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Neurochemistry International
journal homepage:www.elsevier.com/locate/neuint
Glial smog: Interplay between air pollution and astrocyte-microglia interactions
Mireia Gómez-Budia
a, Henna Konttinen
a, Liudmila Saveleva
a, Paula Korhonen
a, Pasi I. Jalava
b, Katja M. Kanninen
a, Tarja Malm
a,∗aA.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
bDepartment of Environmental and Biological Sciences, University of Eastern Finland, Kuopio, Finland
A R T I C L E I N F O Keywords:
Intercellular communication Priming
Microglia Astrocyte Air pollutants Neuroinflammation
A B S T R A C T
Every second we inhale a danger in the air; many particles in the atmosphere can influence our lives. Outdoor air pollution, especially particulate matter is the largest environmental risk factor and has been associated with many cardiovascular and lung diseases. Importantly, air pollution has recently been discovered to also impact the brain. Here, we review the effects of air pollution on glial cells of the brain, astrocytes and microglia, and the tightly controlled interplay between these cell types. We focus on how traffic related air pollutants which include both gaseous and particulate emissions and their secondary products influence the intercellular communication of microglia and astrocytes. Finally, we place these air pollution and glial interactions in a larger context by discussing their impact on neurodegeneration.
1. Introduction
Traffic-related air pollutants (TRAP) are major contributors to global air pollution. The adverse associations between TRAPs and neurological diseases are increasingly indicated in clinical and epide- miological studies (The Lancet Neurology, 2018). Air pollutants may contribute to brain effects through two main routes: the nasal and the respiratory intake pathways (Genc et al., 2011) (Fig. 1). In the case of nasal pathway, air pollutants enter through inhalation and cross the olfactory mucosa to reach the brain directly. In the respiratory intake pathway, the particles may enter the brain by passing from the lungs into the bloodstream and through the blood-brain barrier (BBB), or they may have secondary effects through systemic inflammation. Neurode- generative brain diseases, including Alzheimer's disease (AD), Parkin- son's disease (PD), Amyotrophic Lateral Sclerosis (ALS), and Hunting- ton's disease, as well as acute neurological diseases such as cerebral stroke, are characterized by progressive or sudden loss of neurons and their connections (Price et al., 2007). The main characteristics of these diseases could be triggered by the effects of air pollution.
Traditionally, central nervous system (CNS) studies have primarily focused on neurons: how they work and how they are affected in dis- ease conditions. Indeed, neurons are extremely sensitive to any dis- turbances in brain homeostasis and even the slightest external insult is
likely to affect their functions (Kulkarni et al., 2018). However, during the recent years, the interest of the neuroscience field has turned to- wards other types of cells in the brain, including astrocytes, microglia, oligodendrocytes, NG-2 glia, radial glia and ependymal cells. Glial cells, being extremely sensitive to any external stimuli, are the first sensors and responders to brain penetrating pollutant particles and systemic inflammation (Yang and Zhou, 2019). Indeed, glial cells are severely impacted by TRAPs, and on the other hand, their malfunctions promote and gear the neurodegenerative processes in the brain diseases (Genc et al., 2011). In this review, we focus on the interaction between mi- croglia and astrocytes, and how exposure to TRAPs influence this tightly controlled communication, sensitizing the brain to neuronal malfunctions or aggravating the pathology of various brain diseases.
2. Microglia
Microglia are the resident immune cells of the CNS that constantly survey the surrounding environment with their long cellular processes.
Unlike other brain cells, microglia originate from the yolk sac as pri- mitive hematopoietic stem cells during the early development and migrate to the CNS, where they expand in numbers and maturate to microglia (Ginhoux et al., 2013). After birth, the number of microglia decreases and then stabilizes to adult levels, accounting for 5–15% of
https://doi.org/10.1016/j.neuint.2020.104715
Received 14 January 2020; Received in revised form 5 March 2020; Accepted 6 March 2020
∗Corresponding author. Virtanen Institute for Molecular Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland.
E-mail addresses:mireiag@student.uef.fi(M. Gómez-Budia),henna.konttinen@uef.fi(H. Konttinen),liudmila.saveleva@uef.fi(L. Saveleva), paula.korhonen@uef.fi(P. Korhonen),pasi.jalava@uef.fi(P.I. Jalava),katja.kanninen@uef.fi(K.M. Kanninen),tarja.malm@uef.fi(T. Malm).
Available online 10 March 2020
0197-0186/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T
all brain cells (Allen and Lyons, 2018).
Microglia shape the developing neuronal network by modulating developing synapses together with astrocytes. Microglia-secreted fac- tors such as interleukin 10 (IL-10) and brain-derived neurotrophic factor (BDNF) have been shown to induce synapse formation (Lim et al., 2016; Parkhurst et al., 2013). In addition, microglia eliminate un- needed synapses based on the activation status of the neurons, and participate in the formation of the permanent neuronal network (Bian et al., 2015; Chung et al., 2013; Lee and Chung, 2019). Microglia eliminate synapses via the classical complement pathway; where the pathway initiation component C1q is congregated into unnecessary synapses directing them under microglial complement-mediated pha- gocytosis (Schafer et al., 2012). In addition, the fractalkine pathway mediates microglial engulfment of synapses: knocking out microglial fractalkine receptors increases the number of immature synapses, leads to improper neuronal activity and disturbances in the behavior of the animals (Hoshiko et al., 2012; Zhan et al., 2014). In neurodegenerative diseases such as AD, abnormal synaptic loss driven by glial cells is one of the earliest pathological features (Hong et al., 2016; Litvinchuk et al., 2018).
