INTRODUCTION

Parkinson´s disease (PD) is the second most common neurodegenerative disease associated with aging and the fastest-growing neurodegenerative disease. It is expected that there will be 12 million cases by 2040¹. Worldwide, more than 8.5 million people were reported to have PD in 2019^2,3. This pathology is characterized by movement disorders due to the loss of dopaminergic neurons and degeneration of the substantia nigra pars compacta⁴. The main pathognomonic symptoms are tremor, bradykinesia, rigidity, and postural instability. However, non-motor symptoms such as depression, cognitive changes, neuropsychiatric manifestations, constipation, and fatigue may precede motor symptoms by up to 20 years^5,6. The therapeutic strategy to be used depends on the age of the patient, the progression of the disease, the severity of the symptoms, and the risk/benefit assessment of the chosen treatment⁷. Carbidopa/levodopa, dopaminergic agonists, and monoamine oxidase B inhibitors are the most widely used drugs as initial therapy; however, these treatments are palliative and there is no cure or drug to halt the progression of neurodegeneration^8,9. Recently, efforts have been made to investigate therapies that focus on reducing neuroinflammation and promoting neuroprotection. Cell therapy for neuronal regeneration with pluripotent neural cell-inducing mesenchymal stem cells and mesenchymal neural progenitor cells accounted for 12.2% of all phase-I clinical trials in 2021, whereas immunotherapy (anti-alpha-synuclein antibodies or alpha-synuclein immunogenic peptides) to prevent neurotoxicity accounted for 10.2% of trials¹⁰.

Neuroinflammation, the activation of an inflammatory immune response within the brain or spinal cord, is an active area of research related to central nervous system (CNS) disorders. This process is mediated by both soluble and cellular factors secreted by the CNS and peripheral tissues. Soluble factors include proinflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor (TNF), and interleukin-6 (IL-6); chemokines such as CCL2 and CXCL1; and molecules such as prostaglandins and nitric oxide (NO)¹¹. High serum levels of TNF have been linked to increased severity of depression, fatigue, and cognitive problems¹². Cellular factors involved in neuroinflammation include microglial cells, astrocytes, endothelial cells, and perivascular macrophages¹³. These cells detect signals such as an accumulation of modified alpha-synuclein, increased oxidative stress, neuron damage, and secrete pro-inflammatory mediators (IL-1β, IL-6, TNF, and NO) that aggravate the process. The adaptive immune response is also activated, with cells migrating into the CNS and exacerbating neurodegeneration¹⁴.

Immunomodulatory therapy aims to regulate the neurotoxic inflammatory response by reducing it or preventing an exacerbated neuroinflammatory phenomenon. It has been described that PD patients exhibit decreased counts of immunosuppressive regulatory T cells and increased counts of effector T cells, correlating with the severity of clinical manifestations¹⁵. Immunomodulatory therapy aimed at boosting regulatory T cells could modulate the immune response in these patients and thereby have a beneficial effect on symptoms and/or slow disease progression. Studies of this therapeutic approach range from passive immunization with anti-alpha-synuclein antibodies to the use of soluble factors such as vasoactive intestinal peptide or granulocyte-macrophage colony-stimulating factor (GM-CSF). Similarly, therapeutic agents have been tested in animal models with results that suggest they may be successful in humans. For example, in mice, the GM-CSF produces increased levels of CD4 + CD25 + FOXP3 + regulatory T cells, which inhibit the proliferation of CD3/CD28-activated effector CD4 + CD25 T-cells. These regulatory cells exerted a neuroprotective effect when transfected into MPTP-treated mice. In a rat model of human alpha-synuclein overexpression, regulatory T cells generated by elevated plasma levels of GM-CSF increased the survival rate of tyrosine hydroxylase-positive (TH+) dopaminergic neurons¹⁶. Similarly, Alzheimer´s disease patients treated with sargramostim (GM-CSF) 250 μg/m²/day 5 times/week for 3 weeks showed altered immune profiles with increased monocyte, lymphocyte, and neutrophil counts, as well as IL-10, IL-6, and TNF secretion, and improved plasma biomarkers of cognition and neuropathy without significant adverse effects¹⁷.

