Background
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder and is caused by degeneration of dopaminergic neurons in the substantia nigra pars compacta. Loss of these neurons, their projections to the striatum, and dopamine neurotransmitter leads to resting tremor, postural instability, bradykinesia, and rigidity [
1]. While a minority of PD cases result from defined mutations that initiate PD, the majority of cases result from unknown events [
2].
Both, the innate and adaptive arms of the immune system affect the neuropathology in PD. In PD patients [
3,
4] and animal models [
5‐
7], dopaminergic neuron loss is associated with increased numbers of activated microglia in the substantia nigra. These microglia produce proinflammatory mediators, such as interleukin-6 (IL-6), IL-1β, and nitric oxide that are suggestive of a chronic inflammatory state in PD [
8‐
10]. In addition, PD patients exhibit aberrant adaptive immunity and increases in CD4+ and CD8+ T cells infiltrating the substantia nigra [
3,
5,
11‐
16]. In addition, percentages of CD4+ T cells are diminished in PD patients relative to controls without disease [
11‐
16], but frequencies of Th1 and Th17 effector T cells (Teffs) are increased within the CD4+ population [
17]. These Teffs recognize nitrated-α-synuclein (N-α-Syn) as modified self-protein and exacerbate neuroinflammation and neurodegeneration [
18,
19]. Furthermore, depletion of CD4+ T cells, but not CD8+ T cells, inhibits susceptibility to MPTP, neuroinflammation, and neuronal loss, thus underscoring the importance of CD4+ T cells in affecting progressive neurodegeneration [
18,
20]. In addition to increased numbers of proinflammatory Teffs, frequencies of Th2 and regulatory T cells (Tregs) as well as Treg activity are diminished in PD patients compared to healthy subjects [
13,
17]. Thus, for CD4+ T cells, opposite roles for CD4+ T cells include proinflammatory, neurotoxic processes and anti-inflammatory, neuroprotective functions; both of which can dictate the tempo of PD disease progression. Moreover, the translational implications of these findings are noteworthy as pro-inflammatory immune functions in PD patients can be controlled by CD4+ T cells for potential clinical benefit [
21,
22].
Dendritic cells (DCs) are the antigen presenting cell (APC) responsible for induction of both Teffs and Tregs [
23‐
27]. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces tolerogenic DCs from bone marrow cells, which in turn increase Treg number and function [
28‐
32]. Furthermore, adoptive transfer of GM-CSF-induced tolerogenic bone marrow-derived DCs (BMDCs), diminish autoimmune responses and protect from development of autoimmune sequelae. In autoimmune disorders, Treg induction by tolerogenic BMDCs is mediated by OX40L/Jag-1-dependent mechanisms [
33,
34]. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD model, administration of GM-CSF increases numbers of Tregs without increasing the CD4+ T cell pool and attenuates neuroinflammation and neurodegeneration [
35]. Additionally, Tregs from GM-CSF-treated mice are anti-inflammatory and neuroprotective in MPTP-intoxicated mice. In a recent phase 1 clinical trial, administration of sargramostim (recombinant human GM-CSF) to PD patients increased Treg number and activity, improved UPDRS, III scores, and normalized motor initiation signaling deficits [
36]. Together, these data demonstrate that GM-CSF diminishes neuroinflammation and loss of dopaminergic neurons by increasing Treg numbers and function. As T cells express few, if any, receptors for GM-CSF, Treg induction by GM-CSF likely proceeds through indirect mechanisms. Moreover, those mechanisms must be achieved amid conditions of chronic exposure to GM-CSF, inflammation, and modified, misfolded proteins.
Our current study was designed to assess the effects of GM-CSF and modified α-synuclein (α-Syn) on tolerogenic DCs and Treg induction. Herein, we show that GM-CSF-induced tolerogenic BMDCs lead to induction of Tregs from CD4+ T cells pools. Stimulation of BMDCs with N-α-Syn upregulates expression of cytokine and chemokine genes and proteins, yet Treg induction capability, while diminished, remained at 88% of media controls. Further culture of BMDCs in GM-CSF significantly diminishes Treg induction to ≤55% of controls, regardless of stimulation with N-α-syn or intensity of proinflammatory mediator expression by BMDCs. In MPTP-intoxicated mice, adoptive transfer of tolerogenic BMDCs diminished numbers of reactive microglia and spared dopaminergic neurons along the nigrostriatal axis. While numbers of Tregs were not significantly increased after adoptive transfer of tolerogenic BMDCs to naïve mice, MPTP-intoxication after adoptive transfer significantly induced Treg transformation suggesting the necessity of acute inflammatory signaling to potentiate Treg induction. Together, the data provide a mechanism by which GM-CSF induces tolerogenic BMDCs that can drive T cell-to-Treg transformation, whereas further differentiation by continued exposure to GM-CSF and/or proinflammatory stimulation, diminish DC tolerogenic capacity. We posit that these processes provide a therapeutic strategy for BMDC-mediated control of Treg number and activity that lead to diminution of neuroinflammation and neurodegeneration in PD.
