Background
Mesenchymal stem cells (MSC) are a group of clonogenic, non-haematopoietic, plastic adherent multi-potent stromal cells that possess potential to differentiate into cells of mesodermal lineage [
1],[
2]. MSC have been shown to ameliorate disease in numerous clinical trials and animal models. Approximately 400 clinical trials using MSC are currently underway according to the clinical trials registry of the US National Institute of Health (
http://clinicaltrials.gov). It is noteworthy that a good proportion of these studies are exploring the immunomodulatory properties of MSC. This aspect of MSC is widely explored in managing graft-versus-host disease [
3] and in a variety of experimental disease models, including autoimmune encephalomyelitis [
4], stroke [
5], amyotrophic lateral sclerosis [
6], spinal cord injury [
7], diabetes [
8] and myocardial infarction [
9]. These advances at the therapeutic level are a result of detailed descriptions on the potential of MSC to modulate a range of immune cells including T cells, B cells, dendritic cells, monocytes and macrophages [
10]-[
15].
Microglia are resident macrophages of the central nervous system (CNS), derived from primitive myeloid progenitors that migrate from the embryonic yolk sac to the brain rudiment [
16]. In adults, microglia survey the entire brain every couple of hours through concerted movements [
17] for damaged neurons, endogenous disease proteins, and infection [
18]. Microglia assume functions beyond being immune sentinels by maintaining a healthy CNS environment through synaptic pruning [
19]-[
21] and secretion of neurotrophic factors [
22],[
23]. In response to disturbances in CNS homeostasis microglia undergo morphological, phenotypic and functional changes. These changes include an increase in numbers through proliferation, a shift from ramified to amoeboid morphology, secretion of inflammatory mediators such as cytokines, chemokines and reactive oxygen and nitrogen species, and an increase in phagocytic activity. Experiments have shown that these changes which microglia undergo upon activation can cause deleterious effects within the CNS microenvironment and play a key role in pathogenesis of neurodegenerative disease [
24]-[
27].
Modulating microglia responses is being considered as an effective approach to manage progression of neurodegenerative diseases. Recently, with the rise in reports on immunomodulatory prospects of MSC, we and others have explored and described the potential of MSC to dampen inflammatory responses of microglia [
28]-[
32]. However, the mechanisms underlying these effects are poorly understood. The present study determined the mechanisms through which MSC confer an anti-proliferative effect on microglia by examining apoptosis and cell cycle. We also determined the role of nitric oxide, IL-6 and TNF-α in conferring the modulatory effects of MSC. It was also demonstrated that MSC experience growth arrest in co-culture while modulating microglial responses.
Materials and methods
Mouse bone marrow mesenchymal stem cell culture
MSC previously isolated from ICR mouse bone marrow were obtained from the culture collection of the Immunology Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. Frozen vials stored in liquid nitrogen were thawed at 37°C and cultured in MSC complete medium (high glucose DMEM (GIBCO Invitrogen, CA, USA), supplemented with 15% (v/v) MesenCult® Mesenchymal Stem Cell Stimulatory Supplements (Mouse) (Stemcell™ Technologies, Canada), 1% penicillin and streptomycin (iDNA), 250 μg/ml fungizone (GIBCO Invitrogen, CA, USA), 2.0 mM GlutaMaX and 1.5 g sodium bicarbonate) at 37°C, 5% CO
2 in a humidified incubator. Cells were routinely sub-cultured using 0.25% Trypsin-EDTA (GIBCO Invitrogen, CA, USA) before reaching 90% confluency and immunophenotyped using a panel of markers comprising CD106, CD45, CD44, CD11b, Sca-1, MHC-I and MHC-II (all from Becton Dickinson, BD, San Jose, CA, USA) [
1]. MSC phenotype was confirmed by positivity to CD106, CD44, Sca-1 and MHC-I and negativity to CD45, CD11b and MHC-II.
