Counteracting neuroinflammation in experimental Parkinson’s disease favors recovery of function: effects of Er-NPCs administration
Parkinson’s disease (PD) is the second most common neurodegenerative disease, presenting with midbrain dopaminergic neurons degeneration. A number of studies suggest that microglial activation may have a role in PD. It has emerged that inflammation-derived oxidative stress and cytokine-dependent toxicity may contribute to nigrostriatal pathway degeneration and exacerbate the progression of the disease in patients with idiopathic PD. Cell therapies have long been considered a feasible regenerative approach to compensate for the loss of specific cell populations such as the one that occurs in PD. We recently demonstrated that erythropoietin-releasing neural precursors cells (Er-NPCs) administered to MPTP-intoxicated animals survive after transplantation in the recipient’s damaged brain, differentiate, and rescue degenerating striatal dopaminergic neurons. Here, we aimed to investigate the potential anti-inflammatory actions of Er-NPCs infused in an MPTP experimental model of PD.
The degeneration of dopaminergic neurons was caused by MPTP administration in C57BL/6 male mice. 2.5 × 105 GFP-labeled Er-NPCs were administered by stereotaxic injection unilaterally in the left striatum. Functional recovery was assessed by two independent behavioral tests. Neuroinflammation was investigated measuring the mRNAs levels of pro-inflammatory and anti-inflammatory cytokines, and immunohistochemistry studies were performed to evaluate markers of inflammation and the potential rescue of tyrosine hydroxylase (TH) projections in the striatum of recipient mice.
Er-NPC administration promoted a rapid anti-inflammatory effect that was already evident 24 h after transplant with a decrease of pro-inflammatory and increase of anti-inflammatory cytokines mRNA expression levels. This effect was maintained until the end of the observational period, 2 weeks post-transplant. Here, we show that Er-NPCs transplant reduces macrophage infiltration, directly counteracting the M1-like pro-inflammatory response of murine-activated microglia, which corresponds to the decrease of CD68 and CD86 markers, and induces M2-like pro-regeneration traits, as indicated by the increase of CD206 and IL-10 expression. Moreover, we also show that this activity is mediated by Er-NPCs-derived erythropoietin (EPO) since the co-injection of cells with anti-EPO antibodies neutralizes the anti-inflammatory effect of the Er-NPCs treatment.
This study shows the anti-inflammatory actions exerted by Er-NPCs, and we suggest that these cells may represent good candidates for cellular therapy to counteract neuroinflammation in neurodegenerative disorders.
KeywordsParkinson’s disease Erythropoietin Adult stem cells Neural stem cells transplantation Neuroinflammation Regenerative medicine
Analysis of variance
Brain-derived neurotrophic factor
Central nervous system
Erythropoietin releasing neural precursors cells
Fetal bovine serum
Green fluorescent protein
Nerve growth factor
Normal goat serum
Phorbol 12-myristate 13-acetate
Roswell Park Memorial Institute medium
Substantia nigra pars compacta
Human leukemia monocytic cell line
Tumor necrosis factor
Parkinson’s disease (PD) is characterized by dopaminergic (DA) denervation of the striatum and progressive death of DA neurons in the substantia nigra pars compacta (SNpc) . Neuroinflammation has a role in several neurodegenerative diseases; though it may not be considered the primary cause, it contributes to the symptomatic phase . Several lines of research suggest that neuroinflammation is the major central event in dopaminergic neural cell death in PD [3, 4, 5]. In postmortem SN from human PD brains, microglia results activated, lymphocytes are infiltrated [2, 6], and in cerebrospinal fluids, there are high levels of pro-inflammatory cytokines such as tumor necrosis factor (TNF), cyclooxygenase-2 (COX-2), interleukin-1beta (IL-1beta), and IL-18 [7, 8]. It has been suggested that in CNS neurodegeneration, neuronal damage may lead to the activation of microglia and astrocytes that in turn amplify the inflammatory response through chemokine secretion. This enhances CNS infiltration by peripheral immune cells. Studies in the acute neurotoxic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model provided evidences that neuroinflammatory processes can contribute to nigral DA neuronal death [9, 10, 11]. Both the genetic deletions of microglial effectors  and the suppression of T lymphocytes  reduced neuronal loss suggesting that neuroinflammation in PD may actively participate in neuronal death. The “principal mechanism” that links the inflammatory response with neurodegeneration remains to be fully clarified. However, it is recognized that neuronal degeneration itself and in particular the accumulation and release of alpha-synuclein aggregates by the injured DA neurons early in the disease process , may act as a signal, and can activate glial cells to produce and release a variety of pro-inflammatory molecules, exacerbating microglia activation and neuronal cell death [14, 15, 16, 17, 18]. Within this scenario, the major players are the microglia, the reactive astrocytes, and the infiltrating monocyte-derived macrophages . Upon injury, activated M1-like microglia proliferates and participates in clearing cell debris in the early stages but may exacerbate brain injury through the production of neurotoxic substances, especially when it is overactivated for prolonged periods . In the M2-like phenotype, microglia has anti-inflammatory and neuron-reparative roles, protecting the damaged tissue by removing cell debris and releasing anti-inflammatory cytokines needed for tissue repair . Current treatments for PD are only symptomatic and have no effects on the ongoing neurodegeneration . The ideal therapeutic treatment for PD should have both symptomatic and restorative effects aimed at preserving midbrain DA neurons from degeneration [23, 24]. In this sense, adult neural stem cells grafting into animal experimental models of neurodegenerative diseases have shown beneficial effects promoting both trophic and anti-inflammatory actions [25, 26, 27, 28, 29, 30, 31]. Recently, we reported the therapeutic potential of erythropoietin-releasing neural precursors cells (Er-NPCs) intrastriatally infused in a preclinical model of PD, obtained upon the administration of MPTP [30, 32]. After the unilateral transplantation into the striatum of MPTP-treated C57BL/6 mice, Er-NPCs were vital and capable of engrafting into the recipient’s brain. Er-NPC-treated animals improved their typical motor deficits within 3 days of cell transplantation, and this was accompanied by significant sparing of SN neurons. All these features and effects are likely dependent on erythropoietin’s (EPO) release since all of these were abolished by the co-injection of Er-NPCs with anti-EPO (aEPO) or anti-EPO-receptor (aEPO-R) antibodies. Little is known about the anti-inflammatory actions of Er-NPCs transplant in PD brains. Here, we focus on the study of this aspect and report that a rapid anti-inflammatory effect was evident 24 h after Er-NPCs transplant with the decrease of early pro-inflammatory cytokines mRNA levels (e.g., IL-1alpha, TNF). This effect was maintained for the following days, and IL-6 mRNA levels were significantly reduced as far as 7 days after transplantation. At the end of the 2-week observational period, histological data confirmed the reduction of activated microglia marker expression (GFAP and Iba1) and macrophages infiltration (CD68). Moreover, at the same time point, we observed the increase of markers associated with the M2-like protective phenotype. Co-injection of Er-NPCs with aEPO antibody neutralizes the Er-NPCs anti-inflammatory activity, strongly indicating that this effect is mediated by EPO released from Er-NPCs.
Animals and study approval
Procedures involving animals and their care were conducted in conformity with the Italian Guidelines for Laboratory Animals, which conform to the European Communities Directive of September 2010 (2010/63/UE), and the Review Committee of the University of Milan gave its approval to the study (No 2/2013). Male C57BL/6 mice (Charles River, Milan, Italy), 12–16 weeks old and weighing 20–24 g, were kept for at least 7 days before the experiments and housed in standard conditions (22 ± 2 °C, 65% humidity, and a 12-h light-dark cycle) with food and water ad libitum. Moreover, in order to make them amenable, the animals were accustomed to the behavioral tests (horizontal and vertical, see below for details) daily for 1 week prior to MPTP injection.
Erythropoietin-releasing neural precursors cells (Er-NPCs) expressing green fluorescent protein (GFP) were isolated 6 h postmortem from adult C57BL/6-Tg(UBC-GFP)30Scha/J mice weighing 25–30 g (Charles River) as previously described [29, 33, 34].
Number of animals used for experiments. All animals were tested for behavioral performances
MPTP + Er-NPCs
MPTP + Er-NPCs + aEPO
Real-time RT PCR
Real-time RT PCR
Real-time RT PCR
To investigate the recovery of motor dysfunction after cell transplantation, two different behavioral tests were performed: horizontal and vertical grid tests [30, 32, 37, 38]. Each animal was tested twice at each time point.