Methods
Animals
Male 6–8 week old mice C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were used for all experiments. All procedures were performed in agreement with the National Institutes of Health guidelines and were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Animals were maintained on a 12 h light/dark cycle and given food and water ad libitum.
BMDC differentiation in vitro
Femurs from 6 to 8 week old male C57BL/6J mice were removed and washed two times in Hanks’ balanced salt solution (HBSS, Gibco, Waltham, MA) on ice and single cell suspensions of bone marrow cells (BMCs) were prepared by trituration. Red blood cells (RBCs) were lysed in ACK lysis buffer (Gibco) and washed in HBSS by centrifugation at 200 xg for 10 min. Cells were resuspended in R10 media [RPMI 1640 (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Sigma, St. Louis, MO), 100 U penicillin, 100 μg/ml streptomycin (Gibco), 10 mM HEPES (Hyclone, Logan, UT), 2 mM L-glutamine (Gibco), and 55 nM 2-mercaptoethanol (Sigma)], and 4 × 10
6 cells were cultured in 4 mls of R10 with 20 ng/ml of mouse recombinant GM-CSF (PeproTech, Rocky Hill, NJ) at 37 °C, 5% CO
2 for 4 days. On days 4 and 6, used media was removed, the non-adherent cells harvested, and returned to culture with adherent cells in 4 mls R10 media supplemented with 20 ng/ml GM-CSF. On day 8, media was removed, cells washed and cultured in either R10 media alone or supplemented with 40 ng/ml recombinant mouse GM-CSF. On day 10, half of the BMDCs cultured in R10 media alone or R10 supplement with GM-CSF were stimulated with 30 μg/ml recombinant N-α-Syn [
18] and cultured for 6 h for RNA isolation or 24 h for flow cytometric analysis of cells and supernatants for cytokine and chemokines. Thus, groups include GM-CSF-induced BMDCs further treated with 1) R10 media, 2) 20 ng/ml GM-CSF in R10 media, 3) R10 media followed by stimulation with 30 μg/ml N-α-Syn, and 4) 20 ng/ml GM-CSF in R10 media followed by stimulation with 30 μg/ml N-α-Syn. The timing of GM-CSF culture and N-α-Syn stimulation was chosen to affect maximal immune responses (data not shown).
Isolation RNA, cDNA conversion and PCR arrays
Six hours after N-α-Syn stimulation, BMDCs were harvested, washed, and RNA isolated using the Rneasy mini kit (Qiagen, Germantown, MD) by the manufacturer’s protocol. RNA concentration was determined by UV spectroscopy at 260 nm and 280 nm (ND-100 Nanodrop spectrophotometer, Thermo Scientific, Waltham, MA). Five hundred nanograms of RNA was converted to cDNA using the RevertAID first strand cDNA synthesis kit (Thermo Scientific) following the manufacturer’s protocol. cDNA was added to molecular grade water (Invitrogen, Carlsbad, CA) and 2× RT2 SYBR green mastermix (Qiagen), and 25 μl of the mixture was added to each well of a Mouse Inflammatory Response and Autoimmunity array (PAMM-077ZA). PCR was performed in Eppendorf Realplex 2S Mastercycler starting at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. After 40 cycles, melting curve analysis was performed. The Ct values were determined and the ΔΔCt method was used to determine fold changes relative to cDNA derived from media cultured, unstimulated BMDCs samples using RT2 Profiler PCR array data analysis version 3.5.