BVmicroglia culture
BV2 cells were a generous gift from Dr Thameem Dheen of the National University of Singapore. BV2 is a murine microglial cell line immortalised with v-raf/v-myc genes carrying retrovirus J2 [
33]. Cells were cultured in DMEM with 5% heat-inactivated foetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 g/ml gentamicin (all Invitrogen), 1 × non-essential amino acids and 6.25 μg/ml insulin (Sigma-Aldrich, St. Louis, MO, USA). Cells were either sub-cultured or used for downstream assays before reaching 90% confluency by harvesting with 0.25% trypsin containing 1 mM EDTA for 5 minutes at 37°C.
MSC/BVco-cultures
BV2 and MSC were seeded simultaneously at a ratio of 1:0.2 and incubated overnight at 37°C in a 5% CO2 incubator to allow cells to adhere. Co-cultures were then stimulated with 1 μg/ml lipopolysaccharide (LPS; E. coli serotype O26:B6; Sigma Cat. No. L2762). This culture set-up will be described as `activated co-cultures hereafter. The time point of LPS addition was considered as 0 hour for all experiments. Cell culture inserts with a 1 μm polyethylene terephthalate membrane pore size (Falcon, BD Biosciences, Erembodegem, Belgium) were used for transwell experiment set-up.
3H-TdR incorporation assay
BV2 cell proliferation was determined by assessing tritiated thymidine (3H-TdR; Perkin Elmer, Boston, USA) incorporation. In 96-well plates, 1 × 103 MSC were seeded in triplicate and allowed to adhere overnight. The following day, MSC were treated with 10 μg/ml mitomycin-C (Sigma) for 2 hours to halt their proliferation. Plates were washed thoroughly with DMEM to remove any traces of the mitotic inhibitor and BV2 cells were then seeded at 5 × 103 cells/well. Co-cultures were activated with 1 μg/ml LPS for 48 hours and 3H-TdR (0.037 MBq/well (0.5 μCi/well)) was added to wells at the final 6 hours of incubation. Plates were exposed to a freeze/thaw cycle at -20°C to ease cell harvesting. Cells were harvested onto a filter mat by using an automated cell harvester (Harvester Mach III M, TOMTEC, CT, USA Thymidine incorporation was measured by liquid scintillation spectroscopy on a beta counter (MicroBetaTriLux, Perkin Elmer Boston, USA) after the addition of scintillation fluid (OptiPhaseSuperMix Cocktail; Perkin Elmer Boston, USA) and readouts were in counts per minute (cpm).
Griess assay
Nitric oxide (NO) was detected in the supernatant of cultures using the Griess assay. For this, 50 μl culture supernatant from each sample was transferred to a 96-well plate in triplicate and an equal volume of Griess reagent added (1% sulphanilamide/0.1% N-1-napthylethylenediamine dihydrochloride/2.5% phosphoric acid; all from Sigma). Absorbance was read at 530 nm (MRX II microplate reader, Dynex, VA, USA) after 10 minutes incubation. Nitrite concentration was calculated with reference to a standard curve of freshly prepared sodium nitrite (0 to 100 μm). The results are displayed as concentration of NO2
- in μm.
Apoptosis assay
Apoptosis of cells in co-culture was determined by flow cytometry after double staining with FITC-Annexin-V and propidium iodide (PI). BV2 cells and MSC were co-cultured overnight at a 1:0.2 ratio, stimulated with 1 μg/ml LPS the following day, and left in culture for 48 hours. Cells were then harvested using 0.25% trypsin-EDTA. Cells were washed twice in ice-cold PBS and suspended in 100 μl of 1X binding buffer at a concentration of 1 × 106 cells/ml. Cells were stained for CD45 by incubating with 0.5 μl antibody (Rat anti-mouse CD45, BioLegend®, San Diego, CA, USA ) at 4°C for 15 minutes followed by 15 minutes incubation with secondary antibody (DyLight™ 649 Goat anti-rat IgG, BioLegend®). CD45 staining was performed to distinguish BV2 microglia from the MSC population during flow cytometry analysis. Five microlitres each of FITC-conjugated Annexin-V and PI was added to each tube and incubated for 15 minutes at room temperature. Four hundred microlitres of 1X binding buffer was added to each tube before acquisition and analysis using a flow cytometer.
