Parkinson’s disease (PD) is a common movement disorder and the second most prevalent neurodegenerative disorder worldwide, that affects nearly 2% of the elderly population. PD is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and consequently reduced dopamine (DA) levels in the basal ganglia, causing motor dysfunction (Figure 1). Lewy bodies, intracellular proteinaceous inclusions, are the pathological hallmark within PD brains. Lewy bodies contain fibrillar α-synuclein among other proteins (Spillantini et al., 1997). It is evident that the immune system is involved in PD risk by genome-wide association studies (GWAS), implicating the human leukocyte antigen (HLA) locus with sporadic PD (Hamza et al., 2010) and, as well, by the neuropathology in PD brains demonstrating highly activated microglial and T-cells (McGeer et al., 1988; Imamura et al., 2003). Several inflammatory markers have been identified in SNpc of PD brains including cytokines and neurotrophins (Nagatsu et al., 2000; Hunot and Hirsch, 2003). Also, widespread microglial activation is a concomitant in PD neuropathology (Gerhard et al., 2006). A meta-analysis of anti-inflammatory drug trials revealed an association between non-steroidal anti-inflammatory drugs (NSAIDs) use and reduced risk for developing PD possibly implicating neuroinflammatory processes in the disease (Gagne and Power, 2010). Evidence supports the conclusion that microglia, the brain resident macrophage-like immune cells, participate in the inflammatory response of the disease (Qian and Flood, 2008; Long-Smith et al., 2009; Moehle and West, 2015). In addition, other observations implicate peripheral immune cells in PD (Saunders et al., 2012; Funk et al., 2013; Chen et al., 2015). Together these data indicate that inflammation and microglial activation contribute to the in pathogenesis of PD. Hence, immunomodulation might be a possible therapeutic avenue for PD.

The distribution of microglial M1/M2 phenotypes depends on the stage and severity of the disease. Understanding stage-specific switching of microglial phenotypes and the capacity to manipulate these transitions within appropriate time windows might be beneficial for PD therapy. In this review, we will outline different microglial activation states and provide evidence of M1/M2 activation states in PD. We will also discuss how manipulation of M1/M2 activation may be of potential therapeutic value.

In the central nervous system (CNS), the innate immune response is predominantly mediated by microglia and astrocytes. Microglia play a vital role in both physiological and pathological conditions. Tissue-specific macrophages can be found in most tissues of the body, whereas microglia are present distinctly in the brain. Microglia are derived from primitive yolk sac myeloid progenitors that seed the developing brain parenchyma (Alliot et al., 1999; Ginhoux and Jung, 2014). Microglia represent 10–15% of the total population of cells within the brain and manifest different morphologies across anatomic regions (Lawson et al., 1990; Mittelbronn et al., 2001). Microglia appear to be involved in several regulatory processes in the brain that are crucial for tissue development, maintenance of the neural environment and, response to injury and promoting repair. Similar to peripheral macrophages, microglia directly respond to pathogens and maintain cellular homeostasis by purging said pathogens, as well as dead cells and pathological gene products (Gehrmann et al., 1995; Bruce-Keller, 1999; Stevens et al., 2007; Tremblay et al., 2010; Olah et al., 2011; Paolicelli et al., 2011). In addition, microglial function can be altered by interactions with neurons, astrocytes, migrating T-cells, and the blood–brain barrier itself (Gonzalez et al., 2014).

