Dopaminergic Receptors as Neuroimmune Mediators in Experimental Autoimmune Encephalomyelitis

The dopaminergic system plays an essential role in maintaining homeostasis between the central nervous system (CNS) and the immune system. Previous studies have associated imbalances in the dopaminergic system to the pathogenesis of multiple sclerosis (MS). Here, we examined the protein levels of dopaminergic receptors (D1R and D2R) in different phases of the experimental autoimmune encephalomyelitis (EAE) model. We also investigated if the treatment with pramipexole (PPX)—a dopamine D2/D3 receptor-preferring agonist—would be able to prevent EAE-induced motor and mood dysfunction, as well as its underlying mechanisms of action. We report that D2R immunocontent is upregulated in the spinal cord of EAE mice 14 days post-induction. Moreover, D1R and D2R immunocontents in lymph nodes and the oxidative damage in the spinal cord and striatum of EAE animals were significantly increased during the chronic phase. Also, during the pre-symptomatic phase, axonal damage in the spinal cord of EAE mice could already be found. Surprisingly, therapeutic treatment with PPX failed to inhibit the progression of EAE. Of note, PPX treatment inhibited EAE-induced depressive-like while failed to inhibit anhedonic-like behaviors. We observed that PPX treatment downregulated IL-1β levels and increased BNDF content in the spinal cord after EAE induction. Herein, we show that a D2/D3 receptor-preferred agonist mitigated EAE-induced depressive-like behavior, which could serve as a new possibility for further clinical trials on treating depressive symptoms in MS patients. Thus, we infer that D2R participates in the crosstalk between CNS and immune system during autoimmune and neuroinflammatory response induced by EAE, mainly in the acute and chronic phase of the disease.


Introduction
Multiple sclerosis (MS) is an autoimmune and chronic disease of the central nervous system (CNS), recognized as the most common neuroinflammatory cause of nontraumatic neurological disability arising in young adults between age of 20 and 40 years [1,2]. Indeed, MS affects more than 2.5 million worldwide, mainly women (ratio of 3:1 over men) [2,3]. It is a neurodegenerative disorder characterized by chronic demyelination with concomitant oligodendrocyte death, axonal, and neuronal loss [4,5]. Although it is an idiopathic disease, MS seems to result from a complex and dynamic interaction between deregulation of immune homeostasis, genetic predisposition, and environmental factors. Possible risk factors for the disease development include infections, especially Epstein-Barr virus (EBV) [6], lower levels of vitamin D [7], smoking [8], childhood obesity [9], and variations in the HLA-DRB1 gene [10].
The pathophysiology of MS possibly comprises crosstalk between the innate and adaptive immune system, which allows the activation of potentially autoreactive CD4 + T lymphocytes in the periphery against protein constituents within the myelin sheath [11,12]. Immune cells-mainly Th1 and Th17-migrate across the blood-brain barrier (BBB), triggering the emergence of demyelinating plaques within the white and gray matter of the CNS, which consequently harms synaptic transmission [13][14][15]. Clinical manifestations comprise visual and sensory disturbances, urinary and intestinal system disorders, fatigue, weakness, ataxia, and cognitive deficits, which could be related to neurotransmitters imbalance, such as dopamine [16].
Dopamine is a monoaminergic neurotransmitter [17] produced in the substantia nigra, ventral tegmental area, and hypothalamus of the brain [18] with an essential role in the modulation of sexual behavior, memory, learning, voluntary movement [19,20], motivation, and reward [21]. Besides its role as a neurotransmitter, DA also exerts multiple functions in peripheral tissues [22]. It can mediate the crosstalk between CNS and immune system, since immune cells can synthesize and utilize dopamine as an autocrine/paracrine transmitter, which gives it an immunomodulatory character [23,24].
Previous findings reported that preventive treatment with bromocriptine, a D2-dopamine receptor agonist, via subcutaneous pellet implantation significantly reduced the severity of experimental allergic encephalomyelitis (EAE) in rats [25]. However, an open pilot study conducted in 18 diagnosed patients with MS demonstrated that treatment with bromocriptine (2.5 mg/day for one year) was ineffective in inhibiting disease progression in humans [26]. On the other hand, Lieberknecht and colleagues showed that the preventive treatment of C57BL/6 mice with pramipexole (PPX) (D2-like (D2/3) receptor agonist) administered at 1 mg/kg, i.p., day 0-40 post-immunization (p.i.) abolished the development of EAE. Moreover, the effects of dopamine on T cells, for example, are pretty dynamic since they are subjected to the immune activation status, the local levels of this neurotransmitter, as well as they can express different types of DA receptors [19]. In this way, the dopaminergic signaling could vary among pro-or anti-inflammatory pathways [27,28]. Therefore, due to conflictual literature about dopaminergic receptor-mediated signaling during the autoimmune response in EAE and MS, we investigated the role of DA receptors, mainly D1R and D2R, during different phases of EAE development in C57BL/6 mice. Furthermore, we also performed additional experiments to characterize the oxidative stress levels and the axonal damage in different phases of EAE development.