Cell cycle analysis
Distribution of cells through the three distinct phases of cell cycle (G0/G1, S and G2/M phase) was analysed using PI staining. Co-cultures were harvested using 0.25% trypsin-EDTA and washed once in PBS by centrifugation at 1,000 rpm for 7 minutes. Cells were then suspended in 1 ml of ice-cold PBS at a density of 0.5 × 106 cells per tube. The cell suspension was subsequently added drop wise to 3 ml ice-cold 95% ethanol for fixation. The tubes were incubated at -20°C for at least 2 hours. The tubes were removed from -20°C and spun at 1,200 rpm for 10 minutes and washed once using ice-cold PBS to remove traces of ethanol. The cells were then suspended in 500 μl of 50 μg/ml PI staining solution and incubated at 37°C for 15 minutes. Stained cells were washed once with PBS to remove excess PI and suspended in 400 μl PBS. The cell cycle data for individual samples were acquired using the BD LSR Fortessa™ flow cytometer equipped with BD FACSDiva™ software (BD) and analysed using ModFit LT™ software (Verity Software House, ME, USA).
Cytokine bead array/protein array for TNF-α and IL-detection
Co-culture supernatants were assayed at 24 hours using a custom RayBio® mouse cytokine array kit (RayBiotech, Inc., GA, USA ), according to manufacturer’s instructions. The results are expressed as relative protein levels compared with LPS-stimulated BV2 cells. Absolute quantity of IL-6 and TNF-α in culture supernatants were then determined at 24 hours by using a multiplex bead array kit (BD Cytometric Bead Array mouse inflammation kit; BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. Samples were assayed on a FACS Fortessa flow cytometer (BD Biosciences) and analysed with FCAP array software (BD Biosciences). Concentration of cytokines in samples was calculated using individual standard curves and expressed as pg/ml.
Cytokine blocking studies
Specific blocking antibodies to TNF-α and IL-6 were used to elucidate the functional importance of modulation of these cytokines in co-culture. Blocking antibodies against TNF-α (XT3.11), IL-6 (MP5-20 F3) and isotype (Rat IgG1) control (all from Bio X Cell, NH, USA) were reconstituted in 1X PBS to 1.0 mg/ml. The antibodies were then serially diluted in culture media to obtain working stock concentrations of 20.0, 2.0 and 0.2 μg/ml. Cells were plated either in 12-well plates (for Griess assays) or in 96-well plates (for proliferation assays) as described earlier. Cells were stimulated with 1 μg/ml LPS after overnight incubation. Equal volumes of diluted antibody were added to cultures to obtain a final concentration of 10.0, 1.0 and 0.1 μg/ml. Culture supernatants were collected at 24 and 48 hours and assayed for NO production using Griess assay. Proliferation was analysed at 48 hours by pulsing the cultures with 3H-TdR for the final 6 hours of incubation.
Statistical analysis
Mean values and standard deviations (SD) were calculated from three independent experiments and significance was assessed using one-way analysis of variance followed by the Tukey post hoc test or student’s t test using GraphPad Prism version 6 (GraphPad software, San Diego, CA, USA).
Discussion
Here, for the first time, we demonstrate the role of TNF-α in MSC-mediated immunomodulation of microglia. Treatment of BV2 microglia cultures with TNF-α blocking antibodies reduced LPS-induced proliferation while addition of recombinant TNF-α to co-cultures abolished the anti-proliferative effect of MSC on microglia. Microgliosis is a common response towards injury in the CNS, including Alzheimer’s disease [
39], Parkinson’s disease [
40], multiple sclerosis [
41] and stroke [
42], and the findings here may prove to be beneficial for these conditions.