In addition to synaptic plasticity, microglia maintain CNS home- ostasis and support neuronal functions by secreting survival signals such as nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) (Subhramanyam et al., 2019; Ueno et al., 2013). Microglia ra- pidly sense any disturbances in the CNS, including pathogens, apoptotic neurons, other cell debris, misfolded proteins or amyloid β (Aβ) de- posits, as well as brain entering air pollutants (Block and Calderón- Garcidueñas, 2009; Dani et al., 2018; Hoogland et al., 2015; Kaur et al., 2019). Damaged neurons, on their part, secrete apoptotic signals to promote microglial phagocytosis by activation of the phagocytotic triggering receptor expressed on myeloid cells 2 (TREM2)-mediated pathway (Takahashi et al., 2007). In literature, microglia are often di- vided into proinflammatory and anti-inflammatory phenotypes (Tang and Le, 2016). However, the current understanding is that microglial activation is far more versatile than previously acknowledged and highly dependant on their surroundings. Thus, there are a variety of different microglial phenotypes and subtypes with different functions and thus, this simplified classification is not accurate (Stratoulias et al., 2019; Subhramanyam et al., 2019; Tang and Le, 2016). Resting mi- croglia have small cell somas and long thin processes that they use for the surveillance of their microenvironment (Kettenmann et al., 2011).
Upon stimuli, microglia change their morphology and heavily activated microglia resemble somewhat peripheral macrophages in their amoe- boid morphology: their somas increase in size, their processes become shorter and they start to migrate towards the disturbance attracted by the chemotactic molecules (Eyo et al., 2014; Subhramanyam et al.,
2019).
Microglial cells elicit proinflammatory activation in response to bacterial lipopolysaccharide (LPS), beta-amyloid (Aβ), α-synuclein, and importantly, to air pollution particles (Block and Calderón- Garcidueñas, 2009; Dani et al., 2018; Hoogland et al., 2015; Kaur et al., 2019; Zhang et al., 2017). This is characterized by secretion of proin- flammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor α (TNFα), reactive oxygen species, and other factors that may be harmful for normal neuronal functions (Cherry et al., 2014; Tang and Le, 2016). In the healthy brain, microglial activation is temporary and the cells turn back to the homeostatic state once the disturbance is eliminated (Kettenmann et al., 2011). However, if the clearance fails, as occurs in neurodegenerative diseases, this inflammatory activation may turn from acute to chronic, causing neuronal destruction (Tang and Le, 2016). In the resolving phase of neuroinflammation, microglial alter- native activation is thought to be anti-inflammatory and is elicited by cytokines such as interleukin-4 (IL-4) and interleukin-13 (IL-13), after which they start to secrete IL-10 and other factors, including Arginase-1 and Chitinase-3 which mediate tissue repair and wound healing and are considered to be beneficial (Cherry et al., 2014; Tang and Le, 2016).
Proper regulation of the various microglial functions is extremely im- portant since prolonged proinflammatory activation of microglia is harmful for the CNS tissue (Cherry et al., 2014; Tang and Le, 2016).
Modulation of microglial activation is currently a popular approach as a treatment strategy for several diseases, including cerebral stroke (Korhonen et al., 2015) spinal cord injury (Pomeshchik et al., 2015) and AD (Michaud et al., 2013).
3. Astrocytes
Astrocytes take part in many key functions in the CNS tissue, in- cluding homeostasis, maintenance and defense. Astrocytes can be di- vided into several subtypes based on their morphology, markers they express and their location in the CNS. According to the traditional classification astrocytes are divided into four major types. Protoplasmic astrocytes have several stem branches and thin highly branched pro- cesses and they are located in the grey matter. Fibrous astrocytes with long fiber-like processes are found in the white matter, velate astrocytes in the cerebellum and radial astrocytes in several parts of the CNS (Sofroniew and Vinters, 2010; Verkhratsky et al., 2017). More recently it has become evident that like microglia, astrocytes also display diverse activation states (Liddelow and Barres, 2017).
During the development, astrocytes participate, together with mi-
croglia, in elimination of excess synapses from neurons. This function is
extremely important as impairments in this process may lead to over-
activation of neurons and hyperactivated neuronal circuits, and in the
Fig. 1. A depiction of the potential route and effect of traffic related air pollution (TRAP) derived particulate matter (PM) on the neu- rons and glia in the brain.PM consists of large (PM10), fine (PM2.5) and ultra-fine (UFP, PM0.1) particles. PM10s are trapped in the upper re- spiratory tract and PM2.5s in the lungs, whereas UFPs can reach the interstitium and blood through alveoli. All fractions can induce sys- temic inflammation which in turn may con- tribute to effects seen in brain. UFP can also enter the CNS directly across the olfactory epi- thelium and through BBB from bloodstream. In the brain, PM-induced effects are demonstrated in the neuron-glia cross-talk manifested with microglial and astrocytic activation accom- panied with reactive oxidative stress (ROS) and neuronal death.worst case, to neurological diseases such as epilepsies (Lee and Chung, 2019). Astrocytic elimination of synapses is mediated via Multiple EGF Like Domains 10 (MEGF10) and Mer tyrosine-protein kinase (MERTK) phagocytic pathways (Chung et al., 2013) and via inositol 1,4,5-tri- phosphate receptor type 2 (IP3R2) and P2Y purinoreceptor 1 (P2RY1) dependent signaling (Yang et al., 2016). It also continues during adult life: astrocytes engulf both excitatory and inhibitory active synapses and are thus involved in memory formation and learning (Chung et al., 2013; Lee and Chung, 2019). In addition, astrocytes contribute to the formation of new functional synapses by secreting several synaptogenic factors, including thrombospondins 1 and 4 (Christopherson et al., 2005) glypicans 4 and 6 (Allen et al., 2012) and apolipoprotein E (Mauch et al., 2001).