Animals
Experiments were performed using female C57BL/6 mice (20-35 g, 6-10 weeks old; n total = 145 animals) at the Laboratory of Autoimmunity and Immunopharmacology, obtained from the Universidade Federal de Santa Catarina. Mice were kept in groups of four to six animals per cage, maintained under controlled temperature (22 ± 2 °C) with a 12 h light/dark cycle (lights on at 07:00 a.m.), and food and water ad libitum. Animals were acclimatized to laboratory settings for at least 1 h before testing and were used only once throughout the experiments. Behavioral experiments were performed during the light cycle of the day (07:00 a.m.-05:00 p.m.) in a soundproof room. All behavioral data were measured manually, and the observer was fully blinded to the experimental protocol for all tests. Mice were randomly assigned before treatment or behavioral evaluation, and to avoid cage-effect, animals were not co-housed. All experimental procedures in this study were strictly performed with relevant ethical regulations, including the National Institute of Health Guide for the Care and Use of Laboratory Animals [29], and were approved by the Animal Ethics Committee of the Universidade Federal de Santa Catarina (CEUA-UFSC, protocol number: 3914220319).

EAE Induction
Female C57BL/6 mice were immunized subcutaneously in both flanks with 200 µg of myelin oligodendrocyte glycoprotein peptide (MOG  ) (EZBiolab, NJ, USA) emulsified in Incomplete Freud's Adjuvant (Sigma Chemicals, St. Louis, MO, USA), which was supplemented with 500 μg of Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA). On days 0 and 2, 300 ng of Pertussis toxin was administered intraperitoneally (i.p.). At the end of the experiment, animals were euthanized by decapitation followed by spinal cord and striatum removal. The tissues were snap-frozen or fixated in a 4% formalin solution for posterior analysis.

Forced Swimming Test and Sucrose Preference Test
The forced swimming test (FST) was used to evaluate depressive-like behavior in rodents. The animals were placed into an inescapable transparent water tank while the time spent in an escape-related mobility behavior was registered [31]. Another way to assess the depressive-like behavior was the sucrose preference test (SPT), a reward-based test used as an indicator of anhedonia that consisted of giving two bottles of choice, one containing a sucrose solution (200 mL, 1%) and another one containing filtered tap water. First, each animal habituated to be housed individually and had access to ad libitum to two bottles containing only tap water for 48 h. Then, one of the bottles was changed for a 1% sucrose solution for 24 h [32], and to avoid a place preference behavior, the bottles were swapped after the initial 12 h. The bottles were weighed before and after the test to determine the amount of solution consumed, and the consumption was calculated as the total amount of liquid intake [33]. This behavioral method was adapted from Pazini et al. [33]. The animals were evaluated to FST and SPT at different time points-11-and 12-day p.i. (during the pre-motor phase of EAE) since after this day, the clinical signs of EAE, such as locomotor deficit, were visible [34].

Bielschowsky's Silver Staining
The spinal cord samples were extracted, weighed, and stored in a buffered fixing solution (PBS/Formalin 4%) at 4 °C to perform morphometric analyses. After this process, they were removed from the fixative solution, washed in deionized water for 30 min, progressively dehydrated in an increasing series of alcohols, diaphanized with xylol for 30 min (3x), bathed in paraffin at 56 °C for 30 min (3x), and included in paraffin blocks. Subsequently, the samples were cut into 10-μm thick sections in a microtome. The sections obtained were mounted on slides previously treated with 1% gelatin solution and remained for 24 h in an oven at 56 °C and, posteriorly dewaxed and hydrated in deionized water, 3 times, for 3 min. The sections were placed in ammoniacal silver in the dark at 37 °C for 20 min, keeping the solution for later use, and washed again in deionized water, 3 times, for 2 min. Ammonium hydroxide (28-30%) was added dropwise to the silver nitrate solution (20%) while stirring sufficiently to dissolve the precipitated content. Then, another 2 drops of ammonium hydroxide solution (28-30%) were added for effective silver solubilization. Working solution (0.2 mL of 37% formaldehyde, 12 mL of distilled water, 12.5 μL of 20% nitric acid, and 0.05 g of citric acid) was added to the silver hydroxide solution, and the samples remained in this solution for 10 min until the development of black color. After this, the samples were washed with 0.1% ammonium hydroxide solution, 3 times, for 2 min, and then with distilled water 3 times for 2 min. In the next step, the samples were conditioned in auric chloride solution for 15 min, fixed in sodium thiosulfate, washed in distilled water for later dehydration in alcohol and diaphanization with xylene, cleaned, and mounted in assembly medium. The slides were analyzed using an Olympus® AX 70 light microscope using × 40, × 100, and × 200 magnifications with a Hitachi VK-C150 video camera attached to the microscope to capture the images [35].