Flow cytometry
Twenty-four hours after culture with GM-CSF and/or N-α-Syn stimulation, BMDCs were detached by scraping, washed, and resuspended in 10 μg/ml rat gamma globulin in flow staining buffer (FSB) (0.5% bovine serum albumin (BSA) and 0.1% sodium azide in DPBS) for 40–60 min on ice to block Fc receptors. BMDCs were stained with the following mixture of AlexaFluor 488-anti-CD11c, PECy7-anti-CD11b, PE-anti-Jagged-1, APC-anti-OX40L, AlexaFluor 700-anti-MHC II, eFluor 450-anti-CD86, eFluor 710-anti-CD39 (eBioscience, San Diego, CA) and APC-Vio 770-anti-CD73 (Miltenyi Biotec, Auburn, CA) for 30 min at 4 °C. Cells were washed two times in FSB and were fixed with 1% formaldehyde in DPBS. Samples were analyzed with a BD LSR II flow cytometer and FACSDiva software (BD biosciences, San Jose, CA) at the University of Nebraska Medical Center Flow Cytometry Research Facility. From the single cell-gated population, the percentages of positive for CD11c and CD11b were determined by drawing a gate that comprised 98% of the isotype control as negative. The geometric mean fluorescent intensity (MFI) of each surface marker was determined for CD11c+ cells.
Luminex array
After 24 h of culture with GM-CSF and/or N-α-Syn stimulation of BMDCs, the supernatant was removed and clarified by centrifugation at 10,000 xg for 5 min. Cytokine and chemokine concentrations were determined by Luminex xMAP Mouse cytokine and chemokine magnetic bead kit (Millipore, Billerica, MA) according to the manufacturer’s protocol. Briefly, 25 μl of supernatant from each of the 7 replicates for all treatment groups was added to a 96-well plate in duplicate or triplicate. To each sample or standard was added 25 μl containing antibodies to IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, Lix, IL-15, IL-17, IP-10, MIP-2, MIG, RANTES, and TNF-α and incubated at 4 °C overnight. The plates were washed two times, detection antibodies added, incubated at room temperature (RT) for 60 min, streptavidin-PE was added, and incubated at RT for 30 min. The plate was washed two more times and analyzed with the Millipore Magpix system with Luminex Xponent 4.2 software. The concentration for each protein was determined from a standard curve.
Isolation of CD4+ cells and co-culture
For BMDC and CD4+ T cell co-culture, GM-CSF-induced BMDCs were harvested after culture in R10 media alone or R10 media supplemented with GM-CSF, unstimulated, or stimulated with 30 μg/ml N-α-Syn. CD4+ cells (93% CD4+CD25-Foxp3- and 2% CD4+CD25hiFoxp3+) were isolated from spleens of C57BL/6J mice using the Miltenyi CD4+ negative selection kit (Miltenyi Biotech). Into the wells of a 24-well plate, 2 × 106 CD4+ cells and 1 × 106 BMDCs were combined and incubated at 37 °C with 5% CO2 for 5 days. Non-adherent cells were removed and stained with PECy7-anti-CD4 and PE-anti-CD25 (eBioscience) and cells were permeabilized and fixed using the eBioscience Foxp3/transcription factor staining buffer set according to the manufacturer’s protocol for 1 h prior to staining with APC-anti-Foxp3(eBioscience). Cells were analyzed with a LSR II flow cytometer (BD). Using FACSDIVA software (BD), single cells were gated to include CD4+ T cells, quadrants were set to include 98% of the isotype control, and the percentages of CD25hiFoxp3+ Tregs were determined.
Treg functional assay
The ability of Tregs to suppress the proliferation of CFSE-labelled CD4+CD25- T responder cells (Tresps) was performed as described previously [
13,
37]. Briefly, CD4+CD25- and CD4+CD25+ T cells were isolated using the Treg isolation kit (Miltenyi) from mouse spleens and from non-adherent cells after 5 days of co-culture of media-cultured, unstimulated BMDCs and CD4+ T cells. Splenic CD4+CD25- Tresps were labelled with CFSE (CellTrace Cell Proliferation Kit, Thermo Fisher, Waltham, MA) according to the manufacturer’s protocol. In a 96-well U bottom plate, 50,000 CD4+CD25+ Tregs were diluted by serial 2-fold dilutions. To each well was added 50,000 CFSE-labelled CD4+CD25- Tresps, to yield Tresp:Treg ratios of 1:1, 1:0.5, 1:0.25, and 1:0.125. Each well received 50,000 Dynabeads mouse transactivator CD3/CD28 beads (Gibco) and the 96-well plate was incubated at 37 °C, 5% CO
2 for 3 days. The cells were fixed by removing half the media and adding in 1% formaldehyde in DPBS prior to flow cytometric analysis using a LSR II flow cytometer and FACSDIVA software (BD).
