The role of inflammation in sporadic and familial Parkinson’s disease

Inflammation in animal models of PD

Activated microglia and astrocytes, increased production of cyclooxygenase-2 (COX-2), pro-inflammatory cytokines and NO, recruitment of CD4+ and CD8+ T cells, and involvement of TLR-dependent pathways have been reported in many toxin-based models of PD [38–43]. Interestingly, the early blockade of neuroinflammation by minocycline, non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 or cytokine inhibitors attenuates the PD-like disease process in these models of DA neuron degeneration, supporting the hypothesis that immune cells and inflammatory molecules are important contributors to the initiation of PD [40, 44–48]. Such experimental models also support the role of astrocytes in PD pathology. Astrocytes effectively endocytose α-synuclein secreted from neurons, and initiate inflammatory responses. Moreover, S100B, a protein highly produced in, and secreted from, astrocytes, has proinflammatory properties and induces neurodegeneration through the RAGE and tumor necrosis factor-alpha (TNF-α) pathway in a mouse model of PD [49]. Neuro-immune interactions also modulate DA neuron degeneration, as shown in mouse models of CX3CR1 and CD200R deficiency [23, 50]. The relationship between inflammation and neurodegeneration remains largely unknown. Transgenic animal models of PD show that inflammation is an early event that can even precede neurodegeneration [51]. Inflammation alone can precipitate PD-like pathology and initiate neurodegenerative events in the nigrostriatal system, which correspond to changes observed in early neurodegeneration. Inflammatory cytokines released from activated microglia and astrocytes alter synaptic transmission and axonal transport and increase neuronal susceptibility to subsequent neurotoxic triggers [47, 48]. Inflammation could therefore prime neurons and immune cells in the brain, rendering neuronal populations vulnerable to degeneration in the face of subsequent insults.

Post-mortem studies in PD patients

The first evidence for inflammatory reactions in PD is shown by the work of McGeer et al. [52]. This study showed for the first time the presence of activated HLA-DR-positive reactive microglia within the SN of PD patients, along with LB and free melanin [52]. Reactive microglial cells can therefore be considered a sensitive index of disease pathology [52]. The presence of microglial activation and reactive astrogliosis was then confirmed by several other post-mortem studies [22, 53–56]. Elevated numbers of microglial cells have been found not only in the SN and putamen but also in other brain regions, including the hippocampus, transentorhinal cortex, cingulate cortex, and temporal cortex [54]. Post-mortem studies also show that several pro-inflammatory cytokines and chemokines (including IL1-β, IL-2, IL-6, TNF-α, TNF-α, IFN-γ, CXCL12, CXCR4, ICAM-1) are elevated in the DA nigrostriatal system and other brain regions outside the SN in PD patients [57–63]. In addition, levels of inducible nitric oxide synthase (iNOS), NFκB, COX-1 and COX-2, and prostaglandin E2 are increased in parkinsonian brains [40, 62, 64].

Reactive astrogliosis is absent or generally described as mild or moderate in PD brains [53, 56, 65]. Studies in animal models show that astrocytes express pro-inflammatory molecules that recruit microglial cells in the early phases of neurodegeneration [66, 67]. However, the exact role of astrocytes in PD pathogenesis is still poorly understood. In PD, the distribution of α-synuclein-positive astrocytes parallels the distribution of LB [68], but no correlation between GFAP staining and disease severity has been found [69]. This finding suggests that astrocytes may also have a potential neuroprotective role. Astrocytes secrete a number of neurotrophic factors for DA neurons, including glial cell-line-derived neurotrophic factor, brain-derived neurotrophic factor [70, 71], and protease-activated receptor-1, and this may have a neuroprotective effect by increasing the activity of glutathione peroxidase [72].

Finally, increased numbers of CD4+ and CD8+ T cells are present in post-mortem human brain specimens of PD patients [42, 52]. The dysfunction of the blood–brain barrier (BBB), which has been described in PD patients [73, 74], may not be sufficient to allow such lymphocyte extravasation. Active mechanisms involving the expression of adhesion molecules on endothelial and glial cells are likely implicated in the regulation of T cell recruitment.

In vivo imaging studies

In vivo PET scan studies with [(11)C](R)-PK11195, a ligand of the peripheral binding site of benzodiazepine, provide evidence for significant microglial activation in the midbrain and putamen of PD patients [75]. Imaging studies have yielded mixed results regarding the correlation between microglial activation and disease severity and duration [75–77]. Such differences are likely due to the fact that microglia are activated early in the disease course. Moreover, there are some limitations associated with the use of 11C-PK11195 due to its high level of non-specific binding, indicating the need for better tracers to follow microglia activation and effects of anti-inflammatory treatments in patients [78].

Peripheral immune activation in PD

Levels of several cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, INF-γ, RANTES) are elevated in the serum of PD patients [79]. Several studies support a role for the adaptive immune system in the aetiology of PD (reviewed in [79]). Peripheral alterations have been reported in the blood of PD patients including a lower ratio of CD4+:CD8+ T lymphocytes, higher expression of MHC II molecules on monocytes in the cerebrospinal fluid (CSF) and peripheral blood, and higher numbers of CD4+CD25+ activated lymphocytes [80–84]. Further evidence in support of the involvement of the adaptive immune system in PD comes from recent studies that show increased levels of autoantibodies against α-syn in serum and CSF in PD patients [85, 86].