Enzyme-Linked Immunosorbent Assay
Spinal cord was collected at the end of experiment and homogenized in lysis buffer (0.05% Tween 20, 0.1 mM benzethonium chloride, 10 mM EDTA, 0.5% bovine serum albumin, 0.4 M NaCl, 0.1 mM PMSF, and 2.0 μg/mL aprotinin). Homogenates were centrifuged at 3000 g, at 4 °C, for 10 min, and the supernatant was collected and stored at − 80 °C until use. The levels of interleukin-1β were evaluated using an ELISA kit from R&D Systems (Minneapolis, MN, USA) and performed according to the manufacturer's protocol.

Statistical Analysis
Data were analyzed using GraphPad Prism 8.2.1 software (GraphPad Software Inc., USA) and expressed as mean ± standard error of the mean (SEM) of two independent experiments. Statistical analyses were performed utilizing one-way ANOVA, followed by Neuman-Keuls or Bonferroni post hoc test. P < 0.05 was considered significant.

Protein Levels of Dopaminergic Receptors-D1R and D2R-in the Spinal Cord and Peripheral Lymphoid Organs of EAE Mice
The immune and nervous systems are tightly integrated through a bidirectional pathway, which allows neural pathways to regulate peripheral immunity, and conversely, immune mediators to affect neuronal activity [37][38][39][40]. Different immune cells express receptors for neurotransmitters released by neurons such as DA [41], with a pivotal role for neuroimmune communication [42]. First, we investigated the immunocontent of dopaminergic receptors in the peripheral lymphoid organs of EAE mice-where T and B cells are initially activated upon development (i.e., 30-day p.i.) [43]. Herein, we observed a significant increase in the protein levels of both receptors-D1R and D2R-in the lymph nodes of EAE mice when compared to the naïve group (p < 0.05; Fig. 1A, B). The immunocontent of dopaminergic receptors in the spinal cord of mice exposed to EAE was also evaluated-since the autoreactive T cells begin infiltrating the CNS in the lumbar region-in different phases of the disease development, such as (i) induction (up to day 7 p.i.); (ii) acute (between days 7 and 14 p.i.); and (iii) chronic (from day 30 p.i. upwards) [43,44]. Our results showed no statistically significant difference in the protein levels of D1R in the spinal cord of EAE mice versus naïve animals in the different phases of the experimental model ( Fig. 2A). However, during the acute phase, the spinal cord of immunized mice showed a significant increase in the immunocontent of D2R when compared to the control group (p < 0.05; Fig. 2B).