MPTP and adoptive transfer
Media-cultured, unstimulated tolerogenic BMDCs were prepared by culture of BMCs in 20 ng/ml GM-CSF for 8 days followed by 3 days of culture in R10 media alone. To male C57BL/6J mice, 1.5 × 10
6 BMDCs in 250 μl of DPBS was injected intravenously (i.v.) into the tail vein at two and one week prior to MPTP intoxication. Mice were treated with 4 doses of either 10 ml/kg DPBS (Gibco) or 16 mg free base MPTP (MPTP-HCl, Sigma-Aldrich, St. Louis, MO)/kg with each dose administered subcutaneously (s.c.) every 2 h. MPTP safety and handling protocols were followed [
38].
Perfusion and immunohistochemistry for Mac-1 and tyrosine hydroxylase (TH)
Mice were sacrificed 2 days post MPTP intoxication to assess neuroinflammation and 7 days post MPTP to assess neuronal survival [
35,
37]. Briefly, mice were terminally anesthetized with pentobarbital (Vortech, Dearborn, MI), transcardially perfused with DPBS, and fixed with 4% paraformaldehyde/DPBS. Brains were removed, post-fixed for 24 h in 4% paraformaldehyde, cryoprotected for 2 days in 30% sucrose/DPBS, and snap-frozen. Tissues were cryosectioned at 30 μm sections through the striatum and midbrain containing the substantia nigra and processed for immunohistochemistry [
35,
37]. Free-floating sections were blocked of endogenous peroxidases with 3% hydrogen peroxide and non-specific activity in 5% normal goat serum (Vector Laboratories, Burlingame, CA). Blocked sections were reacted with anti-TH (EMD/Milipore, Burlington, MA) at 1:2000 dilution for striatal sections and 1:1000 dilution for substantia nigra sections. After 48 h, sections were washed and reacted with 1:400 dilution of goat-anti-rabbit secondary antibody (Vector Laboratories) followed by ABC biotin-avidin peroxidase solution (Vector Laboratories) prior to color generation with 3,3′-diaminobenzidine (DAB, Sigma). Sections containing substantia nigra were counter stained for Nissl substance. For Mac-1 staining, sections were incubated with a 1:500 dilution of anti-Mac-1 primary antibody (Bio-Rad, Hercules, CA) overnight. Sections were washed and reacted with 1:500 dilution of biotinylated rabbit anti-rat antibody (Vector laboratories) followed ABC biotin-avidin peroxidase solution (Vector Laboratories) prior to color generation with DAB. Numbers of dopaminergic neurons (TH+Nissl+), non-dopaminergic neurons (TH-Nissl+), and reactive microglia (amoeboid Mac-1+) were estimated by stereological analyses analysis using the optical fractionator module of StereoInvestigator (MBF Bioscience, Williston, VT) [
35,
37]. Densities of TH+ striatal termini were determined using digital densitometry using ImageJ as described [
35,
37].
Isolation of RNA from midbrain for PCR array
Two days after MPTP intoxication, mice were sacrificed, brains quickly removed, hemisected, midbrain dissected, placed in RNAlater (Thermo Fisher), tissues weighed, and flash frozen at − 80 °C. To isolate RNA, midbrains were homogenized in 350 μl β-mercaptoethanol-supplemented RLT buffer (Qiagen) for every 30 mg tissue. Tissues were sequentially drawn up and down through 18, 20 and 27 Ga needles. RNA was isolated, converted to cDNA and assessed using Mouse Inflammatory Response and Autoimmunity PCR arrays as described.
Statistics
Statistics were performed using Prism GraphPad version 6. Means and SEM were determined for release of cytokines from BMDCs, relative changes in mean fluorescent intensity (MFI) in the flow markers on BMDCs, Treg frequency in the CD4+ population, and Mac-1+ microglia. For TH+ neuron counts and striatal densities, means and SEM were determined for values within the 99% confidence interval. For all analyses, one-way ANOVAs were performed followed by the appropriate post-hoc test adjusted for multiple comparisons. A p value less than or equal to 0.05 was selected as significant.
