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Increasing reports support that air pollution causes neuroinflammation and is linked to central nervous system (CNS) disease/damage. Diesel exhaust particles (DEP) are a major component of urban air pollution, which has been linked to microglial activation and Parkinson's disease-like pathology. To begin to address how DEP may exert CNS effects, microglia and neuron-glia cultures were treated with either nanometer-sized DEP (< 0.22 μM; 50 μg/mL), ultrafine carbon black (ufCB, 50 μg/mL), or DEP extracts (eDEP; from 50 μg/mL DEP), and the effect of microglial activation and dopaminergic (DA) neuron function was assessed. All three treatments showed enhanced ameboid microglia morphology, increased H2O2 production, and decreased DA uptake. Mechanistic inquiry revealed that the scavenger receptor inhibitor fucoidan blocked DEP internalization in microglia, but failed to alter DEP-induced H2O2 production in microglia. However, pre-treatment with the MAC1/CD11b inhibitor antibody blocked microglial H2O2 production in response to DEP. MAC1−/− mesencephalic neuron-glia cultures were protected from DEP-induced loss of DA neuron function, as measured by DA uptake. These findings support that DEP may activate microglia through multiple mechanisms, where scavenger receptors regulate internalization of DEP and the MAC1 receptor is mandatory for both DEP-induced microglial H2O2 production and loss of DA neuron function.
Accumulating evidence links air pollution exposure to central nervous system (CNS) pathology and disease (Block and Calderon-Garciduenas 2009; Guxens and Sunyer 2012). Epidemiology studies have shown that exposure to high levels of air pollution is associated with a deficit in neuropsychological development in children (Guxens et al. 2012; Vrijheid et al. 2012), cognitive decline in the elderly (Calderon-Garciduenas et al. 2008a; Suglia et al. 2008; Chen and Schwartz 2009; Ranft et al. 2009; Power et al. 2011; Weuve et al. 2012), behavioral deficits (Wang et al. 2009), autism (Volk et al. 2011), and an elevated stroke risk (Donnan et al. 1989; Villeneuve et al. 2006; Henrotin et al. 2007). Human studies have also revealed that individuals living in highly polluted cities show Alzheimer's disease (AD)-like and Parkinson's disease (PD)-like pathology, when compared to individuals living in cities with less pollution (Calderon-Garciduenas et al. 2004, 2010, 2012; Block and Calderon-Garciduenas 2009; Morales et al. 2009). More specifically, high levels of air pollution were associated with elevated markers of neurodegenerative disease in humans, including tau phosphorylation, diffuse β amyloid plaque deposition, and α synuclein aggregation (Calderon-Garciduenas et al. 2004, 2010, 2012; Morales et al. 2009). Human reports also reveal that air pollution causes oxidative stress, neuroinflammation, and microglial activation in the brain (Calderon-Garciduenas et al. 2008b). Consistent with human reports, animal studies have found that exposure to air pollution causes lipid peroxidation (Zanchi et al. 2010), DNA damage (Calderon-Garciduenas et al. 2003), protein nitration (Levesque et al. 2011b), elevated cytokines (Gerlofs-Nijland et al. 2010; Levesque et al. 2011b; Bos et al. 2012; Cassee et al. 2012), chemokine increases (Levesque et al. 2011b), aggregated α synuclein (Levesque et al. 2011a), increased expression of Aβ-42 in the brain (Levesque et al. 2011a), and activation of microglia (Levesque et al. 2011b; Morgan et al. 2011; Bolton et al. 2012). However, the underlying mechanisms responsible for how air pollution may cause neuroinflammation, impact neuropathology, and lead to CNS disease are largely unknown.