Oxidative Parameters and Axonal Degeneration in Central Tissues During Induction, Acute, and Chronic Phase of EAE
The spinal cord has extensive projections of dopaminergic neurons involved in the modulation of sensory, motor, and autonomic functions [45]. Previous reports demonstrated that excess DA levels-not degraded through catabolic enzymatic pathways-can induce neuronal death mainly due to ROS generation in the auto-oxidation of L-DOPA and DA [46,47]. In this set of experiments, we characterized the levels of oxidative stress and the activity of the antioxidant system 7, 15, and 40 days after EAE induction. Our data showed that GPx levels were significantly increased in the spinal cord of EAE mice 7-day p.i. compared to naïve animals (p < 0.01; Fig. 3A). In contrast, GPx was significantly downregulated 40 days after EAE induction (chronic phase) (p < 0.01; Fig. 3A). Additionally, the enzymatic activity of GPx was significantly decreased in the spinal cord of EAE mice compared to the naïve group during the acute and chronic phase of the disease (p < 0.01; Fig. 3B).
The striatum integrates motor behavior, cognition, emotion, and limbic information processing [48,49]. According to Gentile and colleagues, the striatum of EAE mice showed impairment of DA neurotransmission characterized by an imbalance in dopaminergic receptors signaling [50]. For this reason, we also evaluated some parameters that are indicative of oxidative stress in the striatum, such as ROS formation and the antioxidant activity of GPx. Our data demonstrated that the levels of GPx in the striatum of EAE mice were significantly increased 7-and 14-day p.i. when compared to the control group (naïve) (p < 0.01; Fig. 3D). Surprisingly, during the chronic phase of the disease (40day p.i.), the striatum of the EAE group showed a significant decrease in the levels of GPx compared to naïve animals (p < 0.01; Fig. 3D), as well as the enzymatic activity (p < 0.01; Fig. 3E). Next, we performed DCF fluorescence to measure ROS in both tissues. Our data demonstrated a significant reduction in ROS levels in the spinal cord of EAE animals 7-day p.i. (p < 0.01; Fig. 3C). However, 15and 40-day p.i., EAE animals had an increase in the production of ROS in the spinal cord when compared to the naïve group (p < 0.05) (Fig. 3C). Meanwhile, in the striatum of EAE mice, we identified a significant increase in the levels of ROS compared to the control group (naïve) 40-day p.i. (chronic phase of EAE) (p < 0.01; Fig. 3F).
In order to characterize the axonal damage during the development of EAE, the results of Fig. 3 illustrate that EAE D2R representative immunoelectrophoresis with densitometry. EAE upregulated immunocontent of D2R in the spinal cord of mice during the acute phase of the disease (14-day p.i.). Each column represents the mean ± SEM of 5 animals/group. Asterisks indicate the levels of significance compared to the naive group. *p < 0.05 using one-way ANOVA followed by Bonferroni's post hoc test induced significant axonal damage from 7-day p.i. (p < 0.05), with greater damage after 30 days p.i. (p < 0.01) when compared to the naïve control group (Fig. 3G, H).

Effect of Treatment with PPX, Dopaminergic D3/D2R Agonist, on the Progression of EAE
As aforementioned, there is still no consensus in the literature about the effects of dopaminergic agonist drugs in treating MS and EAE [25,26,30]. Lieberknecht and colleagues provided evidence about the beneficial effects of PPX (administered from day 0 to 40 p.i.) in preventing EAE-induced motor symptoms [30]. In this particular set of experiments, we assessed the effect of PPX treatment in EAE-induced mice from day 15 to 40 p.i. Figure 4 illustrates that the treatment with PPX failed to inhibit the progression of EAE (Fig. 4A), confirmed by analyzing the area under the curve (AUC) (Fig. 4B). Furthermore, the animals treated with PPX had significantly higher scores when compared to the EAE group, which received Panel F. DCF levels were significantly upregulated in the striatum of EAE mice 40-day p.i. with EAE. Panels G and H. The axonal density in the spinal cord of EAE mice was significantly reduced in the pre-motor phase of disease development (induction phase of EAE, 7-day p.i.). Each column represents the mean ± SEM of 12 animals/ group. Asterisks indicate the levels of significance compared to the naive group. *p < 0.05, **p < 0.01 using one-way ANOVA followed by Newmann Keuls post hoc test vehicle (Fig. 4C). Therefore, our results suggest that PPX therapeutic treatment worsened the severity of EAE motor symptoms.

The Effect of PPX on Depressive-Like Behavior in Mice with EAE
Studies had provided direct evidence about dopaminergic dysfunction in clinical manifestations of MS and EAE, including the occurrence of depressive symptoms [50][51][52]. Considering these findings, we investigated if PPX would modulate the depressive-like and anhedonic-like behavior illustrates that the animals, which were given PPX, showed increased scores compared to the control group EAE. Panel C. This panel confirms that mice that received therapeutic treatment with PPX showed an increase in the severity of EAE-induced motor symptoms compared to the EAE group (untreated). Each column represents the mean ± SEM of 12 animals/group. Asterisks indicate the levels of significance compared to naive group ***p < 0.001; ###p < 0.001 versus EAE-group using one-way ANOVA followed by Newmann Keuls post hoc test Fig. 5 The effect of PPX on depressive like-behavior induced by EAE. Animals were given repeated treatment with pramipexole (PPX) (1 mg/ kg, 1x/day, i.p.)-a dopamine D2/D3 receptor-preferring agonist-from day 0 to 11 and 12 p.i. Panel A. PPX treatment reduced the total immobility time of mice submitted to forced swim test (FST) 11-day p.i. Panel B. PPX treatment did not modulate the EAE-induced anhedonic-like behavior in mice evaluated through the sucrose preference test (SPT) 12-day p.i. Each column represents the mean ± SEM of 12 animals/ group. #p < 0.05 versus EAEgroup using one-way ANOVA followed by Newmann Keuls post hoc test in EAE mice. Mice were given PPX (1 mg/kg, i.p.) from days 0 to 11 and 12 and then subjected to the SPT and FST, respectively. Our results showed that PPX treatment significantly reduced the immobility time in comparison to the untreated EAE group (p < 0.05; Fig. 5A), in the FST. Conversely, no differences between the groups were observed when the mice were submitted to SPT (Fig. 5B).