Discussion
We and others showed that GM-CSF increases Treg numbers and function [
28,
30,
35]. In models of neurodegeneration such as PD, Alzheimer’s disease (AD), traumatic brain injury (TBI), and stroke, administration of GM-CSF has been shown to be neuroprotective [
36,
52]. Additionally, adoptive transfer of Tregs from naïve or GM-CSF treated donors is neuroprotective [
35]. In a recent phase 1 clinical trial, treatment of PD patients with human recombinant GM-CSF (sargramostim) increased Treg numbers and Treg-mediated suppression of CD3/CD28-induced proliferation compared to placebo-treated PD patients [
36]. Moreover, sargramostim improved motor deficits and initiation of motor activity as determined by diminished UPDRS, III scores and β-ERD signals by magnetoencephalography. These data underscore the potential for immune transformation by GM-CSF to diminish neurodegeneration in PD.
As T cells express few, if any, GM-CSF receptors, intermediaries are needed to affect Treg function. Several possible intermediary cell types include dendritic cells (DCs), macrophages, microglia, astrocytes, or myeloid-derived suppressor cells [
53‐
57]. Based on recent data supporting the involvement of tolerogenic DCs in regulation of Treg activity, we chose to first examine the effects of GM-CSF on DCs to combat neuroinflammation and protect dopaminergic neurons. We also assessed the effects on DC-induced Treg formation by proteins that are oxidatively-modified and/or misfolded in PD, such as N-α-Syn.
First, bone marrow cells propagated in GM-CSF produce CD11b+CD11c+ cells with low expression of MHC II and CD86. The relative lack of cytokines and chemokines and relatively high levels of CD39 and CD73 associated with purinergic signaling that is important for switching immune cells to anti-inflammatory phenotypes, suggested these cells were of an immature DC phenotype and putatively tolerogenic capable of inducing Tregs [
41]. To test that possibility, we found that co-culture of BMDCs with CD4+ T cells induced T cells with a CD4+CD25
hiFoxp3+ Treg phenotype. Additionally, those phenotypic Tregs were shown to have Treg function as they inhibited CD3/CD28-induced proliferation of CD4+CD25- Tresp co-cultures in a dose-dependent fashion. Moreover, the function of BMDC-induced Tregs was significantly greater than that observed from fresh splenic isolates, and supported the tolerogenic capacity of the BMDCs. While GM-CSF can yield a heterogeneous mixture of myeloid cells, including BMDCs and macrophages, the tolerogenic capability to induce Tregs has not been ascribed to GM-CSF-generated macrophages [
58,
59], thus the BMDCs described herein are congruent with prior results that demonstrate Treg-inducing capacity by tolerogenic DCs [
31,
60]. Notably, tolerogenic BMDCs further exposed to media, GM-CSF, N-α-Syn, or both GM-CSF and N-α-Syn were capable of inducing significant increases in Treg numbers compared to starting isolates of primary CD4+CD25- T cells or those stimulated via CD3/CD28.
In clinical trials of GM-CSF in PD, Crohn’s Disease, or AD [
36,
61,
62] (NCT01409915), treatment regimens use extended or chronic GM-CSF administration to maintain selective pressure on the anti-inflammatory response or Treg induction and function. In our model of extended exposure and/or proinflammatory stimulation, tolerogenic BMDCs were cultured in the presence of media or GM-CSF alone and remained unstimulated or were stimulated with N-α-Syn, an acute proinflammatory stimulus. Compared to media alone, continued culture in GM-CSF yielded few significant effects on surface expression of co-stimulatory molecules or expression of cytokine and chemokine genes and proteins. The few effects included increased expression of Jag-1, IL-10, an anti-inflammatory cytokine released from tolerogenic DCs [
31,
39,
40], and IL-9 as well as decreased production of MIG (CXCL9) and MIP2 (CXCL2), suggesting that continued exposure to GM-CSF may maintain the tolerogenic state, particularly in light of increased Jag-1 which has been shown necessary for Treg induction [
33,
34,
63]. However, the tolerogenic capacity for inducing Tregs was significantly diminished by 45% indicating that continued exposure to GM-CSF may eventually lessen the tolerogenic capacity and reduce Treg production.