Astrocytes are present throughout the CNS and they are connected with each other by connexin gap junctions (Nielsen et al., 2017). Via their end feet the astrocytes participate in the formation of the BBB, which is a layer of pericytes and endothelial cells surrounding the blood vessels in the brain connected by tight junctions. This enables astro- cytes to control the transport of a variety of molecules across the BBB (Nielsen et al., 2017), providing nutrients and physical support for neurons. The activity of neurons and adjacent changes in glutamate levels control the flow of nutrients and glucose, the most important energy source of the CNS, from the blood vessels to neurons via as- trocytes (Rouach et al., 2008). One of the most important functions of astrocytes is a removal of excess neurotransmitters such as glutamate, γ- butyric acid (GABA) and glycine from the synaptic cleft, using specific transporters, such as excitatory amino acid transporter 1 (EAAT-1) and EAAT-2 (Rose et al., 2018). Astrocytes metabolize the internalized neurotransmitters and recycle the metabolites back for neuronal use (Kojima et al., 1999; Mahmoud et al., 2019; Rothstein et al., 1996).
Glutamate is one of the most common excitatory neurotransmitters, up to 90% of which is circulated by astrocytes (Kojima et al., 1999;
Mahmoud et al., 2019; Rothstein et al., 1996). When present in excess, glutamate causes hyperactivation of neurons, also known as ex- citotoxicity, which is a common feature in several neurological dis- eases.
In addition, astrocytes can remove metabolic products from neurons back into the circulation, and control the fluid, ion and pH balance of the CNS via their aquaporin channels (Simard and Nedergaard, 2004).
There is also evidence that astrocytes control the blood flow in the brain vessels by secreting mediators that are able to adjust the diameter of the blood vessels according to changes in neuronal activity (Gordon et al., 2007; Koehler et al., 2009).
4. Microglia - astrocyte interaction
Microglia and astrocytes are in a constant intimate crosstalk, thereby influencing the activity of one another. This interplay is ex- tremely complex and not completely understood. However, to date, several single molecules responsible for specific effects in the crosstalk have been elucidated. The different mechanisms and pathways med- iating this crosstalk will be briefly introduced in this section.
Microglia-astrocyte crosstalk is largely mediated by a variety of small molecules. Microglia have been known for a long time to be mediated by LPS, yet the mechanism behind LPS-mediated astrocytic activation
in vivoremained unknown for a long time (Sun et al., 2016).
Since astrocytes do not express receptors for LPS, it became evident that microglia are responsible for LPS induced astrocytic responses. The capability of microglia to influence astrocytic activation states was ni- cely demonstrated by Liddelow et al. who showed that microglia-re- leased interleukin 1α (IL-1α), TNFα and C1q direct astrocytes towards a more proinflammatory, synapse stripping and neuron-killing state (Liddelow et al., 2017). On the other hand, astrocytes are similarly capable of influencing the function of microglia (Liu and Quan, 2018).
The released molecules with the ability to modulate the function of their counterpart is not restricted to the cytokines mentioned above.
Indeed, in addition to interleukins and other proinflammatory cyto- kines, the released molecules also include proteases and complement proteins, growth factors and mitogenic proteins all of which aid the mounting of the immune responses and the remodeling of the CNS in a tightly regulated, context-dependent manner (Jha et al., 2019).
Whereas most of the studies concentrate on single molecules re- sponsible for specific effects, it is important to keep in mind that the crosstalk between the glial cells is likely to be extremely complex.
Interleukin 1 (IL-1) can mediate a great variety of functions in the CNS and in many different cell types (Quan et al., 2011). Microglia appear to be the major source of IL-1 release. Astrocytes express the receptor for IL-1 and this is one way how microglia regulate astrocytic activation (Liu and Quan, 2018; Pinteaux et al., 2002). Other microglial released molecules can act in a completely opposite manner; for ex- ample IL-1ß together with TNFα can trigger the inhibition of the glial crosstalk (Abudara et al., 2015). Apparently, even the astrocytic inner communication via gap junctions, hemichannel interactions formed by six connexin monomers, can be downregulated by microglial cells (Retamal et al., 2007; Yin et al., 2018). In fact, it has actually been proposed that the main component of the hemichannel interaction, connexin 43 (Cx43), is down-regulated in co-cultures of microglia and astrocytes. This impairment will clearly lead to an inflammatory re- sponse by the opening of the hemichannels thereby the increasing the levels of intracellular calcium and extracellular glutamate (Abudara et al., 2015). Moreover, not only the inflammatory response but also other astrocytic functions like astrogenesis or even differentiation can be sensed and changed by microglial derived molecules, such as IL-6, nitric oxide (NO) or leukemia inhibitory factor (LIF) (Jha et al., 2019).
Microglial and astrocytic functions can also be interlaced by ade- nosine triphosphate (ATP)-dependent calcium dynamics. Activated as- trocytes have increased intracellular calcium levels; indeed, the calcium wave is propagated through hemichannels and purinergic receptors.
These glial cells are capable of regulating their fellows’ ATP production by secreting transcription growth factor ß (TGFß) (Liu et al., 2011;
Orellana et al., 2013; Pascual et al., 2012). The bidirectional fine communication between the glial cells can extend to many other small molecules released by astrocytes. Concrete examples are the acute- phase protein orosomucoid 2 (ORM2) which can by blockage of mi- croglial C–C chemokine receptor 5 (CCR5) affect microglial migration and activation, leading to an anti-inflammatory outcome (Jha et al., 2019; Jo et al., 2017). Dumping of microglial phagocytic function as well as enhanced migration are mediated by astrocytic plasminogen activator inhibitor type 1 (PAI-1) and neurotrophic glial cell line-de- rived neurotrophic factor (GDNF) (Jeon et al., 2012; Rocha et al., 2012). Astrocytes and microglia can also synergically increase their intracellular calcium levels via SDF-1α and CXCR4 ligand (Bezzi et al., 2001).