PPX Modulates the Protein Levels of BDNF and Proinflammatory Cytokine IL-1 ˇ in EAE Mice
Gentile and colleagues reported that IL-1β impairs dopamine neurotransmission in the striatum of EAE mice triggering behavioral changes [50]. Considering that exogenous peripheral administration of IL-1β produces depressivelike symptoms [53,54], we evaluated whether PPX could modulate IL-1β levels in the spinal cord of EAE mice, justifying its antidepressant-like effect observed in the FST (Fig. 5A). Herein, EAE mice showed a significant increase of IL-1β immunocontent when compared to the naïve group (p < 0.001) (Fig. 6A). Meantime, PPX treatment (1 mg/kg, once a day, i.p., during 40 days after EAE induction) significantly reduced the amount of IL-1β in the spinal cord (p < 0.001). According to Qu and co-authors, the severity of the disease can be mitigated by preventing the loss of the brain-derived neurotrophic factor (BDNF)-the most prevalent growth factor in the CNS-in the spinal cord of EAE mice [55]. Considering that preventive PPX administration prevented EAE development, we further assessed whether PPX effectively modulates the levels of BDNF in the spinal cord of EAE mice. Interestingly, 40-day p.i. EAE mice showed downregulation of BDNF protein levels in the spinal cord when compared to the control group (naïve) (p < 0.01), an effect abolished by PPX treatment (p < 0.01; Fig. 6). Total BDNF immunocontent in the spinal cord of EAE mice that received PPX (during 40 days) was upregulated compared to the control group EAE. Each column represents the mean ± SEM of 12 animals/ group. Asterisks indicate the levels of significance compared to naive group **p < 0.01, ***p < 0.001; ##p < 0.01 and ###p < 0.001 versus EAE-group using one-way ANOVA followed by Newmann Keuls post hoc test