N-α-Syn is an oxidatively modified and readily misfolded protein found in neuronal inclusions and extraneuronal environments of the brain in patients with PD, Lewy body disease, and multiple system atrophy [
64,
65], and is also detected in the cervical lymph nodes by 20 h after MPTP intoxication [
18]. Misfolded N-α-Syn is known to activate microglia as well as other myeloid and APCs of the innate immune system [
18,
66]. While the function of native α-Syn remains largely unknown, modified or misfolded α-Syn can serve as a neoantigen. Thus, N-α-Syn activation of APCs that process and present modified epitopes of α-Syn and transmit proinflammatory signals during T cell activation, would support breaking or evasion of immunological tolerance leading to induction of effector T cells and antibody responses to the modified self-protein [
18,
66]. Further, tolerogenic signals with or without modified α-Syn not only play a role in Treg induction, but also may influence the emergence of M1/M2 microglia phenotypes [
67].
To assess the effect of N-α-Syn on phenotype and function, tolerogenic BMDCs were stimulated with N-α-Syn after culture in media or GM-CSF. Stimulation with N-α-Syn showed significant responses as determined by phenotype and expression of cytokines and chemokines regardless of extended culture in media or GM-CSF. Compared to non-stimulated BMDCs, stimulation with N-α-Syn increased surface expression of MHC II, CD86, and Jag-1 co-stimulatory molecules and genes for the maturation markers Cd40, Cebpb, and Ccr7. Additionally, proinflammatory gene expression and protein secretion were increased for IL-1β, IL-23α, IL-6, IFN-γ, TNF-α, IP-10 (CXCL10), LIX (CXCL5), MIG (CXCL9), MIP2 (CXCL2), and RANTES (CCL5). Together these data show that N-α-Syn stimulation drives BMDC pathways to a proinflammatory profile and suggests that differentiation might favor production of mature type 1 DCs that preferentially lead to induction of effector T cells, such as Th1 or Th17. Unexpectedly, co-culture of N-α-Syn-stimulated BMDCs with CD4+ T cells induced levels of Tregs that approached those attained upon culture in media alone, and significantly greater than those after culture GM-CSF alone. The possibility exists that acute or intense inflammatory responses may provide yet another signal or mechanism by which tolerogenic BMDCs induce conversion of conventional T cells to Tregs despite an otherwise proinflammatory environment.
Continued culture of BMDCs with GM-CSF followed by N-α-Syn stimulation diminished several phenotypic and functional responses, but not others. Of note, surface expression of CD86 and MHC II were diminished, but Jag-1 was increased to the highest levels of all culture conditions. Expression of proinflammatory cytokine and chemokine genes by BMDCs were overall elevated compared to culture in media alone, though were slightly diminished compared to those stimulated with N-α-Syn alone. More importantly, production and secretion of proinflammatory proteins were significantly increased over levels induced by N-α-Syn alone included IL-1α, IL-1β, IL-2, IL-5, IL-9, IL-12p70, and IL-17, but decreased LIX (CXCL5), MIG (CXCL9). Few anti-inflammatory mediators, such as IL-10, were increased. Together, these data suggest that BMDCs in the presence of GM-CSF and N-α-Syn induce an overall proinflammatory environment and may not be conducive for Treg development. Indeed, co-culture of CD4+ T cells with BMDCs previously treated with GM-CSF and stimulated with N-α-Syn, proved to induce the least number of Tregs with the exception of stimulating with anti-CD3/CD28 beads alone.
Overall, GM-CSF-induced BMDCs acquire an immature phenotype and exhibit a high degree of tolerogenic activity based on the ability to induce Tregs. That activity is maintained even after stimulation with a proinflammatory stimulus such as N-α-Syn and an elevated proinflammatory state. However, continued exposure to GM-CSF and possibly other mediators, result in a semi-mature state with diminished tolerogenic capability regardless of stimulation status. Both semi-mature and tolerogenic DCs have the ability to support transformation of CD4+ T cells to become Tregs [
53], but at different levels depending on the state of activation. Indeed, while Treg induction capacity was least robust with semi-mature BMDCs produced by culture with GM-CSF and stimulated with N-α-Syn, levels of Tregs inducted were significantly greater than those within the initial isolation of CD4+ T cells or after CD3/CD28 stimulation. Previous work demonstrated that Jag-1 and OX40L are necessary for BMDC induction of Tregs [
33]. However, under the conditions tested, no changes in the surface expression of OX40L were detected. This would suggest that expression of OX40L at the reported levels is sufficient to support Treg induction. Continued culture of tolerogenic BMDCs in GM-CSF increases surface expression of Jag-1, but diminishes their capacity to induce Tregs. This seemingly contrasts reports that Treg induction is dependent on Jag-1 dose and Notch interactions as determined by blocking antibodies or notch-signaling inhibitors [
33]. Our data indicate that increases in Jag-1 surface expression were not sufficient to promote additional Treg induction, but were indirectly correlated (Pearson
r = 0.55,
p = 0.06, F = 4.42, DFn = 1.0, DFd = 10.0). This suggests that i) Jag-1 expression, albeit at low levels, is sufficient to promote Treg induction, ii) Jag-1 expression beyond a specific threshold provides negative signaling to inhibit Treg induction, or iii) other BMDC cell surface or internal signaling interactions are involved.