Under physiological conditions, microglia acquire a neural-specific, relatively inactive phenotype (Schmid et al., 2009) where they sample and inspect the local environment and other brain cells types (Davalos et al., 2005; Nimmerjahn et al., 2005). In a healthy brain, resting quiescent microglia exhibit a ramified morphology, with relatively long cytoplasmic protrusions, a stable cell body and little or no movement (Figure 2). Quiescent microglia extend processes into their surrounding environment (Nimmerjahn et al., 2005). This resting stage is partly maintained by signals conveyed by neuronal and astrocyte-derived factors (Neumann et al., 2009; Ransohoff and Cardona, 2010). The maintenance of this inactive state is regulated by several intrinsic factors like Runx1 (Runt-related transcription factor 1) and Irf8 (Interferon regulatory factor 8), and extrinsic factors such as TREM2 (triggering receptor also expressed on myeloid cells-2), chemokine CX3CR1 and CD200R (Kierdorf and Prinz, 2013). In the normal CNS environment, healthy neurons provide signals to microglia via secreted and membrane bound factors, such as CX3CL1, neurotransmitters, neurotrophins and CD22 (Sunnemark et al., 2005; Frank et al., 2006; Lyons et al., 2007; Pocock and Kettenmann, 2007). In addition, microglia express elevated levels of microRNA-124 which in turn reduces CD46, MHC-II (major histocompatibility complex II) and CD11b, to maintain the quiescent state (Conrad and Dittel, 2011).

As peripheral macrophages respond to endogenous stimuli promoting both pathogenic and protective functions, so do microglia. Upon exposure to endogenous stimuli microglia become activated. Among the gene products released by microglia are various substances including pro-inflammatory cytokines, neurotoxic proteins, chemokines, anti-inflammatory cytokines, and neurotrophic factors (Mahad and Ransohoff, 2003; Block et al., 2007; Benarroch, 2013; Nakagawa and Chiba, 2015). Microglia also display signaling immunoreceptors such as Toll-like receptors (TLRs), scavenger receptors (SRs), nucleotide binding oligomerization domains (NODs) and NOD-like receptors (Ransohoff and Brown, 2012). Fundamentally, the two polar states of microglia, the M1 and M2 phenotypes are associated phenomenologically with injury and homeostasis, respectively (as described below). Differential states of microglial activation within an injured tissue evolve during an inflammatory epoch (Graeber, 2010).

The involvement of innate immunity in PD was first proposed by McGeer et al. (1988) when brain from PD patients showed high levels of reactive microglia that were positive for human leukocyte antigen-D related (HLA-DR) in the substantia nigra and putamen. GWAS indicate that variants in the HLA region are linked to sporadic PD (Hamza et al., 2010; Hill-Burns et al., 2011). Activated microglia in PD brain appear responsible for exacerbating neurodegeneration (McGeer and McGeer, 2004), and the exposure of human neuromelanin discharged from dead DA neurons cause chemotaxis and increases the pro-inflammatory substances in microglial cultures (Wilms et al., 2007). M1 activation-associated inflammatory markers such as MHC-II (Imamura et al., 2003), TNF-α and IL-6 (Boka et al., 1994; Imamura et al., 2003) have been reported in patients with PD. Recent positron emission tomography (PET) studies show that PD patients have cortical microglial activation and lower brain glucose metabolism early in the disease, and imply that microglial activation may be a contributing factor in the disease progression (Edison et al., 2013). PET with inflammatory ligands show elevation in several areas of the basal ganglia involved in PD pathology (Gerhard et al., 2006; Edison et al., 2013; Iannaccone et al., 2013). TLR2 is increased in postmortem PD brain tissue, which correlates with pathological α-synuclein deposition. The neuronal TLR2 rather than glial expression of TLR2 is significantly elevated in PD brain and correlates with disease progression. In addition, TLR2 is strongly localized in α-synuclein positive Lewy bodies (Dzamko et al., 2017). These observations highlight the crucial role of neuroinflammation in PD pathogenesis.

