinical syndrome between the JEV and JEV + MSC groups from 6 dpi. Subsequently, new models and treatments were conducted and brains were collected at 6 dpi for further experiments.
Quantitative real-time polymerase chain reaction (qRT-PCR)
At 6 dpi, mice were anesthetized with 2% pentobarbital sodium (0.1 ml/10 g body weight) and perfused with 1× PBS, and then the whole brain of each mouse from all the groups was harvested and stored at –80 °C. Viral burden and the expression of cytokines and chemokines were determined by relative qRT-PCR (see Additional file 2: Table S1 for the primer sequences). Total RNA from each whole brain and the in vitro cultured cells was extracted with RNAfast1000 (PIONEER, China). The first-strand cDNA was prepared by reverse transcription with total RNA as the template using a PrimeScript RT reagent Kit (TaKaRa, Japan). All qRT-PCR experiments were performed using SYBR Green Real-Time PCR Master Mix (TaKaRa, Japan) according to the manufacturer’s instructions.
Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining
At 6, 10, and 22 dpi, mice were anesthetized with 2% pentobarbital sodium (0.1 ml/10 g body weight) and perfused with 1× PBS followed by 4% paraformaldehyde (PFA) for 30 min. The brain was removed, fixed in 4% PFA for 12 h, and then cryoprotected in 30% sucrose. Tissue sections of 10 μm thickness were prepared with a vibratome. The standard H&E staining protocol was followed for tissue staining. For IHC staining, primary antibodies were incubated at room temperature for 16 h. Corresponding secondary antibodies were then incubated for 4 h at room temperature protected from light (see Additional file 3: Table S2 for the antibodies). The nuclei were counterstained with DAPI (100 ng/ml; Sigma, St. Louis, MO, USA), and coverslips were placed on the samples with 50% glycerol in PBS.
TUNEL staining of the sections was performed using an In Situ Cell Death Detection Kit (Roche, Germany) according to the manufacturer’s instructions. The nuclei were counterstained with DAPI.
Total protein from the brain of each mouse was extracted with RIPA buffer and quantified using a Protein Reagent Assay BCA Kit (Thermo, Waltham, MA, USA). Thirty micrograms of protein from each sample was loaded and electrophoresed using 15% SDS-PAGE gels and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After the membranes were blocked with 3% bovine serum albumin (BSA) at room temperature for 60 min, they were incubated with primary antibodies overnight at 4 °C. Then, the blots were incubated with secondary antibody for 2 h at room temperature (see Additional file 3: Table S2 for the antibodies used). The blots were visualized using an Infrared Imaging System (Odyssey, LI-COR, NE Lincoln, USA).
BBB permeability assay
BBB permeability was determined by visualizing and quantifying the levels of extravasated Evans blue dye (EB) in the brain. Mice were injected intraperitoneally with 0.4 ml 2% (w/v) EB (Sigma, St. Louis, MO, USA) followed by euthanization and intracardiac perfusion with PBS 1 h later. The brains were subsequently removed and photographed. For quantification, brain tissues were weighed and homogenized in 1 ml PBS, and then 1 ml 100% trichloroacetic acid (Sangon, China) was added. The mixture was vigorously shaken for 2 min to precipitate the proteins and cooled for 30 min at 4 °C. After the mixture was centrifuged (30 min at 6000 g), the absorbance of the supernatant was measured at 620 nm using a spectrophotometer. The content of EB was quantified as nanograms of dye per gram of brain tissue using a standard curve.
Neuro2a cells and MSC co-culture experiment
Neuro2a cells were plated in the basolateral chamber of a six-well transwell (3.0 μm, polycarbonate membrane tissue culture-treated polystyrene; Costar, Corning, NY, USA) at a density of 2 × 105/well, and 1 × 105 MSCs were added to the inserts and separately incubated overnight. The Neuro2a cells were then infected with JEV (multiplicity of infection (MOI) = 1). After adsorption for 1 h, the old virus suspension was removed, and fresh DMEM was added to the Neuro2a cells; subsequently, the transwell inserts containing either MSCs or DMEM alone were placed into their corresponding wells. The cells were harvested for qRT-PCR and flow cytometry after 48 h.