It is important to take into account that apart from microglia-as- trocyte communication these cells also intimately communicate with neurons. Indeed, fractalkine (CX3CL1) is one of the most important chemokines produced by neurons and can be sensed by microglial CX3CR1 receptors (Cardona et al., 2006). Deletion of CXCR1 has been shown to exacerbate demyelination and neuronal damage as deficient microglia are incapable of acquiring an anti-inflammatory phenotype (Cardona et al., 2018; Garcia et al., 2013). Although many molecules have been identified to be the mediators of the crosstalk between mi- croglia and astrocytes, their interconnection is much more complicated and still warrants further studies. A global overview of this linkage is needed to efficiently harness the cells for therapeutic benefit.
5. Traffic related air pollutants (TRAP)
Traffic is a major source of air pollution in urban areas across the
world. TRAP usually refers to air pollution derived from combustion of
fuel in motor vehicles. TRAP include both gaseous and particulate
emissions and their secondary products resulting from atmospheric
processes (Giles et al., 2011). In this review, we have focused especially on the particulate form of TRAP.
Outdoor air pollution, especially particulate matter (PM) is the largest environmental risk factor for deaths and disability-adjusted life- years, causing approximately 4.2 million premature deaths annually (Cohen et al., 2017) and this estimate may even be substantially larger (Burnett et al., 2018). It is expected that by 2050 the contribution of outdoor air pollution to premature mortality can be even doubled due to urbanization and severe air pollution problems (Lelieveld et al., 2015). Both diesel and gasoline vehicle exhaust have been in associa- tion with hospital admissions (Samoli et al., 2016) as well as daily mortality (Tobías et al., 2018). In addition to premature deaths, parti- culate air pollution is associated with respiratory diseases (Cohen et al., 2017), cardiopulmonary diseases (Chen et al., 2017a,b) and recently also with cognitive disorders and CNS diseases (Calderón-Garcidueñas et al., 2019; Maher et al., 2016; Power et al., 2011). In a very recent publication, it was found that even low levels of air pollution are not safe. Traffic related black carbon emissions were reported to be in stronger association with adverse cardiac effects and stroke than any other type of emission sources (Ljungman et al., 2019).
Air pollutants, their sources and concomitant adverse health con- sequences have considerable variation in different parts of the world (Li et al., 2019). It has been estimated that 33–55% of premature deaths may be linked with emissions from traffic, powerplants and domestic heating in developed countries (Lelieveld et al., 2015). Living near to road traffic has been associated with higher risk for various adverse health outcomes and TRAP has been shown to cause cardiovascular diseases, asthma, chronic obstructive pulmonary disease (COPD), other impaired lung functions as well as premature deaths from these reasons.
The near vicinity of traffic seems to enhance the observed adverse health effects up to hundreds of meters from roads (HEI, 2010). In fact, people who already have asthma or diabetes are in greater risk of de- veloping COPD when living near busy traffic (Andersen et al., 2011).
Living near to major traffic may also increase the risk of premature death (Finkelstein et al., 2004). Furthermore, increased prevalence of heart attacks has been observed while commuting in traffic (Peters et al., 2004) and, in addition to these rather commonly known adverse effects, it has been shown that living near to traffic may cause dementia and poor level of cognition (Chen et al., 2017a,b; Power et al., 2011).
In densely populated urban areas traffic is a major source of at- mospheric aerosol particles (Pey et al., 2009). Ultrafine particles (par- ticles smaller than 100 nm in diameter) derived from traffic are rather short lived and there exists a strong assumption that these particles may be of the greatest importance when considering the relationship be- tween TRAP and adverse health outcomes. Indeed, it has been observed that smaller particles may be in stronger connection with adverse health effects than larger particles (Yang et al., 2018). Moreover, it has been observed that the lung deposition and retention of smaller nano- particles from exhaust emissions are higher than for larger particles (Muala et al., 2015).
6. Toxicity of exhaust particles
The toxicological potency of diesel and gasoline exhaust is stronger than most of the other emission sources (Park et al., 2018). The Inter- national Agency for Research on Cancer (IARC) has categorized diesel exhaust as carcinogenic to humans (Group 1) and gasoline exhaust to possibly carcinogenic to humans (Group 2B) (IARC Working Group on the Evaluation of Carcinogenic Risks to humans, 2013). Most of the toxicological studies, conducted with TRAP have been conducted with collected particles from exhaust, and the majority of studies include the exhaust from old technology engines (Hesterberg et al., 2011). Thus, there is now a need to study emissions from engines with modern technologies. Along with the new emission technologies, the mass concentrations from exhaust have in recent times become lower.
However, at the same time, ultrafine or nanoparticles have become a
major concern in traffic environments and recent findings indicate that modern diesel and gasoline engines produce emissions in the nano- particle size range (Liati et al., 2018; Platt et al., 2017; Wu et al., 2017).