“The dual hit theory” of PD development states that a neurotropic pathogen enters the brain by nasal and/or gastric route by axonal transport, the latter via the vagus nerve (Braak et al., 2003; Hawkes et al., 2009). There is evidence that some forms of α-synuclein can be transmitted from the gut to the brain (Pan-Montojo et al., 2010; Ulusoy et al., 2013; Holmqvist et al., 2014). Instillation of rotenone into the rodent stomach exhibits progressive pathological α-synuclein inclusions in the enteric nervous system, the vagus nerve and subsequently in the brain stem (Pan-Montojo et al., 2010). Vagotomy prevents transport of pathological proteins from the gut to CNS (Phillips et al., 2008; Pan-Montojo et al., 2012). A recent study in Danish patients reveals that those who underwent full truncal vagotomy had lower risk for PD, suggesting that the vagus nerve might be critically involved in PD pathogenesis (Svensson et al., 2015). Another clinical study reports that serum levels of the pro-inflammatory cytokine IL-1β discriminated asymptomatic LRRK2-G2019S carriers from controls and suggests that peripheral inflammation is greater in a percentage of subjects carrying LRRK2-G2019S mutation (Dzamko et al., 2016).

The major peripheral immune cells, T-lymphocytes and B-lymphocytes, are not found in the CNS in normal biological conditions. However, with peripheral inflammation such as infection or injury, blood monocytes, and tissue-resident immune cells are activated and secrete variety of pro-inflammatory mediators including TNF-α, IL-6, and IL-1β. These pro-inflammatory mediators cross the blood–brain barrier leading to the activation of brain resident microglia, which then triggers a neuroinflammatory cascade. The blood–brain barrier is considered to be impermeable to external pathogens and circulating macrophages, hence serving as an additional line of defense to the CNS. Nevertheless, damage to the integrity of the blood–brain barrier renders the brain vulnerable. PET studies on patients with PD reveal dysfunctional blood–brain barrier (Kortekaas et al., 2005). Damage to the blood–brain barrier in rats induce degeneration of dopaminergic neurons in the substantia nigra and activate glial cells (Rite et al., 2007). CD8⁺ and CD4⁺ T cells are observed in the postmortem human PD brain and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of PD during its neurodegenerative phase suggesting T cell-mediated dopaminergic toxicity as a putative mechanism (Brochard et al., 2009). Moreover, rats with ulcerative colitis are more susceptible to LPS-induced dopaminergic neuron loss, blood–brain barrier permeability, microglial activation, and generation of pro-inflammatory mediators suggesting that peripheral inflammation may increase the risk of PD (Villaran et al., 2010). A recent study in the acute MPTP mouse model where nigrostriatal pathologies are not robust show that administration of chemokines [regulated on activation, normal T cell expressed and secreted (RANTES) and eotaxin] that facilitate T cell trafficking can lead to marked nigral α-synuclein pathology, loss of dopaminergic neurons and striatal neurotransmitter depletion, glial-associated inflammation, and motor impairments. However, systemic administration of pro-inflammatory cytokines, TNF-α and IL-1β, did not induce such disease pathologies in this acute MPTP model (Chandra et al., 2017). Taken together these studies suggest a more direct link between peripheral inflammation and, potential to elicit and affect the timing of PD onset.

Mutations in leucine-rich repeat kinase (LRRK2, PARK8) are linked with autosomal dominantly inherited PD and is the greatest known genetic contributor to PD. LRRK2-G2019S mutation in the kinase domain is the most common mutation in both familial and sporadic form of the disease (Goldwurm et al., 2005). GWAS show that the genetic locus containing the LRRK2 gene presents a risk factor for sporadic PD (Singleton and Hardy, 2011). Interestingly, GWAS implicate LRRK2 as a major susceptibility gene in inflammatory bowel diseases that involve chronic inflammation (Barrett et al., 2008; Liu and Lenardo, 2012). LRRK2 is reported to be a target gene for IFN-γ, a M1-activation-associated pro-inflammatory cytokine. LRRK2 expression is elevated in human intestinal tissue of patients with Crohn’s disease inflammation (Gardet et al., 2010). LRRK2 shows high expression in immune cells including microglia and inhibition of LRRK2 function reduces M1-associated inflammation and PD neurodegeneration (Moehle et al., 2012; Daher et al., 2014, 2015; Russo et al., 2015). Collectively, these studies point toward the importance of LRRK2 function in M1-activation responses in PD animal models (Moehle and West, 2015).