For mixed cultures of Neuro2a cells and MSCs, the Neuro2a cells were plated in six-well plates at a density of 2 × 105/well with ten-fold diluted numbers of MSCs (ranging from 200 to 2 × 105). After the cells were incubated in DMEM overnight, they were infected with JEV at an MOI = 1; 48 h later, the cells were harvested for detection of the viral load. Additionally, Neuro2a cells were plated in six-well plates at a density of 2 × 105/well with MSCs (2 × 104). After the cells were incubated in DMEM overnight, they were infected with JEV at a MOI = 0.1 and then harvested at 24, 48, 72, and 96 h post-infection for detection of the viral load.
N9 cell and MSC mixed culture experiment
N9 cells were plated in six-well plates at a density of 2 × 105 with or without MSCs (2 × 104) and incubated overnight, after which JEV (MOI = 5) was inoculated as described above, and the cells were harvested after 48 h for flow cytometry and qRT-PCR analysis.
Flow cytometric analysis
For annexin V/PI staining, an Annexin V-FITC Apoptosis Kit (BD Biosciences, Franklin lakes, NJ, USA) was used. The cells were washed and incubated for 15 min at room temperature with annexin V labeled with FITC and propidium iodide (PI).
Both primary cells from mouse brains and N9 cells were stained with FITC anti-mouse F4/80 and PE anti-mouse MR (CD206) for 50 min at 4 °C. After the cells were washed, they were permeabilized and stained with APC-anti-mouse iNOS for 50 min. Analysis was conducted using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
All statistical analyses were performed using GraphPad Prism version 6.01 software (La Jolla, CA, USA). Data represent the mean ± standard error of the mean (SEM). Significant differences between the experimental groups were determined using Student’s t test. P values < 0.05 were considered significant.
MSC treatment protects mice from JEV infection-induced lethality
MSC transplantation attenuates the inflammatory response in JEV-infected mouse brains
MSC transplantation inhibits the activation of microglia and regulates M1-to-M2-like phenotypic switching
It has been reported previously that different stimulation could polarize the microglia into the M1 phenotype (pro-inflammation) marked by iNOS and CD86 or the M2 phenotype (anti-inflammation) marked by Arg1 and CD206. To verify the effect of MSCs on the activation of microglia, mixed culturing of MSCs and the microglia cell line N9 was performed in vitro. The expression of iNOS and CD86 in N9 cells was induced by JEV infection, while it was decreased in the presence of MSCs. In contrast, the expression of arginase-1 (Arg1) and CD206 (which contribute to the phagocytosis of pathogens and presentation of antigen) was upregulated in N9 cells when co-cultured with MSCs (Fig. 3c and d). Thus, MSC transplantation inhibited JEV-induced overactivation of microglia as well as regulated the M1-to-M2-like phenotypic switching.
MSCs reduce neuronal death in vivo and in vitro
MSC transplantation alleviates virus-induced BBB destruction and improves the expression of ZO-1 in the mouse brain
MSCs prevent JEV propagation via interferon expression
To verify the role of MSCs on JEV proliferation, a series of mixed cultures of Neuro2a cells and MSCs were conducted. The viral load was decreased as the number of co-cultured MSCs increased (Fig. 6c, left). Meanwhile, there was significantly lower viral copy and slower proliferation in Neuro2a cells when co-cultured with MSCs compared with the control group (Fig. 6c, right). Besides, through transwell co-culture system, the viral copy number in the Neuro2a cells was reduced accompanied with increased expression of IFN-β and IFN-α in MSCs (Fig. 6d). Therefore, these data indicated that MSCs could inhibit the propagation of JEV in Neuro2a cells.
Recently, there have been an increasing number of studies on the therapeutic effect of MSCs on CNS diseases and virus infection. It was reported that MSCs promoted neurological recovery via regulating neuroinflammation and secreting neurotrophic factors. In addition, exogenous MSC transplantation has been demonstrated to reduce influenza virus-induced acute lung injury [32, 34]. In our study, we found that MSC transplantation improved the survival rate and alleviated neurological symptoms in a JEV-infected mouse model. These findings are the first evidence that MSCs could serve as a potential adjunctive therapy for JE.