Therefore, the administration of immunomodulators could help control neuroinflammation in PD (Fig. 1). Immunomodulators and immunotherapy could be used in different approaches. For example, pioglitazone, a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist used to treat type 2 diabetes by reducing insulin resistance, inhibits microglial activation and reduces NO and proinflammatory cytokine levels in a rat model of PD¹⁸. On the other hand, cinpanemab and prasinezumab, monoclonal antibodies of human origin that binds to alpha-synuclein aggregates, have been shown to reduce motor impairment¹⁹.

Figure 1 Proposed mechanism of action for Parkinson´s disease (PD) treatments included in this meta-analysis. Several therapeutic strategies are used to modulate neuroinflammation. A: prasinezumab and cinpanemab are humanized monoclonal antibodies that selectively bind alpha-synuclein. In murine models, prasinezumab reduced the propagation, aggregation, and accumulation of alpha-synuclein by binding to its C-terminal residue, preventing its transport to other neurons and microglial cells and improving protein clearance. B: cinpanemab selectively binds to extracellular alpha-synuclein. Both therapies could prevent neurotoxicity and inflammation following glial activation. However, this mechanism has not been extensively studied in animals and not at all in humans; it is possible that the presence of the alpha-synuclein-antibody complex could activate the immune response. C: dysfunction of the innate and adaptive immune response in PD is characterized by the production of proinflammatory cytokines, proinflammatory glia, presence of effector T cells, and decreased Treg cell counts. Sargramostim is a recombinant human granulocyte-macrophage colony-stimulating factor that increases CD4 + CD127loCD25hi Treg cell expression and induces tolerogenic dendritic cells in humans. By regulating the suppressor response, sargramostim could modulate glia toward an M2 phenotype, attenuating neuroinflammation and promoting neuron survival and neuroprotection, as observed in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in patients with Alzheimer´s disease, stroke, and traumatic brain injury^20-23. D: PD03A is a 10-amino-acid peptide designed to mimic an epitope of the C-terminal region of alpha-synuclein. The peptide is conjugated to a Th-cell antigen to elicit a long-lasting antigenic response. PD03A is a B-cell epitope that promotes a significant humoral response that neutralizes extracellular alpha-synuclein and prevents glial activation²⁴. E: pioglitazone is an antidiabetic thiazolidinedione (TZD), a synthetic ligand of PPAR-γ. TZDs have been shown to regulate inflammation by decreasing IL-1β and TNF production by macrophages and microglia; in mice, pioglitazone inhibited nitric oxide (NO) production. In neuron-microglia cocultures, pioglitazone inhibited microglial activation and reduced NO production^25-27. F: tetracyclines such as minocycline have shown pharmacological effects in addition to antibiotic activity, such as inhibition of superoxide production in neutrophils and iNOS expression in macrophages. In a mouse MPTP model of PD, minocycline prevented dopaminergic neuron degeneration and loss of striatal dopamine. In primary cultures of astrocytes and microglial cells, treatment decreased the expression of iNOS and caspase 1 and reduced the expression of caspase 1 in neurons. However, the mechanism has not been studied in humans^28,29. G: finally, GPI-1485 is a tacrolimus-derived neuroimmunophilin ligand (NIL) that has demonstrated neuroprotective activity by reversing neurodegeneration and preventing cell death. NILs bind to a group of proteins called FK506-binding proteins, which are involved in the regulation of glucocorticoid receptors, heat shock proteins, and, in immune cells, the inhibition of the expression of cytokines that alter calcineurin. Exactly how NILs act in models of Alzheimer´s disease, PD, and trauma-induced neuropathies is unclear, and there is some doubt as to whether such an effect is due to immunosuppression or immune alterations, as no immune cells have been identified as targets of GPI-1485, and NILs do not appear to affect calcineurin^30-32. 

In addition to evaluating the effect of immunomodulators and exploring their mechanism of action, the current studies in animal models and clinical trials assess the risks and adverse effects of these drugs in patients. The objective of this meta-analysis is to review recent evidence on the evaluation of immunomodulators in randomized controlled clinical trials measuring the improvement of motor symptoms in PD patients.