Diesel Exhaust (DE) has received significant attention as a human health concern in both ambient and occupational exposure conditions (Pronk et al. 2009; Hesterberg et al. 2010). DE is a major component of pollution near roadways and urban pollution (Ma and Ma 2002; Hesterberg et al. 2010), where several studies have documented the CNS effects of DE. For example, acute DE exposure has been shown to affect electroencephalogram parameters in adult human subjects (Cruts et al. 2008). Animal research also points to the prenatal period as a critical period of vulnerability, as maternal DE exposure has been shown to decrease brain DA levels and cause motor deficits in offspring (Yokota et al. 2009; Suzuki et al. 2010). Mice exposed to nanoparticle-enriched DE show elevated neuroinflammation and performance deficits in hippocampal-dependent spatial learning and memory tasks (Win-Shwe et al. 2011). Short-term studies (up to 1-month exposure) show pro-inflammatory factors, such as TNFα, in the adult brain with DE exposure, using month-long inhalation models (Gerlofs-Nijland et al. 2010; Levesque et al. 2011b; Cassee et al. 2012), intratracheal administration directly into the lung (Levesque et al. 2011b), and a 2 h long exposure by nose-only inhalation (van Berlo et al. 2010). DE exposure also causes elevated neuroinflammation with subchronic (6 month) exposure in certain vulnerable brain regions (Levesque et al. 2011b). In fact, we have previously shown that DE elevates α synuclein levels in the midbrain, indicating that DE may impinge on PD pathology. Thus, while there are clear CNS effects with DE exposure, the underlying mechanisms are poorly understood.
At present, there are several hypotheses regarding how air pollution affects the brain. It has been proposed that soluble peripheral signals in the blood (e.g., circulating cytokines or modified lipids and proteins) (Levesque et al. 2011b), neuronal signals from the periphery, translocation of the particle components of air pollution (particulate matter, PM) to the brain (Gillespie et al. 2011), and the transfer of the chemical constituents adsorbed on the PM (e.g., polyaromatic hydrocarbons) (Cordier et al. 2004) to the brain may all regulate how air pollution cases neuroinflammation and neuropathology (Block and Calderon-Garciduenas 2009; Tonelli and Postolache 2010; Lucchini et al. 2012). While it is likely that these pathways interact to contribute to CNS health effects, post-mortem sampling has identified PM in the human brain (Calderon-Garciduenas et al. 2008b), emphasizing the importance of understanding how PM and its adsorbed chemical compounds affect cells in the brain.
Microglia are the resident innate immune cell in the brain and are activated in response to diverse stimuli, including air pollution (Block and Calderon-Garciduenas 2009). Microglia have been implicated in the progressive nature of diverse neurodegenerative diseases, including PD (Block et al. 2007; Schwab and McGeer 2008; Tansey and Goldberg 2010; Kraft and Harry 2011; Cunningham 2013). Consistent with human studies (Calderon-Garciduenas et al. 2008b), we and others have demonstrated in rodent models that air pollution activates microglia (Levesque et al. 2011b; Morgan et al. 2011). Our previous work also indicates that measures of neuroinflammation in response to DE exposure in vivo are the highest in brain regions with the highest levels of the IBA-1 microglial marker, such as the midbrain, which houses the substantia nigra that is damaged in PD (Levesque et al. 2011b). We have also shown that nanometer-sized DE particles (which are components of air pollution believed to reach the brain) activate microglia in vitro, which is then neurotoxic through the production of reactive oxygen species (Block et al. 2004). Recently, we demonstrated in vitro that low concentrations of DEP amplify the microglial response to pro-inflammatory stimuli (Levesque et al. 2011b). Thus, evidence supports that DE may be a common, chronic source of microglial activation in the environment. At present, how DE causes neuroinflammation, microglial activation, and neuropathology remains unknown.
This study begins to address these issues by focusing on how DEP may be activating microglial cells to impair DA neuron function. Here, using cell lines and primary cultures we test the ability of: (i) components of DEP (ultrafine carbon particles and diesel exhaust extracts) and (ii) pattern recognition receptors (MAC1 and scavenger receptors) in microglial activation and loss of DA neuron function.
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Accumulating evidence points to microglial activation as a contributing factor to neuropathology, neuroinflammation, and oxidative stress in response to air pollution exposure, but the specific responses and the mechanisms driving how microglia become activated are as of yet unresolved. Recent studies have identified PM from urban air pollution in human brains (Calderon-Garciduenas et al. 2008b), and animal studies have revealed that various inhaled nanoparticles translocate to the brain (Lucchini et al. 2012), supporting that inhaled PM interacts with cells in the brain parenchyma. Here, we addressed how the particle components of DE and the chemical compounds contained on the particle itself activate microglia to impact DA neuron function in vitro. We also began to explore the identity of the pattern recognition receptors necessary for microglia to respond to DEP and the consequence for DA neuron function.