Gut inflammation has been recently linked to PD. PD patients show inflammatory reactions within the enteric nervous system (ENS), characterized by increased expression levels of pro-inflammatory cytokines and glial markers [87]. Even though these findings need to be replicated in larger cohorts of patients, studies in animal models of PD further support a link between early inflammatory events in the ENS and PD pathology [88].

The nervous and the immune system influence each other even at long distances in physiological and pathological conditions. The vagus nerve, which originates in the brainstem and innervates visceral organs, controls the immune function via the “inflammatory reflex” [89]. The stimulation of the vagus nerve inhibits cytokine release and attenuates inflammatory reactions in septic shock and post-stroke immunosuppression by activating acetylcholine-producing T cells [90, 91]. Since PD patients show early autonomic dysfunction and the involvement of the vagus nerve, further studies addressing the role of such neuroimmune interactions in neurodegeneration are needed. In principle, it would be possible to target peripheral inflammation and prevent neurodegeneration without affecting the host immune system.

Epidemiological studies

Initial epidemiological data show that chronic use of NSAIDs lowers the risk of development of sporadic PD [92–94]. A large-scale meta-analysis revealed conflicting results regarding the use of NSAIDs and PD risk, indicating that only ibuprofen provides significant protection [95]. Based on a recent Cochrane review, however, there is no evidence for the use of NSAIDs in the prevention of PD [96]. Further studies are therefore needed.

Polymorphisms in immune-related genes and PD risk

Several genetic studies have investigated the link between polymorphisms in immune-related genes and PD (Table 1).

• Genome-wide association studies (GWAS) have identified an association of genetic variation in the HLA region with sporadic PD [9, 97, 98].

• A pathway analysis of GWA data revealed that the T cell receptor signaling pathway is a susceptibility factor for PD and genetic variability in genes regulating inflammatory pathways differentiates PD patients from controls [99, 100].

• Gene polymorphisms of several inflammatory cytokines, including TNF-α, IL-1β, and CD14 monocyte receptor, might be a risk factor for PD [101, 102].

• Three single nucleotide polymorphisms in the CARD15 gene, previously associated with CD, a chronic inflammatory bowel disease, have been shown to be a risk factor for PD [103].

However, the exact role of these polymorphisms in PD pathogenesis still needs to be elucidated and the frequency of these risk alleles strongly depends on the heterogeneity of the population [104].

The role of PD-genes in immune responses

Recent research into the functional genomics of PD has identified PD-genes as regulators of key processes in innate and adaptive immunity (Table 2; Figs. 1, 2). PD proteins may contribute to PD pathogenesis by directly triggering dysfunction in immune cells (e.g., LRRK2, PLA2G6, GBA) or promoting protein misfolding/aggregation and reactive inflammatory responses (e.g., α-syn). As revealed by the pathway analysis shown in Fig. 2, PD-genes are directly or indirectly involved in several immune biological processes including T cell activation (Th1 and Th2 inflammatory responses), B cell responses, and innate immunity responses, such as activation of macrophages, microglia, and astrocytes.

The expression of many PD-genes is not restricted to neurons but also found at high levels in cells of the immune system. In this respect, LRRK2 is highly expressed in APCs including microglia, monocytes and B cells and is involved in the immune responses to pathogens [105–108]. Increasing evidence is accumulating that LRRK2 is involved in innate and adaptive immune responses. Aberrant activation of microglia and kidney inflammatory reactions have been observed in LRRK2-deficient mice [109–112]. LRRK2 negatively regulates the transcription factor NFAT and its deficiency in mice triggers hyperactive immune responses and greater susceptibility to inflammatory bowel disease [113]. These data support the hypothesis that LRRK2 mutations are linked to excessive and poorly controlled immune responses, which may cause neuronal dysfunction and increase neuronal vulnerability to degeneration. The association of LRRK2 with CD and bowel inflammation suggests that peripheral inflammation may be involved in the prodromal stages of the disease.

Heterozygous mutations in the GBA1 gene, which encodes acid β-glucosidase (glucocerebrosidase, GCase), represent the strongest genetic risk factor for PD [114]. The role of GBA mutations in the pathogenesis of PD and other synucleinopathies is not clear. Homozygous mutations in the GBA1 gene cause Gaucher’s disease (GD), the most common lysosomal storage disorder. GD and PD were initially associated because of the observation of Parkinsonism and LB pathology in patients with GD. GBA is highly expressed in cells of the mononuclear phagocyte lineage and GCase deficiency results in the accumulation of glucosylceramide (an intermediate of the sphingolipid pathway) in macrophages and subsequent release of several pro-inflammatory molecules [115]. The onset and progression of GD are in fact associated with a sustained systemic inflammatory reaction and increased levels of pro-inflammatory molecules [115–118]. In the neuronopathic forms of GD, neuronal loss is also accompanied by astrocytosis and microgliosis [119, 120]. Interestingly, GCase deficiency, even in the absence of large amounts of sphingolipid storage, which may mimic the condition observed in PD, triggers inflammatory reactions [116]. Increasing evidence suggests that GBA directly modulates immune cells other than macrophages, including T and B cells [121, 122].