There are many ways to construct a JEV infection mouse model, such as intracerebral injection, intravenous injection, and intraperitoneal injection. Based on feasibility and noninvasion, we adopted intraperitoneal injection for our study. In this study, mice older (8–10 weeks of age) than those previously reported in a JE mouse model (4–6 weeks of age) were used, and the dose of inoculated JEV was slightly higher than the median lethal dose (LD50). In our preliminary experiments, there was no obvious difference in the neurological symptoms and histopathology between the younger and older mouse model. JEV-infected mice generally began to exhibit typical symptoms at 3 dpi. There was an increased risk of pulmonary embolism when the number of intravenously transplanted cells was more than 5 × 105, and thus two intravenous injections of 5 × 105 cells/mouse were conducted at 1 and 3 dpi. In this study, we focused on the effect of MSCs on the acute neuroinflammation; thus, brains from each group were collected at 6 dpi.
The overproduction of inflammatory cytokines is the main cause of neuropathology during JE. Thus, inhibiting the inflammation during the progression of JE has been shown to reduce neuronal death and promote neurogenesis . An appropriate immune and inflammatory response during the early phase of infection is critical for clearance of the virus and regeneration of the nervous system . However, the overproduction of IFN-γ and TNF-α accelerated the neuroinflammation, and neutralization of these inflammatory cytokines could reduce the neuronal damage during JEV infection [37, 38]. Thus, it is important to achieve a balance between immune and inflammatory responses for the treatment of JE. MSCs are characterized by immunoregulation and have shown great potential in the treatment of immune deficiencies and infectious diseases [25, 39]. In this experiment, MSC-treated mice showed decreased level of IFN-γ and TNF-α at 6 dpi. Although the effect of these decreased cytokines in the course of JE is uncertain, it was well demonstrated that the neuroinflammation was alleviated in the MSC-treated mice according to tissue pathological changes. The dynamic regulation of MSCs during JE, which might be involved in the increased immune defenses to speed up the clearance of virus in the early stage of infection, is worth further exploration.
Previous studies have shown that breakdown of the BBB can exacerbate the progression of JE . It has been demonstrated that MSCs can improve the barrier function of epithelial cells through the production of the growth factors Ang1 and KGF, as well as regulating the integrity of the BBB through the expression of TIMP3 (tissue inhibitor of matrix metalloproteinase-3) . Our results indicated that the BBB permeability was ameliorated in MSC transplanted mice compared to the PBS-treated group. One possible explanation is that MSCs could inhibit the excessive production of inflammation cytokines and the overactivation of glia, both of which can lead to the degradation of ZO-1  and the subsequent destruction of the BBB. However, the direct effect of MSCs on the BBB during JEV infection is still being explored.
It is well known that activation of microglia can trigger a secondary pathway of damage and influence the outcome of JEV progression [11, 40]. Our data indicated that MSCs could inhibit the activation of microglia. Meanwhile, when co-cultured with MSCs, JEV-challenged N9 microglia cells showed increased expression of Arg1 and CD206, the markers of the M2 phenotype. These data are consistent with a previous study that MSCs reprogrammed microglia into a M2-like phenotype to inhibit the overproduction of inflammatory mediators and promote the regeneration of tissue .
After JEV infection either in vivo or in vitro, there is a significant production of TNF-α. It has been demonstrated that MSCs can secrete TSG-6 (TNF-α stimulated gene/protein 6) upon the induction of TNF-α to modulate inflammatory responses, inhibit the destruction of the BBB, and ameliorate tissue injury [42, 43]. In vitro, we found that upon co-culturing with JEV-infected Neuro2a cells, MSCs showed increased expression of TSG-6 and TGF-β, and the Neuro2a cells exhibited decreased inflammatory cytokine expression and increased survival. The role and mechanism of TSG-6 from MSCs in the alleviation of neuroinflammation during JE are still in research.