Discussion
MS is the most prevalent chronic neuroinflammatory disease of the brain and spinal cord [50]. This study aimed to investigate mechanisms by which peripheral and central inflammatory signals are integrated, mainly dopaminergic receptors' role in different phases of the EAE model. Firstly, after 30-day p.i., the immunocontent of both dopaminergic receptors in the lymph nodes of EAE mice was significantly increased. We considered the phases according to previous studies, which characterized the induction phase-disease onset beginning-(7 days p.i.), acute phase-acute disease peaking-(14 to 15 days p.i.), and chronic phase (30 days p.i.) [44,56]. Of note, no previous literature has described the expression of dopaminergic receptors in tissues (e.g., spinal cord or lymph nodes) of rodents with EAE or patients with MS. In general, previous published studies aimed to assess the expression of dopaminergic receptors in specific cell subtypes (macrophages, dendritic cells, regulatory T cells) [27,28,57,58]. Therefore, it is necessary to consider the methodological discrepancies between the previously published data and the findings of our study. Our results support the findings of a previous study, which demonstrated that peripheral blood monocytes (PBMCs) obtained from MS patients showed an exacerbated level of all dopamine receptors [58]. In order to remember, during the inflammatory response, monocytes and monocyte-derived cells can also enter draining lymph nodes and promote adaptive immune responses [59], a system that mediates the autoimmune response in MS [60]. In accordance, the increased expression of D1R in lymph nodes of EAE mice we are reporting here may support earlier evidence about the proinflammatory potential of this receptor [58,61]. The activation of D5R (belonging to the dopamine D1-like receptor family) expressed on DCs showed to potentiate the immune response by contributing to CD4 + T cell activation and differentiation to Th17 phenotype, in vivo critical players in MS [27,61,62]. Likewise, the activation of D1R/D5R in human Tregs downregulated Treg-dependent inhibition of effector T-lymphocyte (Teff) proliferation, through inhibition of the expression of IL-10 and transformation of Tregs by growth factor-beta (TGF-β) [63]. Consistent with these findings, deficiency of D5R on the surface of DCs impaired LPS-induced IL-23 and IL-12 production, driving the differentiation of naive T cells into Th1 and Th17 cells [58]. Still, as reported by Prado and colleagues, D5R-deficient DCs prophylactically transferred into wild-type mice mitigated the severity of EAE symptoms [61]. However, it is essential to highlight that the D1-like receptor family might play a dual role in EAE. According to Osorio-Barrios and colleagues, D5R-mediated signaling may vary depending on the cell type involved and disease stage [18]. These authors suggested that D5R-signaling in CD4 + T-cells potentiates T-cell activation and exerts a proinflammatory role, favoring EAE development [27]. While others have proposed that D5R-signaling in Treg cells facilitates Treg-mediated suppressive response, in late stages of the disease [18]. In this context, the activation of D5R-signaling in Treg cells increases glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR)-constitutively expressed in Treg cells-inducing Treg expansion. In turn, the activation of GITR strengthens Treg suppressive activity [27]. Considering the abovementioned, the D1R increased levels in the lymph nodes of the EAE group found in the present study-particularly in the chronic phase-could be related to the maintenance of autoimmune response, or even as an attempt of suppressing the immune response via Treg cells activation. Here we suggest that D1R upregulation is associated to inhibited suppressive activity of Treg cells and subsequently, to Th1 and Th17 cells increased differentiation. However, further studies are needed to identify the cause of dopaminergic receptors upregulation in the CNS after EAEinduction, especially D1R.
In addition, our data demonstrates enhanced D2R expression in the spinal cord of EAE mice during disease acute phase (15 days p.i.), and lymph nodes in the chronic phase (30 days p.i.). Therefore, it is possible to hypothesize that D2R activation, mainly during the acute phase of EAE, could augment disease severity, conferring to this receptor a proinflammatory characteristic. Supporting this, we showed that therapeutic treatment with PPX, a dopamine D2/D3 receptor-preferring agonist, not only failed to prevent EAE progression but in fact worsened EAE symptoms. In turn, our findings support previous evidence, which reported the ineffectiveness of bromocriptine (2.5 mg/day) in inhibiting MS progression in humans [26]. Despite this previous work supporting a negative relation between dopamine agonists and MS, at least during the onset of the motor symptoms, other studies have described beneficial effects related to bromocriptine (D2R agonist) administration in Lewis rats submitted to EAE [25,64]. Likewise, Lieberknecht and colleagues also demonstrated that preventive administration of PPX modulated the inflammatory response induced by EAE induction [30]. Furthermore, the use of atypical antipsychotic agents, including risperidone and clozapine (potent antagonists of a wide range of neuroreceptors, including dopamine and serotonin receptors), have been investigated in mouse models of MS [65][66][67]. The preventive treatment of EAE mice with risperidone (serotonin 2A and C (5-HT2A and 5-HT2C) and D2R antagonist; 3 mg/ kg/day) decreased disease severity [66]. However, authors highlighted that the beneficial effects mediated by risperidone are not due to the antagonism of the dopaminergic receptors [66]. Recently, a study has provided new evidence about the mechanisms underlying the immunomodulatory properties of antipsychotic agents [67]. Robichon and colleagues reported a significant D1R and D5R downregulation and D2R upregulation in microglia and CD4 + -infiltrating T cells, while clozapine treatment upregulated D1R and D2R levels, suggesting that this drug could modify dopaminergic pathways in MS [67]. A recent clinical trial suggested that risperidone and clozapine were beneficial for reducing MS-induced neuroinflammation [68]. However, unlike the D1R, D2R activation has been linked to anti-inflammatory effects. In a astrocytic cell culture, for instance, D2R activation was able to suppress LPS-induced IκK phosphorylation and NF-κB nuclear translocation [69][70][71]. In the same way, D2R-dependet β-arrestin recruitment represses inflammasome activation by binding to NLR family pyrin domain containing 3 (NLRP3), a critical component of the innate immune system that mediates caspase-1 activation and triggers proinflammatory cytokines release, such as IL-1β [71][72][73]. Considering the above exposed, we suggest that D2R possibly has a dual role in the pathogenesis of EAE since we show that the therapeutic treatment with PPX worsened disease symptoms, while the preventive administration inhibited EAE development [30]. Despite that, our findings contradict previous works, which indicated an anti-inflammatory role linked to the D2R. Therefore, additional experiments are required to shed light on the reason for preferential dopamine D2/D3 receptor agonist PPX are harmful in an EAE mouse model, when administered therapeutically.