After the combustion of different fuels, diesel engine exhaust causes increased inflammation, genotoxicity, cytotoxicity and oxidative stress in macrophage cell cultures (Jalava et al., 2012) and epithelial cells (Bengalli et al., 2017). Emissions from second generation biofuel and compressed natural gas (CNG) combustion have induced generally lower responses than traditional fossil diesel fuel combustion (Jalava et al., 2012). However, Skuland et al. (2017) found that biodiesel blends can also increase the toxic potential of the emission exhaust in bronchial epithelial cells. In contrast, in their 28-day rat inhalation study, a similar effect was not seen (Magnusson et al., 2019). The majority of the literature is limited to diesel exhaust, but it has been shown that gasoline engines emit large quantities of nanoparticles, carrying genotoxic polycyclic aromatic hydrocarbons (PAH) com- pounds (Muñoz et al., 2018). Indeed, a recent study reported that particles emitted from Euro 6 car engines had very different tox- icological profiles when diesel, gasoline E10, Ethanol E85 and CNG powered cars were compared (Hakkarainen et al., n.d.). The oxidative potential of the particles from flexifuel vehicle have decreased along with the increase of ethanol in the fuel (Yang et al., 2019). Although engine exhaust emissions have been intensively studied in the past decades, there is very limited information available on the emissions and their toxicity from modern engines, fuels and after treatment sys- tems. Therefore, it is important that these newer technologies are also investigated, and that the toxicity screening also includes other systems in addition to assessment of the respiratory effects of the emitted par- ticles.
7. Evidence for microglial involvement in TRAP effects
A large body of evidence implicates exposure to TRAP in induction of neuroinflammation manifested by microglial activation and oxida- tive stress leading to neurodegeneration across different species.
Human post mortem brain tissues from individuals living in highly polluted Mexico City exhibit abundant PM depositions in olfactory bulb (OB) (Calderón-Garcidueñas et al., 2008b) and neuropathological changes with multiple inflammatory indicators (Calderón-Garcidueñas et al., 2018b, 2012, 2008b, 2004, 2002). These studies suggest that air pollution can cause changes reminiscent to neurodegenerative pa- thology already starting in childhood. Mexico City canines show ad- verse impacts of heavy air pollution evidenced as chronic inflammation accompanied with altered glial cell levels and neurodegeneration (Calderón-Garcidueñas et al., 2008a, 2003, 2002). In line with these studies performed in one research group in a highly polluted area, microglia-related brain pathologies are as well corroborated in adult rat and mouse models and are associated with negative effects on cognition and behavior in animals as described by other research groups (Allen et al., 2013, 2014a; Allen et al., 2017a,b; Bolton et al., 2012, 2013, 2014; Calderón-Garcidueñas et al., 2018a; Cerbai et al., 2012; Fonken et al., 2011; Kulas et al., 2018a; Li et al., 2018; Umezawa et al., 2018;
Woodward et al., 2018). Furthermore, mechanistic
in vitrostudies with organotypic slices (Morgan et al., 2011), primary rodent microglia and neuron-microglia cocultures (Block et al., 2004; Cheng et al., 2016;
Roqué et al., 2016; Wang et al., 2018), as well as murine BV2 (Bai et al., 2019; Chen et al., 2018; De Prado Bert et al., 2018; Lovett et al., 2018;
Sama et al., 2007; Zhang et al., 2016) and rat HAPI (Levesque et al.,
2011b) microglial cell lines have confirmed TRAP involvement in in-
flammatory-related pathways and support the hypothesis that TRAP
exposure mediates adverse effects specifically through microglia
(Table 1). Interestingly, the only
in vitrostudy with human microglia
showed no apparent change in morphology or any other microglial
responses, but this might be explained by the use of a relatively low
concentration < 20 μg/ml or small particle size < 180 nm in diameter
(Campbell et al., 2014).
Table1 InvitrostudiessupportinginflammatoryactivationofmicrogliainresponsetoPMexposure. InvitromodelExposure(source)ConcentrationDurationMajoroutcomeReference BV2CB(Cabot) DEP(NIST)50–100μg/ml24hCytotoxicity,lipidperoxidation,inflammationandautophagy.(Baietal.,2019) PM2.5(Wuhanair)50μg/ml24hIntracellularROSandinflammation.InhibitionofNrf2exacerbatedeffects.(Chenetal.,2018) PM2.5(LosAngelesair)1–20μg/ml30min 1,24hInflammation.MorningPMhadstrongereffectthanafternoonPMandcontainedmoretransition metalsandorganiccarbon.Belowtoxic.(Lovettetal.,2018) PM2.5(LosAngelesair)5–100μg/ml6hInflammationandlipidperoxidation.NF-κBactivationcorrelatedwithvariationinPMbatch toxicity.(Zhangetal.,2019) CAPs2.5(Tuxedoair)6–500μg/ml15min, 1.5,4h,6hInflammation,oxidativestress,mitochondrialmembranedepolarizationanddecreasedATP.NF-κB andinflammationmagnitudecorrelatedNiandVcontent.