To begin to understand how DEP activates microglia, we assessed the role of the chemical extracts from DEP (eDEP) and the nanometer-sized particle (ufCB) on microglial activation and DA function, when compared to DEP. Figure 1 shows that both eDEP and ufCB elevated H2O2 production and induced an ameboid, activated microglia morphology, similar to DEP. Figure 2 demonstrates that similar to DEP, both eDEP and ufCB reduced DA uptake and neuron morphology indicative of damage, indicating that both components impair DA neuron function in vitro. Together, these data indicate that DEP are a complex trigger of microglial activation, where multiple characteristics of DEP have the potential to activate microglia and impair DA neuron function. Notably, there are over 300 chemical compounds adsorbed on DEP (Ma and Ma 2002), many of which have the potential to be neurotoxic. As such, there is a significant need for future research to further refine mechanistic studies by identifying and quantitating the amount of PM and associated chemicals that reach the brain in vivo upon DE exposure.
Microglia actively survey the brain environment (Nimmerjahn et al. 2005) and rapidly respond to large molecular patterns (e.g., α synuclein, LPS, neuromelanin, and Aβ) that trigger a pro-inflammatory response with pattern recognition receptors (Block et al. 2007). Scavenger receptors are pattern recognition receptors expressed on multiple cell types, including microglia, and are broadly defined as a family of molecules that share the ability to bind polyanionic ligands, which include both pathogens/particles and ligands of self-origin (Wilkinson and Khoury 2012). Class A scavenger receptors are a subgroup that are essential for host defense against several bacterial and viral pathogens (Wilkinson and Khoury 2012). For example, class A scavenger receptors have been implicated in microglial activation, internalization, and Reactive Oxygen Species production in response to Aβ (Wilkinson and Khoury 2012), emphasizing the potential role of this receptor subtype in neurotoxic microglial activation. Here, we demonstrate that while scavenger receptors regulate microglial internalization of DEP (Fig. 3), these receptors have no effect on DEP-induced H2O2 production (Fig. 4). The importance of scavenger receptors for DEP clearance without reactive oxygen species production supports a beneficial role for these receptors, similar to what has been found for the microglial response to both LPS (Pei et al. 2007) and α synuclein (Zhang et al. 2007).
The MAC1 pattern receptor is selectively expressed on cells of myeloid lineage, such as microglia (Akiyama and McGeer 1990), binds LPS (Wright and Jong 1986; Wright et al. 1989), and was previously identified as a TLR4-independent receptor for LPS in phagocytes (Perera et al. 1997), including microglia (Pei et al. 2007). We have previously shown that MAC1 regulates neurotoxic reactive microglia microgliosis in response to the DA neurotoxin MPTP (Hu et al. 2008), is responsible for LPS-induced extracellular super-oxide production in microglia (Pei et al. 2007), and is a component of LPS-induced DA neurotoxicity (Pei et al. 2007). In addition, other labs have also shown that microglial MAC1 plays a role in α synuclein-induced (Zhang et al. 2007) and neuromelanin-induced (Zhang et al. 2011) DA neurotoxicity, further supporting a key role for MAC1 in microglia-mediated neurotoxicity. In this study, we demonstrate that MAC1 is essential for DEP-induced H2O2 production (Fig. 5) and loss of DA function (Fig. 6), emphasizing a key role for both microglia and this receptor in the deleterious effects of DEP.
In summary, DEP are a complex trigger of microglial activation that cause activated microglial morphology and reactive oxygen species production (superoxide and H2O2), without cytokine (TNFα and IL-1β) or nitric oxide production. Data support that both the adsorbed compounds (eDEP) and the carbon particles (ufCB) are capable of activating microglia and may contribute to the microglial response to DEP. Further, scavenger receptors were shown to mediate internalization and clearance of DEP without H2O2 production, which is likely beneficial. However, MAC1 was shown to mediate the microglia H2O2 response to DEP and the associated loss of DA neuron function, demonstrating a deleterious role for this pattern recognition receptor. While other pattern recognition receptors may also contribute, these data support that DEP can exert deleterious effects through microglia and that the MAC1 pattern recognition receptors may be key to this process, providing much needed insight into the mechanisms through which air pollution can impact the brain.