In addition to the regulation of inflammation, the immune defense function of MSCs has also been demonstrated in many bacteria- and virus-induced injuries [32, 33, 44]. To our knowledge, there are few reports about the antiviral role of MSCs. Interestingly, we found statistically decreased viral load in MSC-treated mice. In addition, the JEV titer in Neuro2a cells was decreased when co-cultured with MSCs. One reason for this phenomenon is that the immunomodulation of MSCs can improve the innate and adaptive immune responses and contribute to the elimination of virus. Moreover, in this study, MSCs co-cultured with JEV-infected Neuro2a cells showed significantly increased expression of IFN-β and IFN-α compared with control cells. It is well known that type I interferons play a key role in the defense against JEV . Furthermore, a series of changes in genes related to antiviral activity in MSCs were detected after either JEV stimulation or co-culturing with JEV-infected Neuro2a cells through gene expression profiling in our study (data unpublished). It has been reported that MSCs can exert antibacterial effects by secretion of beta-defensin-2 via Toll-like receptor 4 (TLR4) . Whether MSCs have a direct antiviral effect and the exact mechanism is currently being explored.
Recently, engineered MSCs have also been developed to exert more effective antiviral activities. As previously reported, MSCs with a transgene encoding humanized Venezuelan equine encephalitis virus (VEEV) neutralizing antibody (anti-VEEV) had a more efficacious protective effect in nude mice infected with VEEV . However, the protection of sham MSCs transplanted via intramuscular injection was poor in the VEEV infection model . In our study, we found that MSC transplantation via intravenous injection increased the survival and reduced the weight loss of JEV-infected mice. This divergence between the VEEV and JEV results might due to the different pathogenic mechanisms, mouse models used, and transplantation means applied.
In this study, we focused on the effects of MSCs on the morbidity and acute neuroinflammation during JEV infection and harvested mouse brains at 6 dpi. Whether MSC transplantation improves neurological function and prognosis during the late stage of encephalitis is also being explored by our group.
Taken together, this study demonstrated that MSCs exerted beneficial effects against JE in the mouse model. It is the first evidence that intravenous MSCs could serve as a potential adjunctive therapy for JE. Whether this treatment modality can be applied to other types of viral encephalitis warrants further investigation.
B-cell lymphoma-2 protein
Bovine serum albumin
Chemokine (C-C motif) ligand 2
Central nervous system
Evans blue dye
Fetal bovine serum
Fourth Military Medical University
Hematoxylin and eosin
Ionized calcium binding adaptor molecule-1
Inducible nitric oxide synthase
Japanese encephalitis virus
Low-glucose Dulbecco’s modified Eagle’s medium
Multiplicity of infection
Mesenchymal stem cell
Neuron-specific nuclear protein
Quantitative real-time polymerase chain reaction
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Standard error of the mean
Transforming growth factor
Tumor necrosis factor
Terminal-deoxynucleotidyl transferase-mediated nick-end labelling
Venezuelan equine encephalitis virus
We would like to thank Dr. Hong Fan (Department of Neurobiology and Collaborative Innovation Center for Brain Science, School of Basic Medicine, the Fourth Military Medical University), Professor Boquan Jin (Department of Immunology, the Fourth Military Medical University), and Dr. Bei Li (State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Disease, Center for Tissue Engineering, School of Stomatology, the Fourth Military Medical University) for their generous suggestions regarding our study. We appreciate the technical assistance of Dr. Yong Zhao (Laboratory Animal Center, the Fourth Military Medical University).
This work was supported by the National Natural Science Foundation of China (NSFC) (Grant no. 31370193) and the National Key State Science and Technology Projects of China (Grant no. 2013ZX10004609).
Availability of data and materials
All relevant data are included within the paper and supporting information files.
BP conception and design, data collection and assembly, data analysis and interpretation, manuscript writing. YC data collection and assembly, data analysis and interpretation, manuscript writing. ZX data analysis and interpretation, manuscript writing. YJ, YW, WY, ZY, MH, and HP data collection. ZH, ZY, and ZF administrative support, provision of study material. LY: conception and design, financial support, administrative support, data analysis and interpretation, manuscript writing. JZ conception and design, financial support, administrative support, final approval of manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All authors agreed to the submission.
All animal experiments were approved by the Animal Care and Use Committee of the FMMU.
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