Horizontal grid test
The grid apparatus was constructed according to Tillerson and co-workers [30, 32, 37]. The animal was videotaped for 30 s, and the videos were replayed for percentage forepaw fault analysis using a recorder with slow motion option. The number of unsuccessful forepaw steps divided by the total number of attempted forepaw steps was evaluated . The mice were acclimatized to the grid twice a day for 1 week, before MPTP treatment. Three observers (ZG, FR, MM) in blind rated each trial for forepaw faults per step.
Vertical grid test
The vertical grid apparatus was constructed according to Kim and co-workers [30, 32, 38]. For this test, the mouse was placed 3 cm from the top of the apparatus, facing upwards, and was videotaped when turning around and climbing down. The score reported was the time required by the mouse to make a turn, climb down, and reach the bottom of the grid with its forepaw within 180 s [30, 32, 38]. Before MPTP administration, mice were acclimatized to the grid twice a day for 1 week. The analysis was performed by three observers in blind (ZG, FR, MM).
For this test, mice were food deprived for 20 h before the test. A corn chip was buried under their bedding (1 cm) in a corner of the cage. Each mouse was positioned at the center of the testing cage, and the time to retrieve and bite the corn chip was measured . The analysis was performed by three observers in blind (ZG, FR, MM).
Immunohistochemistry and quantitative analysis
THP1 co-cultures with Er-NPCs
THP1 cells were seeded with fresh RPMI medium supplemented with 3% FBS on six trans-well plates at the concentration of 5 × 105 cells/well for co-culture experiment and activated with 50 ng/ml PMA. Twenty-four hours after seeding, the THP1 was stimulated adding 1 μg/ml LPS for 1 h. For co-culture experiments, Er-NPCs were dissociated, counted, and re-suspended in the THP1 medium at the concentration of 4 × 105 cells/well. Co-culture of THP1 and Er-NPCs with or without aEPO antibody (5 μg/ml) was performed using 0.4 μm pore size trans-well inserts (Corning). After 3 h of co-culturing, inserts were removed and the THP1 were isolated for RNA extraction and real-time RT-PCR. Dexamethasone was used (100 μM) as anti-inflammatory positive control .
RNA extraction and real-time PCR
Primer sequences used to study gene expression
Statistical analyses between groups were evaluated using GraphPadPrism 4.00 version, and data are expressed as mean ± SD. Behavioral data were analyzed with a two-way ANOVA model with time and group (CTRL, MPTP, SHAM, MPTP + Er-NPCs, and MPTP + Er-NPCs + aEPO) as factors. The null hypothesis was rejected when p < 0.05. Gene expression data were analyzed in triplicate and results were expressed as the average of six animals. The expression pattern of each gene was analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test to assess statistical significance.
Transplanted Er-NPCs integrate into the damaged host brain and promote a rapid recovery of function
Er-NPCs grafts promote the rescue of endogenous TH-positive projections in the recipient striatum
Er-NPCs transplantation counteracts pro-inflammatory cytokines expression in MPTP-recipient mice
Er-NPCs regulate the expression of neuroinflammatory markers in the damaged striatum of recipient animals
The anti-inflammatory effect of Er-NPCs is mediated by EPO
Er-NPCs override the pro-inflammatory cytokine production by macrophages in vitro
Many reports have shown the ability of transplanted neural stem cells to modify the environment of the recipient tissue and give rise to positive effects in animal experimental models of PD. Here, we uncover the anti-inflammatory action exerted by engrafted Er-NPCs in an MPTP model of PD. Their transplantation promoted functional recovery and resulted in the sparing of dopaminergic nigro-striatal projections. By perceiving signals coming from the microenvironment of damaged tissue, stem cells can migrate to specific sites in the body and respond to a specific signal by releasing cytokines and modifying their own fate . All of these features allow stem cells to be a possible therapeutic approach for many neurodegenerative diseases, including PD.
Here, we used accessible, stably expandable, and well-characterized erythropoietin-releasing neural precursors cells (Er-NPCs) [29, 30, 31, 32, 33, 34]. The aim of this work was to investigate the mechanisms exerted by Er-NPCs transplantation in a mouse model of PD, obtained with MPTP administration, which mimics the inflammatory process related to the pathology [50, 51, 52, 57].