METHODS

RESULTS

Initially, 1532 articles were found with the keywords used (Fig. 2). After reviewing them by title and abstract, 1518 studies were excluded, most of them for not being clinical trials. Next, 19 studies were reviewed; 8 were excluded for not reporting MDS-UPDR III scores; and 4 were replicated. Finally, seven studies were selected for meta-analysis. These trials tested different immunomodulatory treatments: cinpanemab, prasinezumab (PRX 002), sargramostim (GM-CSF), PD03A, pioglitazone, minocycline, and GPI-1485. The baseline demographic and clinical characteristics of the patients are shown in table 1. In each study, the placebo groups were compared to the treatment groups. Subjects in all groups were patients with early-stage PD, with or without background treatment (levodopa, dopaminergic agonists or MAO-B inhibitors, or combinations).

Figure 2 Literature screening flow chart. 

Meta-analysis results

A total of 1212 patients were included in the meta-analysis: 590 patients were treated with immunomodulators and 622 patients received a placebo. Due to the high heterogeneity of the studies (I² = 91%, p < 0.01), a random effects model was used (Fig. 3). The mean standard difference for change in MDS-UPDR III score was −0.46 (CI [95%] = −0.90 - −0.02, p < 0.01). These results show no significant differences in the change in MDS-UPDR III score between the treatment and placebo groups. However, two studies showed a trend toward separation from the mean (Fig. 3).

Figure 3 Forest plot for the meta-analysis of change in Movement Disorder Society-Unified Parkinson´s disease Rating Scale-III score for placebo versus treatment groups. The number of patients in each group is shown in the "Total" column, and the mean change in score for placebo and treatment groups is shown in the "Mean" column. The first author or investigation group, the year of publication, and the doses administered are shown in the "study" column. Doses are given because multiple doses were tested in the same drug trial. 

Sensitivity was assessed to determine the influence of each study on the overall effect estimate. As shown in Fig. 4, no study had a significant overall effect. The results of the meta-analysis did not change dramatically in magnitude or direction, indicating that the meta-analysis is robust.

Figure 4 Forest plot of sensitivity analysis. 

Finally, the risk of bias in the meta-analysis was assessed using a funnel plot. Because the plot was too sparsely populated to visually detect asymmetry (Fig. 5), Egger´s test was also used. As shown in Supplementary Fig. 2, the plot is symmetrical (intercept = −0.27, P = 0.9337), suggesting no publication bias in the included studies; however, this result should be taken with caution due to the small number of included studies.

Figure 5 Funnel plot. 

DISCUSSION

At present, patients with PD receive only symptomatic treatment. However, new therapeutic approaches such as immunomodulatory drugs and immunotherapies hold promise for improving patients´ quality of life and halting disease progression³³. The neuroinflammatory process that accompanies PD is associated with abnormalities in alpha-synuclein aggregation, chronic glial activation, secretion of proinflammatory cytokines, and the presence of activated immune cells. The relevance of neuroinflammation during PD in brain tissue and cerebrospinal fluid underscores the importance of developing and evaluating drugs to modify this inflammatory profile^34,35. Immunomodulatory treatments, including those tested with positive results in animal models of PD, have paved the way for therapies being tested in clinical trials.

Seven clinical trials evaluating immunomodulators or immunotherapies were included in this meta-analysis. Although no significant differences were found in the change in MDS-UPDRS III score between the treated group and placebo, even in studies using higher doses (Fig. 2), 2 studies showed a tendency to depart from the mean.

Pagano et al. reported no difference in the change in MDS-UPDRS total score between prasinezumab and a placebo; however, only Part III of the scale was analyzed in this meta-analysis, so it is possible that prasinezumab has little effect on motor improvement. This result may also be due to the fact that the data were not adjusted for multiple comparisons, as acknowledged by the authors³⁶.

Sargramostim, recombinant human GM-CSF, is the only drug included in this meta-analysis that showed a positive effect on PD. Gendelman et al.²⁰ reported a significant decrease in the MDS-UPDRS III score (Table 1) in the treatment group at the end of the study (8 weeks) from baseline. A mean change of −5 indicates a significant improvement in motor symptoms. However, this result was not significant when compared to the mean change across studies. This may be due to the small number of patients included in this study (nine on placebo and five on treatment). As shown in the forest plot, the confidence interval (CI) of this study was the largest among the included studies. It is noteworthy that sargramostim increased CD4 + CD25 + CD127lo Treg counts after 2 weeks of treatment, accompanied by an alteration in tryptophan pathway: L-kynurenine and quinolinic acid levels were increased, and serotonin levels decreased. These findings suggest that sargramostim could be an effective immunomodulator; however, trials with a greater sample size and the required power to evaluate clinical outcomes should be conducted. This highlights the need for further research and larger clinical trials in this area.