Depressive disorders have a high incidence among MS patients, negatively influencing their quality of life [74]. The present study describes that PPX significantly decreased EAE-induced depressive-like behavior in a model of learned helplessness but without efficacy on the anhedonic-like behavior. In accordance, previous reports have demonstrated that PPX exhibits promising responses on treatment-resistant depressed patients [75], and depressive symptoms in Parkinson's disease patients [76]. Also, repeated PPX administration for 7 days completely prevented LPS-induced depression-like and anhedonic-like behavior in mice [32]. In this work, authors showed that PPX modulated IL-1β and 3-nitrotyrosine levels [32]. The same authors attributed the PPX antidepressant-like effect to its immunomodulatory properties, since the administration of the dopaminergic antagonist haloperidol and sulpiride was unable to prevent the antidepressant-like effect of PPX [32]. Supporting this, we also report that PPX downregulated IL-1β levels in the spinal cord of EAE mice; a proinflammatory cytokine expressed preferably by innate cells, including macrophage, NK cells, monocytes, and neutrophils, which plays a crucial role in the pathogenesis of major depressive disorder [77,78] and possibly mood disturbances in MS patients [50]. As reported by Gentile and colleagues, IL-1β-driven dysfunction of dopaminergic signaling triggers mood alterations in EAE mice [49]. Thus, we initially hypothesize that IL-1β mediates PPX antidepressant-like effect, possibly by modulating D2R/β2-arrestin signaling. Additionally, our findings also demonstrate that PPX upregulated BDNF levels in the spinal cord of EAE mice. This neurotrophic factor plays a critical role in neuronal and oligodendroglial growth and survival [79][80][81] and has an essential role in the pathophysiology of psychiatric diseases, including depressive disorders [82]. Previously, PPX showed to exert protective effects in cell cultures exposed to MPP + [83,84] and lactacystin (proteasome inhibitor), via downstream BDNF recruitment [85]. Still, according to Berghauzen-Maciejewska and colleagues, chronic treatment with antiparkinsonian drug PPX reverted depressive-like behavior induced by bilateral injections of 6-hydroxydopamine (6-OHDA) into the ventral region of the caudate-putamen [86]. Posteriorly, the authors also demonstrated that chronic PPX influences BDNF signaling in limbic structures [87]. Other piece of evidence suggests that PPX neuroprotective effects may involve D3R activation, recruitment of BDNF-TrkB, and mTOR signaling [88]. In this context, the activation of D3R-Giβγ-PI3K signaling subsequently promotes the activation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and rapamycin complex 1 (mTORC1) [89,90]. In turn, mTORC1 is responsible for regulating the survival, differentiation, and development of neurons [91]. Altogether, we suggest that in order to induce neuroprotective effects in EAE, when administered preventively, PPX interacts with BNDF signaling, possibly through D3R activation.
In order to characterize the oxidative stress during different phases of EAE, we evaluated the levels of GPx-a crucial antioxidant enzyme [92,93]-and DCF in the spinal cord and striatum of EAE mice. The imbalance in the antioxidant defense system has been implicated in the pathogenesis of MS. Herein, our findings demonstrate that the levels of GPx are downregulated in the spinal cord and striatum of EAE mice in the chronic phase of the experimental model. In agreement with our results, Conde and colleagues also reported a significant reduction in the protein levels of GPx in the brain and spinal cord of rats with EAE, 65-day p.i. [94]. Additionally, the levels of GPx were reduced in the blood of patients with MS [95]. Nevertheless, we also identified increased GPx levels during the induction phase of EAE (7-day p.i.). Following these results, a previous study reported that thirteen genes were upregulated in acute and chronic MS lesions, among them GPX1 (GPx coding gene) [96]. The demyelinated acute lesions obtained from patients with MS also showed upregulation of superoxide dismutase 1 and 2 (SOD1 and SOD2) and heme oxygenase (HO), which play an essential role in the antioxidant system, suggesting an adaptive defense mechanism that aims to restore the levels of ROS, reducing cell damage related to its accumulation [97]. We also demonstrate a significant increase in the oxidative damage in the spinal cord and striatum of EAE mice during the chronic phase of the EAE model. ROS are naturally produced within biological systems and play an essential role as signaling molecules in a wide variety of physiological processes [98]. However, disruption of redox homeostasis promotes an inflammatory degenerative vicious cycle [99]. The intensity of DCF fluorescence corresponds to the detection of reactive oxygen and nitrogen species. In this way, our data agree with a previous report, which demonstrated increased levels of inducible nitric oxide synthase (iNOS) in the spinal cord of mice submitted to EAE between 32-and 67-day p.i. [100]. According to Espejo and colleagues, during the chronic phase of EAE, the brain, cerebellum, brain stem, and spinal cord of immunized mice showed intense oxidative damage characterized by immunoreactivity of iNOS, nitrotyrosine and malondialdehyde (MDA) [101]. In contrast, we also identified a significant reduction in the fluorescence intensity of DCF, only in the spinal cord of mice, in the initial phase of EAE development (7-day p.i.). Different from our findings, Hasseldam and colleagues showed an increased reactive oxygen species burden in the brain tissue 5 and 7 days after EAE induction in rats, without significant differences in the spinal cord tissue [102]. However, GPx elevated amounts here found during the induction phase of the disease could justify ROS reduction. Indeed, further comprehensive studies regarding the oxidative aspect of dopaminergic receptors in the EAE model will allow a deeper understanding of these findings.
Here, we report a significant reduction in axonal density in the spinal cord of animals submitted to EAE 7-day p.i., suggesting axonal damage in the pre-symptomatic phase of the disease, when no macroscopic motor disturbance is evident. In consonance, the axonal damage not only occurs in lesioned areas but also normal-appearing white matter. According to DeVos and colleagues, the axonal damage characteristic of MS-related lesions is similar to those detected in other neurological conditions unrelated to demyelination, such as amyotrophic lateral sclerosis (ALS) [103]. From this, it is possible to hypothesize that the axonal damage occurs independently of the demyelination process.
Additionally, although axonal damage can be observed already in the acute phase of EAE, there is no correlation between axonal damage and the degree of neurological disability [104]. However, according to the same authors, the number of symptomatic attacks in EAE mice was correlated with axon loss and neurological impairment in the chronic phase of the model [104]. In this way, our data offer additional evidence on damage to neurons in the dorsal horn of the spinal cord and the subsequent appearance of sensory disturbances preceding the emergence of EAE-induced motor deficits.