Transplanted Er-NPCs survived after transplantation, differentiated in the recipient striatum, and induced significant amelioration in functional motor symptoms caused by MPTP intoxication 3 days post-transplantation, as well as reduced pro-inflammatory cytokines 24 h after the infusion. These results support the hypothesis that the transplanted Er-NPCs inhibit the neuroinflammatory phenomena associated with Parkinsonism induced by the administration of MPTP neurotoxin, via an EPO-dependent mechanism. Moreover, although the expression of specific markers of macrophages polarization is not completely categorized , the transplanted neural precursors interact with the recipient microenvironment and stimulate tissue response to inflammation, supporting the transition of macrophages from M1-like phenotype to M2-like, towards a more protective action . It would require specific assays such as RNASq experiments to dissect deeply the macrophages pathways involved. We have observed that there is no direct contact between M2-like macrophages (positive to CD206) and transplanted Er-NPCs, suggesting that the observed transition does not require the direct contact with the cells but can probably be ascribed to a paracrine phenomenon. The mechanism could be related to a local counteraction of inflammation and neuroprotective action mediated by EPO [30, 32, 59, 60, 61] on neurons and neural processes affected by MPTP. Neuroinflammation is a physiologic response to the administration of the MPTP toxin resulting in microglia activation which secretes multi-functional immunoregulatory factors, most notably TNF, IL-1 and IL-6 families, interferon gamma (IFN-gamma), and transforming growth factor beta (TGF-beta), [62, 63]. All of these factors act in context-dependent ways to modulate inflammatory processes and the permeability of the BBB [15, 64, 65]. Anti-inflammatory properties of Er-NPCs are shown by the fact that their transplant downregulates IL-6 and TNF mRNA levels and reduces macrophages invasion in the striatum and in the SNpc of recipient mice.
This is consistent with our previous work performed in experimental traumatic spinal cord injury, which reported the reduction of inflammatory cytokine levels and macrophage infiltration after the treatment with rhEPO  or administration of Er-NPCs [29, 33]. This seems to happen also in the present work, as there is a counter-action of the inflammatory events caused by MPTP, particularly ipsilateral to the injection site. Moreover, we observed here that transplanted Er-NPCs were able to counteract the expression of activated microglia’s markers both in the ipsi- and contra-lateral striatum. This further validates our previous evidences [30, 32] showing that the inhibition of EPO’s release by Er-NPCs influenced the striatum microenvironment by reducing endogenous EPO expression . Thus, we could speculate that the action on contralateral neuroinflammation can be ascribed both to the diffusion of EPO released by engrafted precursors and by the modification of the local microenvironment . The counteractive effects on astrocytes (GFAP staining reduction) are quite relevant since this type of cells are highly activated in PD and produce an elevated level of inflammatory cytokines and reactive oxygen species . Moreover, such an activation is correlated with increased neuronal death [67, 68].
We found that the levels of pro-inflammatory cytokines, such as IL-1alpha, TNF, and IL-6 mRNA levels, are significantly reduced 24 h (IL1-alpha, TNF) and 1 week (IL-6) after Er-NPCs administration in contrast with IL-10, an anti-inflammatory cytokine, which resulted increased in the ipsilateral striatum 1 week after administration of Er-NPCs. The variation in the mRNA levels of cytokines is concordant with the decrease of CD86 (marker of macrophage phenotypes M1-like) and the increase of the level of CD 206 (marker of macrophages M2-like). Moreover, our results suggest that EPO released by Er-NPCs is implicated in the shift from a pro-inflammatory to a positive phagocytic state [58, 59, 69]. Phenotypic M changes are associated with no major changes in overall CD11b immunoreactivity in MPTP-treated animals transplanted with Er-NPCs with respect to CTRL. This could be explained by the fact that both resident microglia and infiltrated macrophages are positive to CD11b staining following central nervous system injuries [47, 48]. It has been shown that both phenotypes have phagocytic functions: under pro-inflammatory conditions M1-like macrophages, being toxic cells, target viable neurons, cause their death, and help in the propagation of the damage. On the other hand, under anti-inflammatory stimuli, M2-like subtype macrophages, being protective, with their phagocytic activity remove toxic cellular debris and dying cells to allow the creation a favorable microenvironment for the recovery [70, 71]. The ability of Er-NPCs to influence and limit the “pro-inflammatory activity” might be a key pathway to confer protection from PD.