(Samaetal.,2007) Primarymurinemicrogliaandneuron-glia co-culturesDEPPM2.5(Diesel engine)25–100μg/cm21–24hMicrogliamediateinflammation,ROSandneurotoxicity.Minocyclineandpioglitazoneattenuated neurondeath.(Roquéetal.,2016) PM2.5(NIST)50–200μg/ml4hNeurotoxicityandinflammationviaincreasingROSandNLRP3inflammasome.Amplified synergisticeffectswithLPSandAβ.(Wangetal.,2018) Primaryratmicrogliaandneuron–gliaco- culturesufCB(Degussa)10–100μg/cm230min 24h,7dRecognizedbymicroglialsurfaceMAC-1receptor.OxidativedamagetoDAneuronsthrough NADPHoxidase.Amplifiedsynergisticeffectswithrotenone.(Wangetal.,2017) nPM0.2(LosAngelesair)12μg/ml2h,24hInhibitionofneuriteoutgrowththroughmicroglialTNFαproduction.BlockingofTNFR1restored neuritelength.(Chengetal.,2016) DEP0.22(NIST)ufCB (Degussa)50μg/ml30min 3h,9h,7dInflammatoryamoeboidmorphology,H2O2productionandlossofDAneuronfunctionviaMAC1, internalizationofDEPviascavengerreceptors.(Levesqueetal.,2013) DEP0.22(NIST)5–50μg/ml30minInflammationandDAneurotoxicitysynergisticallyamplifiedwithLPS.Fractalkineameliorated H2O2productionandDAneurotoxicity.(Levesqueetal.,2011b) DEP0.22(NIST)5–50μg/ml2h–9dDAneurotoxicitythroughphagocyticactivationofNADPHoxidaseandoxidativeinsult.Noeffect onGABAorNeu-NneuronsoronTNFα,PGE2nornitrite.(Blocketal.,2004) Rodentorganotypichippocampalslice culturefPM4(NIST)30μg/ml24hMurine:NoeffectonviabilityincreasedAβ,glialactivationespeciallyinCA1andDGregionsof hippocampus.InhibitionofPARP-1reversedPMeffects.(Jangetal.,2018) nPM0.2(LosAngelesair)1–20μg/ml24–72hRat:glutamatergicneurotoxicity,inflammation,conditionedmediafromgliaimpairedoutgrowth ofneurites.(Morganetal.,2011) HumanmicrogliaUFP0.18(LosAngelesair)2–20μg/ml24hNochangeinmorphologynorviability,noeffectonLDH,ROS,orTNFα.NeuronaldecreaseinROS formationpersistedinthepresenceofglialcells.(Campbelletal.,2014) Aβ,amyloidbeta;CA,cornuammonisregioninhippocampus;CAPs,concentratedambientparticles;CB,carbonblack;DA,dopaminergic;DEP,dieselexhaustparticles;DG,dentategyrus;fPM,fineparticulatematter; NF-κB,nuclearfactorkappaB;NISTSRM,NationalInstituteofStandardsandTechnologyStandardReferenceMaterial;NO,nitricoxide;nPM,nano-sizedparticulatematter;PARP-1,poly[ADP-ribose]polymerase1;PM, particulatematter;ufCB,ultrafinecarbonblack;UFP,ultra-fineparticles.
Table 2
Microglia-selective molecules deregulated as a result of air pollutant exposure in post-mortem human brain samples, animal models and cell cultures.
Main findings are presented as arrows: ↑level of factor was increased; ↓level of factor was decreased; ↔ no statistical differences were indicated in the study.
Factor Biological function Findings and references
Morphological changes
Amoeboid morphology Proinflammatory activation of microglia ↑ (Babadjouni et al., 2018;Block et al., 2004;Bolton et al., 2017(male only);Chen et al., 2018;Coburn et al., 2018;Cole et al., 2016;Mumaw et al., 2017;Roqué et al., 2016)
↔ (Campbell et al., 2014;Levesque et al., 2011b);
↓ (Bolton et al., 2017) (E18 female only)
ACTB Beta actin, cytoskeletal motility and structure ↑ (Araújo et al., 2019)
Cell-surface receptors involved in activation of microglia
IBA1 Ionized calcium binding adaptor molecule, increased upon morphological
change from quiescent ramified to activated amoeboid microglia. ↑ (Allen et al., 2014b,2014a;Babadjouni et al., 2018;Bai et al., 2019;
Bolton et al., 2012,2014;Chen et al., 2018;Cheng et al., 2016;Coburn et al., 2018;Cole et al., 2016;Gasparotto et al., 2019;Levesque et al., 2011b;Li et al., 2018;Woodward et al., 2017a,2018;Zhang et al., 2019);
↔ (Allen et al., 2014a,2014b(female);Bhatt et al., 2015;Bolton et al., 2017,2014(HFD female);Kulas et al., 2018a;Woodward et al., 2017a)
TSPO Translocator protein, increased upon M1 activation ↔ (Cole et al., 2016)
CD68 Cluster of differentiation 68, activation increases ↑ (Cheng et al., 2016;Morgan et al., 2011)
CD14 LPS binding, activation increases ↑ (Calderón-Garcidueñas et al., 2008b(human);Morgan et al., 2011a)
CR3 Complement receptor 3 recognizing many molecules ↑ (Wang et al., 2017)
C5AR1 Complement receptor for C5a ↑ (Babadjouni et al., 2018)
CX3CR1 Chemokine fractalkine receptor, migration towards neurons ↓ (Mumaw et al., 2017) (biodiesel)
Arginase Anti-inflammatory enzyme ↑ (Kulas et al., 2018a)
Intracellular proteins indicating inflammatory activation
COX1 Cyclo-oxygenase 1, biosynthesis of inflammatory prostaglandins ↑ (Bhatt et al., 2015)
COX2 Cyclooxygenase 2, biosynthesis of inflammatory prostaglandins ↑ (Bhatt et al., 2015;Calderón-Garcidueñas et al., 2003,2008c(human), 2008a (dog);Durga et al., 2015;Kulas et al., 2018a;Liu et al., 2019))
↔ (Kulas et al., 2018a) NFKB/p65 Nuclear factor kappa B and subunit p65, control inflammatory signaling
pathway ↑ (Calderón-Garcidueñas et al., 2003,2002;Campbell et al., 2005;Chen