In addition, the present study negated the role of apoptosis and soluble factors such as NO and IL-6 in conferring this effect. We also provide the first evidence that MSC confer the inhibitory effect on microglia proliferation through modulation of microglia cell cycle and MSC themselves undergo a G0/G1 arrest while exerting this effect.
Following co-culture with MSC, microglia proliferation in response to LPS stimulation was dampened and NO levels were increased. Surge in NO levels in MSC co-cultures have been previously identified as the key mechanism conferring anti-proliferative effects on T cells [
35],[
36]. To examine similar prospects, NO production in co-culture was abolished using a specific iNOS inhibitor - L-NAME - and microglia proliferation was analysed. Converse to the reports in MSC/T cell co-cultures, abolishing NO did not restore microglia proliferation in our study. This is contradictory to a recent report which suggested the role of NO in MSC-mediated inhibition of microglia proliferation [
43]. In the mentioned report, conditioned media from MSC cultures treated with an iNOS inhibitor was used to study the anti-proliferative effect, and thus the role of microglial cues that may be required by MSC to exert such effects was not negated. Our laboratory has previously reported that MSC requires microglial cue to induce a NO surge [
34]. The current approach to eliminate NO directly from co-culture by addition of L-NAME efficiently negates such doubts and rules out the role of NO in inhibition of microglial proliferation by MSC.
The inhibition of microglial proliferation reported here could also be contributed by cell death. MSC are shown to induce indoleamine 2,3-dioxygenase-dependent apoptosis of activated T cells [
37]. Through examination using Annexin-V/PI staining, we demonstrate that co-culture with MSC does not induce apoptosis in microglia. Such differences in interaction of MSC with T cells and microglia emphasises diversity in the modulatory functions that MSC exert on different target cell types.
Next, we examined whether co-culture induces cell cycle modulation in microglia. Studies have shown MSC induce cell cycle arrest in dendritic cells [
14] and tumour cells [
38]. Here, for the first time, we provide evidence that MSC exert their anti-proliferative effect through modulation of microglia cell cycle. Co-culture with MSC restored the percentage of BV2 cells in the different phases of the cell cycle to levels comparable to resting microglia. Interestingly, this modulation was dependent on cell-to-cell contact. From a therapeutic perspective, we presume that suppression of the proliferative response of microglia through cell cycle modulation is beneficial, as inducing apoptosis or a permanent cell cycle arrest may perturb the volume of glia and hence affect homeostasis of the brain. Interestingly, MSC entered a G0/G1-phase cell cycle arrest while exerting the anti-proliferative effect suggesting that the inhibitory effect of MSC on microglia activation (at least in terms of microglia proliferation) is not dependent on a need for a proliferative response from MSC. The ability of MSC to modulate the proliferative response of microglia whilst simultaneously entering a cell cycle arrest is presumably a beneficial one. Tumourigenic transformation of MSC has been implicated in several experimental transplantations including osteogenic sarcomas [
44], myocardial infarction and diabetic neuropathy [
45]. Therefore, careful evaluation and understanding of proliferative potential of MSC within the inflamed milieu is necessary for the success of clinical interventions.
We have also demonstrated here that MSC modulate the expression of TNF-α and IL-6. Co-culture with MSC significantly reduced the TNF-α which was upregulated upon LPS stimulation whereas co-culture induced a surge in IL-6. Similar modulation of cytokines was also reported previously by us [
34] and others [
29],[
43]. The impact of differential modulation of IL-6 and TNF-α was further deduced using blocking antibodies and recombinant TNF-α. Through addition of blocking antibodies, we have identified that the IL-6 surge in co-culture does not influence the anti-proliferative effect of MSC or NO modulation. Distinct from IL-6, TNF-α seems to play a context-dependent role on modulation of NO and microglial proliferation in co-culture. Reducing TNF-α level in culture using blocking antibodies did not influence the NO profile of activated microglia. However, addition of recombinant TNF-α induced a dose-dependent NO surge in co-cultures. It has been previously described by our group that soluble factors from activated co-culture induce NO production in MSC and these soluble factors include TNF-α [
34]. It is possible that, in co-culture, the additional TNF-α added to the co-culture acts in tangent with microglial signals to induce a surge in NO, or TNF-α itself acts as the signal to induce NO production by MSC. MSC are known to respond to TNF-α signals and elicit downstream responses including NFκB translocation which is required for iNOS transcription [
46]. However, such postulations need to be further validated.