Cinpanemab (anti-synuclein alpha monoclonal antibody), which had reduced striatal dopamine transporter loss and reversed movement impairment in a mouse model of PD, showed no significant effect compared with the placebo group³⁷. PD03A, a short peptide derived from an epitope of the C-terminal region of alpha-synuclein, showed efficacy in inducing antibodies and activating the immune response but had no effect on the MDS-UPDRS III score^24,38. Pioglitazone, minocycline, and GPI-1485 are drugs currently used in various conditions that have been reported to have neuroprotective effects and evaluated in preclinical models^28,30,39. Neither pioglitazone, a PPAR-γ agonist; minocycline, a tetracycline used to treat bacterial infections with anti-inflammatory properties that reduce levels of proinflammatory cytokines nor GPI-1485, a neuroimmunophilin ligand with immunosuppressive effects, induced a change in MDS-UPDRS III score and did not improve motor problems in PD patients. It is possible that the lack of significance in the differences is due to (a) the duration of treatments, (b) the sample size, (c) the time of treatment initiation (unlike animal models, we do not know when PD started in patients), and (d) the need for concurrent administration of dopamine or other treatments with the tested drug. As for our meta-analysis study, most of the studies lacked the power to analyze clinical outcomes such as MDS-UPDRS III scores, imaging studies, or immunologic profiles, as well as other cellular tests. It would be beneficial to assess the immune profile of patients by measuring cytokine levels and analyzing T-cell function and phenotypes when evaluating drugs with potential immunologic effects.

Regarding the quality of our meta-analysis, most of the included studies have a low risk of bias in the domains assessed by the Cochrane tool (Supplementary Fig. 1), suggesting a good methodological quality of the included studies. As for the sensitivity test, the omission of any study did not substantially change the magnitude or direction of the result (Fig. 4), so the result of the meta-analysis can be considered robust. With respect to publication bias, although the funnel plot shows some asymmetry in the included studies (Fig. 5), Egger´s test (Supplementary Fig. 2) showed no evidence of asymmetry. Thus, it can be assumed that there is no publication bias, although the small number of included studies should also be taken into account.

None of the adverse events were serious and were reported in both the treatment and placebo groups (Table 1). The most common adverse events were related to the route of administration; however, it should be noted that adverse events such as abnormal white blood cell counts or joint pain, which were reported in some of the included trials, could further affect patients who already have a poor quality of life due to PD. Such adverse events may warrant a cost-benefit evaluation of these drugs.

Finally, this study has clear limitations, one of which is the high heterogeneity of the results. This heterogeneity may be the result of methodological differences between the included articles since different drugs were tested and, although they all have immunomodulatory or immunotherapeutic effects, the different molecules were administered by different routes, at different doses and with different treatment durations. In addition, the number of studies found and analyzed was small, and although the total number of patients included in the meta-analysis is adequate, some of the included studies have very small sample sizes. For example, the study by Gendelman et al.²⁰ reported a positive effect with a significant mean change in scores; however, because the sample is very small (14 patients), the confidence interval is very wide, making it less meaningful, this is reflected in a lower weight (influence) of the Gendelman´s study within the meta-analysis. In addition, it should be noted that most of the included studies were not designed to assess clinical outcomes and clinical improvement, so more information and evaluation are required.

In conclusion, idea of controlling neuroinflammation to halt or delay disease progression or improve symptoms in PD is promising, as shown by pre-clinical studies with positive results; however, no significant effects on motor symptoms were found in the clinical trials included in this review. It is worth noting that in our study, we only assessed motor symptom improvement by MDS-UPDRS III score reports. However, several molecules with immune system activity that is currently being tested were not included because they are in phase 1 and/or have not reported MDS-UPDRS III scores. In addition, we should keep in mind that both the disease and the mechanisms of neuroinflammation are complex, so immunomodulatory therapies are also expected to be intricate and not easy to evaluate. Treatments that target neuroinflammation are valuable not only because they might halt a degenerative disease but also because of the insights they might provide into the relationship between the nervous and immune systems.