Three different missense mutations (A530T, A30P, and E46K) within the open reading frame or duplication or triplication of the wild type α-synuclein gene (SNCA, PARK1) are associated with autosomal dominant PD (Polymeropoulos et al., 1997; Kruger et al., 1998; Singleton et al., 2003; Zarranz et al., 2004). Fibrillar forms of α-synuclein are a major component of the Lewy bodies in both sporadic and familial PD. α-Synuclein, a cytoplasmic protein, can be expressed in microglia and may be involve modulation and pre-sensitization of microglial activation (Barkholt et al., 2012; Roodveldt et al., 2013; Zhang et al., 2017). Most activated microglia in PD patient brains are associated with α-synuclein-positive Lewy neurites (Imamura et al., 2003), and there is significant correlation between the expression of M1 activation-associated marker MHC-II and α-synuclein deposition in the substantia nigra of PD patients (Croisier et al., 2005). Previous studies by our group and others using in vitro and animal models show that both wild type and mutant α-synuclein can modulate microglial activation leading to neuroinflammation. Our previous work shows that α-synuclein activates microglia in a dose-dependent manner in cultured cells and an early microglial activation event occurs in mice overexpressing wild type α-synuclein (Su et al., 2008). Another study reveals that mutant α-synuclein can directly interact with cultured microglia releasing pro-inflammatory substances and mice overexpressing mutant α-synuclein exhibit microglial activation at a very early age (Su et al., 2009). In addition, we show that misfolded α-synuclein induces microglial activation and the release of pro-inflammatory cytokines in BV2 microglial cells (Beraud et al., 2013). Overexpression of α-synuclein in BV2 microglial cells increase pro-inflammatory mediators (TNF-α, IL-6, nitric oxide, COX-2) and induce a reactive microglial phenotype (Rojanathammanee et al., 2011). Interestingly, TLR4, which is activated by LPS to induce microglial M1-phenotype, is reported to mediate α-synuclein-induced microglial phagocytosis, upregulation of pro-inflammatory cytokine expression and ROS generation in primary microglial cultures (Fellner et al., 2013). In addition, α-synuclein is reported to play a crucial role in modulating microglial activation states in postnatal brain derived microglial cultures (Austin et al., 2006). Moreover, neuroinflammation with activated microglia and increased pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ) precedes α-synuclein-mediated neuronal cell death in rats delivered with mutant A53T human α-synuclein (Chung et al., 2009). There is a rich literature on the role of α-synuclein in the progression of PD by inducing microglia activation and neuroinflammation which is reviewed elsewhere (Zhang et al., 2017).

In addition, genes linked to familial recessive PD including phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1, PARK6) and DJ-1 (PARK7) are strongly associated with neuroinflammation. Deletion of PINK1 or DJ-1 causes aberrant expression of genes involved in p38 MAP kinase/NF-κB signaling pathways that regulate innate immune responses in disease models (Cornejo Castro et al., 2010; Akundi et al., 2011).

LPS, a bacterial endotoxin from cell wall of Gram-negative bacteria, induces M1-polarization of microglia through activation the pattern recognition TLR4. LPS administration into rodent brains recapitulate certain characteristics of sporadic PD including progressive degeneration of nigrostriatal dopaminergic pathway and motor anomalies. A single injection or 2-week infusion of LPS in the supranigral region in rat brain causes rapid microglia activation followed by dose and time-dependent degeneration of nigrostriatal dopaminergic circuitry (Castano et al., 1998; Gao et al., 2002; Liu, 2006; Dutta et al., 2008). Direct injection of LPS shows dopaminergic neuron loss specifically in SNpc but not in ventral tegmental area which also houses dopaminergic neurons (Kim et al., 2000). This specific neurotoxicity in SNpc may be attributed to the high proportion of microglia in SNpc compared with other brain regions (Lawson et al., 1990), that may trigger inflammatory events leading to the degeneration of nigrostriatal pathway. Moreover, injection of a TLR3 agonist in the substantia nigra of adult rats induces a sustained inflammatory reaction in the substantia nigra (SN) and dorsolateral striatum, and also increases the vulnerability of midbrain dopaminergic neurons to a subsequent neurotoxic trigger (Deleidi et al., 2010).