Conclusion
Dopaminergic signaling has emerged as an essential neuroimmune-mediator in the communication between immune system and CNS, promoting homeostasis [24]. Consequently, imbalances in the dopaminergic system might be affecting both innate and adaptive immunity and contributing to neurodegenerative diseases, including MS. Our data confirms that axonal damage precedes EAE-induced motor deficits and the presence of intense oxidative stress in the spinal cord and striatum of EAE mice, mainly during disease chronic phase. Here, we demonstrate that mice therapeutically treated with preferential D2/D3 dopamine agonist showed increased severity of EAE symptoms. Furthermore, our results disagree with previous literature demonstrating D2R activation-related anti-inflammatory effects. Therefore, considering the limitations of our study, it was not possible to identify for which reason the use of preferential dopamine D2/D3 receptor agonist, PPX, proved harmful in EAE mice when administered therapeutically, as well as the deferential content of dopaminergic receptors following the stages of EAE development (induction, acute, and chronic phase), and the possible specific cell types involved. In addition, we did not perform experiments using CFA-treated group as a control group in order to eliminate possible biases. However, as long as administered preventively, PPX showed to reduce EAE-induced depressive-like behavior, downregulated the levels of IL-1β, and increased the expression of BDNF in the spinal cord of EAE mice. Thus, based on previously published data, we suggest that the protective effect of PPX against depressive-like behavior and the modulatory effect on the expression of IL-1β and BDNF may be associated with the activation of D2R/β2-arrestin signaling, which negatively regulates activation of NLRP3 inflammasome-and D3R-PI3K signaling, which in turn, signals via TrkB and extracellular regulated kinase (ERK) 1/2. Finally, this study points to the possible participation of dopamine and its receptors, mainly D2R, in the crosstalk between the CNS and the immune system response during the genesis and progression of the autoimmune and neuroinflammatory response induced by EAE.
Author Contribution ECDG and VL performed all experiments. ECDG analyzed and interpreted the data and was a major contributor in writing the manuscript. VVH, BDR, FAF, ALSR, and DFM contributed to the methodological analysis and manuscript editing. RCD designed this study, edited, and wrote the manuscript. All authors read and approved the final manuscript.