We also showed distinct responses in microglia proliferation upon altering TNF-α levels in culture. TNF-α has been previously identified as vital for beta-amyloid-induced proliferation of microglia; abolishing TNF signals through addition of anti-TNF-α antibody or a soluble TNF receptor inhibitor both prevented beta-amyloid-induced proliferation of microglia [
47]. Similarly in an MPTP mouse model of Parkinson’s disease, knocking out TNF-α significantly reduced microglial activation as measured by cell number and morphology compared to wild-type controls [
48], suggesting the important role played by TNF-α in microglial activation. Managing TNF-α levels in the inflammatory milieu is thus considered an efficient way to modulate inflammation [
48],[
49]. At present, there is no evidence to suggest that modulation of TNF-α is the mechanism through which MSC confer an anti-proliferative effect on microglia. Likewise, there is no report on how MSC reduce microglial TNF-α. However, earlier reports from our laboratory [
34] and the present study confirms the potential of MSC to downregulate TNF-α production in an LPS-stimulated microglia co-culture paradigm. Similarly, intravenous transplantation of MSC into rat traumatic brain injury models significantly decreased TNF-α levels in the injured cortex and decreased the number of glial cells at the site of injury [
50]. Thus, we hypothesised that modulation of TNF-α by MSC may be vital in conferring their anti-proliferative effect and monitored the effects of neutralising TNF-α and adding recombinant TNF-α on proliferation of LPS-stimulated BV2 microglia in co-culture. Abolishing TNF-α from co-culture did not enhance the anti-proliferative effect of MSC, indicating that a further decrease of TNF-α from that seen in co-cultures does not have an additive effect on MSC anti-proliferation of BV2. Also, the basal proliferation of BV2 cells may not require TNF-α, as addition of anti-TNF onto unstimulated BV2 did not affect their proliferation. However, the proliferation of LPS-stimulated microglia was reduced in a dose-dependent manner upon addition of TNF-α neutralising antibodies, thus confirming the role of TNF-α in inducing proliferation of microglia. It is noteworthy that addition of recombinant TNF-α to LPS-stimulated BV2 culture did not increase their proliferation. This indicates that a critical concentration of TNF-α may be required for microglial activation above which the cells may become non-responsive. Also, other cues may be necessary for a proliferative response. Considering the requirement of cell-to-cell contact in modulating the cell cycle of activated microglia, we suggest the involvement of one or few cell surface molecules that may work in tandem with TNF-α modulation to achieve the inhibition of microglia proliferation by MSC. Recent studies have strongly suggested the role of cell surface molecules such as CD200 [
51], HLA-G [
52] and PD-1 [
53] expressed by MSC on their immunomodulatory potential. It is possible that these cell surface ligands could be involved in the MSC-mediated modulation of BV2 microglia proliferation.
Next, in order to confirm the relation of MSC-mediated reduction of TNF-α and inhibition of microglial proliferation in co-culture, we used recombinant TNF-α. At concentrations as low as 0.5 ng/ml, TNF-α abolished the inhibition of microglia proliferation in co-culture. Moreover, higher concentrations of recombinant TNF-α induced the proliferation of microglia in co-culture to levels beyond that of LPS-stimulated culture. This confirms downregulation of TNF-α in co-culture by MSC as the key mechanism leading to the inhibition of microglial proliferation.
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Competing interests
The authors declare that they have no competing interests.