et al., 2018;Guerra-Araiza et al., 2013;Kleinman et al., 2008;Sama et al., 2007;Zhang et al., 2019)
PGE2 Prostaglandin E2, inflammatory prostanoid fatty acid derivative of
arachidonic acid ↑ (Bai et al., 2019);
↔ (Block et al., 2004)
iNOS Inducible nitric oxide synthase ↑ (Calderón-Garcidueñas et al., 2002,2003;Cheng et al., 2016;Levesque
et al., 2011b;Lovett et al., 2018;Zhang et al., 2019)
↔ (Kulas et al., 2018a) Secretable cytokines and other diffusible factors
CCL2 Monocyte chemoattractant protein 1 (MCP1), recruits immune cells ↑ (Lovett et al., 2018) CCL3 Macrophage Inflammatory Protein 1-alpha (MIP1α), recruits immune cells ↑ (Lovett et al., 2018);
↓ (Levesque et al., 2011b,2011a)
C5, C5α, C1q Complement activation components ↔ (Babadjouni et al., 2018)
↔ (Levesque et al., 2011b)
IL-1α Interleukin 1α, proinflammatory cytokine ↔ (Cole et al., 2016);
↓ (Kulas et al., 2018a)
IL-1β Interleukin 1β, proinflammatory cytokine ↑ (Bolton et al., 2013,2017;Calderón-Garcidueñas et al., 2008c,2008a;
Chen et al., 2018;Cole et al., 2016;Durga et al., 2015;Fonken et al., 2011;
Gasparotto et al., 2019;Gerlofs-Nijland et al., 2010;Guerra-Araiza et al., 2013;Kulas et al., 2018a;Levesque et al., 2011b,2011a;Li et al., 2018;
Lovett et al., 2018;Morgan et al., 2011;Roqué et al., 2016;Sama et al., 2007;Win-Shwe et al., 2008;Zhang et al., 2019)
↓ (Bhatt et al., 2015;Woodward et al., 2018)
IL-2 Anti-inflammatory cytokine ↓ (Kulas et al., 2018a)
IL-3 Growth factor, proliferation ↑ (Cole et al., 2016)
IL-4 Anti-inflammatory cytokine ↓ (Kulas et al., 2018a;Woodward et al., 2018)
IL-6 Proinflammatory cytokine ↑ (Bai et al., 2019;Cole et al., 2016;Durga et al., 2015;Kulas et al., 2018a;
Levesque et al., 2011b;Li et al., 2018;Lovett et al., 2018;Morgan et al., 2011;Roqué et al., 2016;Sama et al., 2007;Zhang et al., 2019)
↔ (Bhatt et al., 2015;Levesque et al., 2011a;Woodward et al., 2018)
↓ (Kulas et al., 2018a)
IL-9 Anti-apoptic cytokine ↓ (Cole et al., 2016)
IL-10 Anti-inflammatory cytokine ↑ (Bolton et al., 2017);
↓ (Bolton et al., 2013;Kulas et al., 2018a;Woodward et al., 2018)
IL-13 Anti-inflammatory cytokine ↓ (Woodward et al., 2018)
NO⁻ ₂ Nitrite, proinflammatory ↔ (Block et al., 2004)
NO2- Nitric oxide, proinflammatory ↑ (Cheng et al., 2016;Levesque et al., 2011b;Lovett et al., 2018;Zhang
et al., 2019)
3-NT Neurotrophin-3, neurotrophic factor of activated microglia ↑ (Cheng et al., 2016) (only in OB) TNFα Tumor necrosis factor-α, proinflammatory cytokine, induce apoptosis
(continued on next page)
8. Microglial activation in response to TRAP
Microglial activation due to TRAP exposure is consistently demon- strated by morphological changes into an amoeboid form, upregulation of inflammatory markers and production of proinflammatory cytokines (Table 2). Exposure to TRAP causes alterations in beta actin (ACTB) levels (Araújo et al., 2019), which could reflect cytoskeleton re- arrangement upon morphology changes. Increased expression of mi- croglial surface markers such as ionized calcium binding adaptor mo- lecule 1 (IBA1), CD68, and CD14 correlates with increased cell amounts in corpus callosum (Babadjouni et al., 2018) and OB (Chen et al., 2018) of adult mice. In other studies, it was not investigated whether in- creased marker expression indicated increased intensity within a cell or whether it was caused by increased numbers of cells (Levesque et al., 2011b). Surprisingly, no changes in morphology were observed after 1 month exposure to very high 2 mg/m
3diesel exhaust particles (DEP) levels in rats (Levesque et al., 2011b) although other typical in- flammatory changes were observed. Furthermore, studies with gesta- tional or postnatal exposures (Bolton et al., 2017; Kulas et al., 2018a) and female rodents (Allen et al., 2014a, 2014b; Bolton et al., 2014) showed no increase in IBA1. Microglial activation was also confirmed by increased intracellular inflammatory mediators such as cycloox- ygenase (COX) enzymes, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and its subunit p65, prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) both
in vitroand
in vivo(references in Table 2).
Microglia are the primary cytokine-producing cells in the brain and, indeed, TRAP consistently induced production of proinflammatory cy- tokines IL-6, IL-1β, and TNFα (references in Table 2). Increases in other cytokines have also been reported to a lesser extent, especially related to chemoattraction (Campbell, 2004; Cole et al., 2016; Kulas et al., 2018a; Levesque et al., 2011b; Lovett et al., 2018; Zhang et al., 2019).
On the contrary, the levels of anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 (Bolton et al., 2013; Kulas et al., 2018a; Woodward et al., 2018), and anti-apoptotic IL-9 (Cole et al., 2016) are decreased in response to TRAP, supporting a proinflammatory role of microglia in response to air pollutant exposure.