It is not known whether PD patients carrying heterozygous GBA mutations also display such immune phenotype. Interestingly, non-motor symptoms, such as dementia and neuropsychiatric disturbances, are very common in GBA-PD patients [123]. Since inflammation has been associated with cognitive decline [124], further immunological investigations are needed both in PD patients and asymptomatic mutation carriers.

Given the crucial role of LRRK2 and GBA in autophagy and lysosomal function, it is likely that the dysfunction of these enzymes not only affects the degradation of misfolded proteins in neurons [125] but also the phagocytic properties of microglial cells with subsequent inflammatory reactions.

Astrocytes also play an important role in protein clearance in the SNC. Interestingly, many PD-genes are expressed at high levels in astrocytes. Among them, DJ-1 is expressed mainly by astrocytes in the normal human brain [126], and is strongly up-regulated in reactive astrocytes in PD brain, MS inflammatory lesions, and stroke [126–129]. DJ-1 plays a key role in the suppression of inflammatory responses in astrocytes by regulating NO and pro-inflammatory cytokines production [130]. In PD patients with DJ-1 mutations, a non-cell autonomous dysfunction of astrocytes and neuroinflammation can therefore be the driving force of neurodegeneration.

α-syn is found at high levels within astrocytes and α-syn-positive inclusions in astroglia have been described in PD and other α-synucleinopathies [68, 131–135]. TLRs likely mediate the clearance of α-syn by microglial cells and astrocytes [136–138]. The current theory is that α-syn released by neuronal terminals is internalized by adjacent astrocytes, where it forms aggregates [68]. Recent evidence confirmed the transfer of α-syn from neurons to astrocytes [67], and an additional study has shown that the accumulation of α-syn within astrocytes initiates DA neuronal death, suggesting a non–cell autonomous component in the pathogenesis of the disease [139]. Early α-syn accumulation in glial cells may therefore trigger inflammatory reactions and the release of pro-inflammatory molecules, which in turn damage vulnerable neurons [51, 66, 140–144]. Extracellular forms of α-syn and its modified forms can in fact activate glial cells promoting harmful inflammatory reactions including the release of inflammatory cytokines, NADPH oxidase and ROS production [145–151]. The pro-inflammatory properties of wild-type and mutant forms of α-syn have been further confirmed in several experimental models of PD [148, 152]. Importantly, neuroinflammation precedes neuronal death in these models [51].

Evidence for a role of genetic variation in the regulation of immune pathways also exists for other rare PD-linked genes (summarized in Table 2). Among these of particular interest is Nurr1 that has been linked to a rare form of familial PD, although such association has not been confirmed after the initial reports [154, 155]. Nurr1 is a transcription factor that regulates expression of the gene encoding tyrosine hydroxylase (TH) [156]. It belongs to the NR4A subfamily (NR4A1/NUR77, NR4A2/NURR1, and NR4A3/NOR1) of orphan NRs, which are key transcriptional regulators of cytokines and growth factors [157]. Nurr1 is also highly expressed in human lymphocytes and macrophages [158]. Interestingly, the rare Nurr1 variants potentially linked to PD affect the transcription of gene that encodes TH and also decrease Nurr1 mRNA level in lymphocytes [154, 159]. In experimental models, the reduction of Nurr1 expression results in exaggerated inflammatory responses in microglial cells that are further intensified by astrocytes, leading to the death of DA neurons [160]. In addition, Nurr1 has an indirect immunomodulatory effect mediated by the regulation of vasoactive intestinal protein [161], which promotes the release of glial-derived trophic factors and inhibits microglia activation [162].

Several transgenic animal models containing mutant or knock-out PD genes do not show overt neuroinflammation. It is unlikely that inflammation alone is the cause of neurodegeneration even in familial forms of PD linked to mutations in “immune genes”. However, it is tempting to speculate that such PD mutations and SNPs in immune genes confer an immune phenotype that contributes to the development of aberrant immune responses upon inflammatory triggers (e.g., ageing, infections, sterile inflammation). Past infections may in fact contribute to cognitive impairment and neurodegeneration [163]. Inflammation can initiate neuronal, axonal, and synaptic dysfunction in susceptible neuronal populations, such as the vulnerable DA neurons, and, over time, increase neuronal vulnerability to subsequent neurodegenerative insults.

It would therefore be interesting to profile the immune responses to selected stimuli (e.g., adjuvants, immune modulators, hormones, cytokines), and to correlate those with differences to the genetic background and clinical features of PD patients, both in manifesting and non-manifesting mutation carriers. The greater homogeneity of these populations would facilitate the identification of immune markers that could eventually be used as disease biomarkers.