A transgenic mouse PD model that overexpresses human wild type α-synuclein, Thy1-aSyn (line 61) (Chesselet et al., 2012), shows microglial activation as early as 1 month and persists until 14 months of age (Watson et al., 2012). Increased levels of TNF-α, TLRs (TLR1, TLR2, TLR4, and TLR8), MHC-II, CD4, and CD8 are observed in Thy1-aSyn mice at different ages. This study also reveals that despite expression of α-synuclein globally in the brain only the regions containing cell bodies and axon terminals of nigrostriatal pathway show early inflammatory response. Another transgenic rodent model overexpressing doubly mutated (A53T and A30P) human α-synuclein show glial mitochondria alterations (Schmidt et al., 2011). In a PD mouse model that overexpresses human α-synuclein by recombinant adeno-associated virus vector, serotype 2 (rAAV2)-mediated transduction in the SNpc, inflammatory responses such as microglial activation and greater infiltration of B and T lymphocytes are observed in addition to dopaminergic neurodegeneration (Theodore et al., 2008). These studies show that animal models overexpressing human or mutant α-synuclein exhibit microglial activation and neuroinflammation.

MPTP, a meperidine analog byproduct, is a neurotoxin that causes acute and irreversible human parkinsonism (Davis et al., 1979; Langston et al., 1983). MPTP is a lipophilic compound that can actively cross the blood–brain barrier and gets oxidized by monoamine oxidase to the toxic cation, MPP⁺ (1-methyl-4-phenylpyridinium) in the glial cells (Langston et al., 1984; Markey et al., 1984). MPP⁺ utilizes the DA transporter (DAT) to get into the dopaminergic neurons. MPP⁺ accumulates in the mitochondria and inhibits the mitochondrial complex I in the electron transport chain (ETC) (Nicklas et al., 1985; Ramsay et al., 1986) resulting in reduced ATP levels and production of ROS (Hasegawa et al., 1990; Chan et al., 1991; Hantraye et al., 1996; Przedborski et al., 1996; Fabre et al., 1999; Pennathur et al., 1999). In animal models, MPTP induces inflammatory responses that lead to neurodegeneration. MPTP causes microglial activation and an increase in M1-associated pro-inflammatory cytokines such as IL-6, IFN-γ and TNF-α. The glial response to MPTP is reported to peak before dopaminergic neuron loss (Czlonkowska et al., 1996; Smeyne and Jackson-Lewis, 2005). In support of these findings, it is reported that mice lacking IFN-γ or TNF-α signaling show resistance to MPTP-induced neurodegeneration (Sriram et al., 2002; Mount et al., 2007). Mice treated with MPTP show T-cell (CD4⁺) infiltration into the substantia nigra and the MPTP-induced dopaminergic neuron loss is attenuated in T-cell deficient mice suggesting a pro-inflammatory role for T-cells (CD4⁺) in MPTP neurotoxicity (Brochard et al., 2009). In addition, treatment with anti-inflammatory agents such as minocycline (Du et al., 2001), ibuprofen (Swiatkiewicz et al., 2013), flavonoid pycnogenol (Khan et al., 2013) and peptide carnosine (Tsai et al., 2010) and inhibition of pro-inflammatory mediators (Watanabe et al., 2004; Zhao et al., 2007), reduce inflammation and prevent neurodegeneration in MPTP animal models.