In PD models, administration of GM-CSF increases Treg number and function, attenuates inflammation in the brain, and protects dopaminergic neurons along the nigrostriatal axis [
35,
51]. In light of our results showing Treg induction mediated by BMDCs, we tested whether tolerogenic BMDCs could also serve as another mechanism to provide protection from MPTP-mediated neurodegeneration. Indeed, adoptive transfer of GM-CSF-induced BMDCs prior to MPTP intoxication attenuated neuroinflammation and protected dopaminergic neurons along the nigrostriatal axis. Most interestingly, gene expression of proinflammatory mediators in the midbrain were overall increased compared to those from midbrains of mice treated with MPTP alone. These data suggested that adoptive transfer of BMDCs alter the chemokine environment, which in turn may change immune cell recruitment in brain. This is consistent with changes in chemokine gene expression in the substantia nigra of GM-CSF-pretreated mice [
35]. However, gene changes also are indicative of decreased inflammation with down-regulation of
Il1r1, the gene encoding the receptor for IL-1α and IL-1β which transduces signals intracellularly [
68],
Cebpb, a transcription factor which induces TNF expression [
69], and chemokine genes
Ccl3l3 and
Ccl4, which promote inflammation [
70] and leukocyte recruitment into the brain [
71].
Ccl24 encodes a chemokine released from M2 macrophages which can recruit T cells, especially Tregs [
72,
73]. In glioblastoma multiforme,
Ccl2 is important for Treg recruitment into the brain and may perform a similar function after MPTP intoxication [
74,
75].
Il1rn prevents the recruitment of IL-1 receptor associated protein to IL-1 receptor which is required for transduction of signals within the cell [
76], therefore its increased expression, combined with downregulation of
Il1r1 would diminish IL-1α and IL-1β signaling. Together with results showing diminished reactive microglia in Fig.
6, further support the notion that BMDCs diminish neuroinflammation.
As GM-CSF increases Treg numbers [
35], we assessed the ability of tolerogenic BMDCs to induce Tregs after adoptive transfer. Unexpectedly, in naïve mice, Treg frequencies diminished after transfer. The causes for these results remain unclear, but may be in part due to limited numbers of BMDCs (1.5 × 10
6) that could be safely transferred. Another possibility is that without inflammatory insult, induction of Tregs may be inactive or suppressed. Our results suggested that BMDCs may not function by the same mechanism(s) as GM-CSF or that GM-CSF has effects independent of DC-mediated Treg induction. The possibility that GM-CSF or BMDCs are protective apart from the induction of Tregs is also suggested. Indeed, GM-CSF can directly promote the survival of PC12 neurons and primary neurons from MPP+ neurotoxicity [
51] and could increase other regulatory immune cells such as myeloid-derived suppressor cells (MDSCs) [
56]. Interestingly, the possibility that endogenous induction of Tregs by BMDCs within the bone marrow cannot be ruled out as a small population (< 0.5%) of CD4+CD25+Foxp3+ Tregs constitute the bone marrow stem cell population which was shown to be neuroprotective in a rat model of experimental stroke [
52]. Such alternative mechanisms should be examined in future studies. Because the adoptive transfer of BMDCs was protective in the MPTP model, it may be possible that the adoptive transfer of tolerogenic bone marrow-derived or monocyte-derived dendritic cells may promote Treg induction and decrease PD-associated motor symptoms. It may be possible to induce, expand or prime autologous DCs ex vivo for adoptive transfer as a personalized therapeutic modality to elicit clinical benefits.