This work confirmed that after the infusion, most of Er-NPCs survive (≥ 75%)  in the striatum and differentiate mainly in neurons . The engrafted precursors migrate in the distal zones from the injection site and show a higher capacity to differentiate: 60% of these cells are positive to MAP2, and more than 80% express the NeuN marker.
This data, if considered together with the results obtained from the transplant of Er-NPCs in traumatic spinal cord injury model [29, 30], shows that these neural precursors are able to interact with the microenvironment where they are infused and differentiate to specific lineages [29, 32].
One of the most encouraging results of the pre-clinic approach that characterizes this work is the study of functional recovery in animals treated with Er-NPCs. Indeed, the motor recovery is already evident at the third day after transplant; it is further improved during the full observational period of 2 weeks, and it is maintained up to 2 months after the transplant . The positive results obtained from the analysis of the motor capabilities are supported by observations obtained via immunohistochemistry analysis that demonstrate that precursor’s transplantation promotes the recovery of TH projections and DAT expression in the recipient striatum associated with the recovery of TH-positive cellular bodies in the SNpc . Aiming to characterize the mechanism of action of injected Er-NPCs, we have studied the role of EPO when released by Er-NPCs  in vivo and in vitro. The inhibition of EPO’s action, caused by the co-administration of the aEPO antibody both in the in vivo and in the in vitro macrophage activation assay, allowed us to validate that the release of this cytokine by the Er-NPCs is one of the mechanisms responsible for their capability to counteract neuroinflammation. EPO’s neuroprotective and anti-inflammatory effects are very well studied and widely supported in the literature . EPO analogs or non-erythropoietic-mutant EPO variants showed neuroprotective actions in MPTP-induced neurotoxicity and had neuro-rescue effects in rodent models of PD [73, 74, 75, 76]. Interestingly, the administration of aEPO antibody in the in vitro macrophages activation assay only partially suppresses the effect of Er-NPCs on TNF mRNA expression levels. This suggests that the feature of Er-NPCs to act as biological disease-modifying agents should not only be due to EPO but possibly to other unknown anti-inflammatory molecules released by these cells.
In conclusion, the data presented in this work suggests that the adult neural precursors releasing EPO represent an interesting model of stem cell therapy approach. Er-NPCs are able to interact with and improve the microenvironment of the damaged tissue thanks to the release of protective factors such as EPO. Moreover, our data suggests that erythropoietin can be a potential pharmacological therapy useful for the treatment of Parkinson’s disease, thanks to its anti-inflammatory properties.
The authors are deeply grateful to Professor Alfredo Gorio (University of Milan, Italy) for his scientific support and unswerving encouragement to the work. Dr. Zuzanna Gombalova participated in the research when she was visiting a student in the Laboratory of Pharmacology, Department of Health Sciences, University of Milan, supported by the Erasmus Program for PhD students. Dr. Federica Rey and Maria Carlotta F. Gorio are PhD student in the Nutritional Sciences, University of Milan. Federica Rey is supported by the Fondazione F.lli Confalonieri.
The authors acknowledge the economic support of the “Neurogel-en-Marche” Foundation (France) and AUS Niguarda Onlus (Italy) to AG and Fondazione “Romeo and Enrica Invernizzi” to AMDG.
Availability of data and materials
The data used in the current study are available if necessary.
SC contributed to the conception and design, data analysis and interpretation, and manuscript writing. TG performed the experiments, data analysis, and manuscript writing. ZG, FR, MCFG, and MM performed the experiments. AMDG contributed to the conception and design, data analysis and interpretation, financial support, and manuscript writing. Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. All authors read and approved the final manuscript.
All animal experiments in this study have been approved by the Institutional Animal Care and Use Committee of Milan University, in accordance with the European Communities Directive of September 2010 (2010/63/UE) for the Care and Use of Laboratory Animals.
Consent for publication
Not applicable (no human data or tissues were used in this paper).
All the contributing authors have seen and approved the manuscript. All authors declare that they have no competing interest.
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