Microglial activation, astrogliosis and invasion of activated peripheral immune cells trigger deleterious events in the brain that lead to neuronal loss and progression of PD (Hirsch and Hunot, 2009). These observations led to several animal studies and clinical trials to test a variety of established anti-inflammatory molecules (see Table 2). Acetylsalicylic acid, a COX-1/COX-2 inhibitor, exhibits neuroprotective effects in in vitro and in MPTP animal models of PD (Teismann and Ferger, 2001). A prospective clinical study shows that consumption of non-aspirin NSAIDs may delay or prevent the onset of PD (Chen et al., 2003). A Cochrane collaboration study which analyzed several prevention trials and observational anti-inflammatory studies reveals that while ibuprofen use might reduce the risk of developing PD, there is no existing evidence that supports NSAID use in PD prevention (Rees et al., 2011). A clinical study in PD cases shows an association between use of ibuprofen and lesser PD risk. However, this association is not shared by other NSAIDs studied (Gao et al., 2011). Similarly, minocycline, a tetracycline antibiotic that showed promising anti-inflammatory properties in PD animal models (Du et al., 2001; He et al., 2001; Wu et al., 2002), did not provide any symptomatic improvement in PD patients (Ninds Net-Pd Investigators, 2008). See Table 2 for the list of anti-inflammatory agents studied in PD. Hence, NSAID use appears to provide benefits in PD susceptibility in some cases (Wahner et al., 2007; Samii et al., 2009; Gagne and Power, 2010; Steurer, 2011), however, this beneficial effect of NSAIDs were not replicated in several other studies (Shaunak et al., 1995; Bornebroek et al., 2007; Samii et al., 2009; Becker et al., 2011). One study even suggests that anti-inflammatory drug treatment may be detrimental if given at the later stage of the disease (Keller et al., 2011). This therapeutic approach aiming to counteract general neuroinflammation has failed in several disease therapies as reviewed elsewhere (Pena-Altamira et al., 2016). Collectively, these studies indicate that the non-specific inflammatory blockade is unlikely to be beneficial for the disease treatment. Concurrently, the data on the dual role of microglial activation has led to the emergence of the novel therapeutic strategy in other inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis, and multiple sclerosis (MS) (Rau, 2002; Braun et al., 2007; Wilms et al., 2010; Noda et al., 2013). This approach of the M1 to the M2 phenotype shift might be beneficial in neuroprotection compared to completely blocking microglial activation through anti-inflammatory drugs. Hence, a more reasonable approach of more specific treatment related to the M1/M2 activation stage by inhibiting the M1 responses and/or promoting the shift of M1 to M2 phenotypic responses is necessary in PD.

Other novel potential microglial targets for immunomodulation are reviewed elsewhere (Pena-Altamira et al., 2016). These approaches include the following targets: (1) 5′ adenosine monophosphate-activated protein kinase (AMPK): a critical enzyme in cellular energy homeostasis, (2) microglia-produced high-mobility group box-1 (HMGB1): an early released pro-inflammatory cytokine, (3) glycogen synthase kinase-3β (GSK3β): an enzyme that mediates microglial migration and inflammation-induced neurotoxicity, and (4) histone deacetylases (HDACs).

The critical role of microglia in most neurodegenerative pathologies including PD is increasingly documented through many studies. Until recently, microglial activation in pathological conditions was considered to be detrimental to neuronal survival in the substantia nigra of PD brains. Recent findings highlight the crucial physiological and neuroprotective role of microglia and other glial cells in neuropathological conditions. Studies on anti-inflammatory treatments targeting neuroinflammation in PD and other diseases by delaying or blocking microglial activation failed in many trials due to the lack of a specific treatment approach, possibly the stage of disease and an incorrect understanding of mechanisms underlying microglial activation. With the updated knowledge on different microglial activation states, drugs that can shift microglia from a pro-inflammatory M1 state to anti-inflammatory M2 state could be beneficial for PD. The M1 and M2 microglial phenotypes probably need further characterization, particularly in PD pathological conditions for better therapeutic targeting. We support targeting of microglial cells by modulating their activation states as a novel therapeutic approach for PD.

HF conceived of the project, SS and HF wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.