9. TRAP induced oxidative stress in microglia
In addition to microglial activation, TRAP exposure causes oxidative stress. The brain is uniquely susceptible to excessive oxidative stress, and microglia together with astrocytes, are the major regulators of oxidative stress in the brain by both generating reactive oxygen species (ROS) and controlling the antioxidant defense pathways. Various TRAP exposures increase levels of oxidative stress products, especially for lipid peroxidation such as malondialdehydes (MDA), thiobarbituric acid reactive substances (TBARs) as well as plain ROS including H
2O
2(references in Table 3). Also, cellular antioxidant machinery proteins including nuclear factor erythroid 2-related factor 2 (Nrf2), superoxide dismutase (SOD), glutathione (GSH), heme oxygenase 1 (HO-1), and catalase (CAT) are commonly elevated, indicating increased demand for
detoxification, but also decreased in some cases which could indicate an impairment in the detoxification machinery (references in Table 3).
Co-culture studies of microglia and neurons demonstrate that mi- croglia are an important source of oxidative stress products that cause neuronal death (Block et al., 2004; Morgan et al., 2011; Roqué et al., 2016; Wang et al., 2018). Furthermore, studies inhibiting oxidative stress with antioxidants N-acetylcysteine (NAC), ascorbic acid (Onoda et al., 2017b) and vitamin E (Liu et al., 2019) or by knocking out NADPH oxidase (PHOX) (Block et al., 2004) reduce the damage induced by TRAPs. Comparably, inhibition of microglial activation with dex- amethasone (Bai et al., 2019), PPAR-γ agonist pioglitazone (PGZ) (Coburn et al., 2018; Roqué et al., 2016), minocycline (Roqué et al., 2016), poly(ADP-ribose) polymerase (PARP-1), and toll-like receptor (TLR) and IL-1β inhibitors (Wang et al., 2018) or TLR4 knockout (Bolton et al., 2017) similarly protect against inflammatory damage.
The TLR4 pathway was indicated in PM-induced microglial activation (Bolton et al., 2017) and suggested to be involved in inflammasome activation/priming (Bilbo et al., 2018). On the contrary, deficiency for Nrf2, the master regulator of antioxidant pathways, exacerbates TRAP- induced injury (Chen et al., 2018). These studies verify that activation of microglia contribute critically to oxidative stress and inflammation observed after exposure to TRAPs.
10. Microglial activation by TRAP induces a metabolic shift
Microglial activation and oxidative stress are closely related to mi- tochondrial energy metabolism. Indeed, the TRAP induced reduction in detoxification of ROS pathways is accompanied by metabolic altera- tions (Araújo et al., 2019). An extensive analysis of the proteome of rat brain homogenates after acute, intermediate and chronic long-term exposure to TRAP reveals a metabolic switch from oxidative respiration to glycolysis (Araújo et al., 2019). To meet the increased energy de- mand upon activation, microglia are known to trigger a similar shift to glycolysis while retaining the oxidative mitochondrial activity (Gimeno-Bayón et al., 2014; Orihuela et al., 2015; Voloboueva et al., 2013). Furthermore, TRAP exposure increases mitochondrial mem- brane depolarization which is associated with impaired mitochondrial function and is a prerequisite for mitophagy (Sama et al., 2007;
Veronesi et al., 2005). Supporting the hypothesis that TRAP induce impairment in cellular metabolism, also levels of ATP and nicotinamide adenine dinucleotide (NADH) were altered (Araújo et al., 2019; Sama et al., 2007). These observations point out that TRAPs could trigger microglial activation accompanied with increased and altered meta- bolism and deleterious oxidative stress.
11. TRAP induced phagocytosis causes oxidative stress
Although microglial activation and oxidative stress are unequivocal, the specific molecular mechanisms of how TRAP induce these phe- nomena are poorly understood. Microglial phagocytosis has been sug- gested to be one source of oxidative stress (Block et al., 2004). In- volvement of phagocytosis has been also implicated by increased
Table 2(continued)Factor Biological function Findings and references
↑ (Babadjouni et al., 2018;Bai et al., 2019;Campbell et al., 2005;Chen et al., 2018;Cheng et al., 2016;Coburn et al., 2018;Cole et al., 2016;
Durga et al., 2015;Fonken et al., 2011;Gerlofs-Nijland et al., 2010;
Guerra-Araiza et al., 2013;Kulas et al., 2018a;Levesque et al., 2011a, 2011b;Li et al., 2018;Lovett et al., 2018;Roqué et al., 2016;Sama et al., 2007;Win-Shwe et al., 2008;Woodward et al., 2017a;Zhang et al., 2019)
↔ (Bhatt et al., 2015;Block et al., 2004;Campbell et al., 2014;Woodward et al., 2018)
↓ (Campbell et al., 2014;Kulas et al., 2018a) (human microglia-neuron co- culture)
physical contact and overlap between microglia and neurons (Bolton et al., 2017), increased levels of complement C5, C5α, and C5a receptor (CD88) (Babadjouni et al., 2018) and increased macrophage-1 antigen (MAC-1) (Levesque et al., 2013; Wang et al., 2017). The MAC-1 re- ceptor promoted rotenone-induced p47phox and p67phox translocation assembling active NADPH oxidase, which induced superoxide produc- tion and toxicity to dopaminergic (DA) neurons (Wang et al., 2017).
The same study demonstrated that ultra-fine carbon black (CB) is re- cognized by the microglial surface MAC-1 receptor. Cytochalasin D inhibited DEP-induced superoxide production in primary rat microglia, implying that DEP must be phagocytosed by microglia to produce su- peroxide (Block et al., 2004). Together, these data indicate that DEP damages neurons through the phagocytic activation of microglial NADPH oxidase and consequent oxidative insult (Block et al., 2004).
Time-course studies indicate that TRAP causes activation of micro- glia and subsequent or simultaneous oxidative stress and neuroin- flammation preceding neurodegeneration (Coburn et al., 2018; Cole et al., 2016; Levesque et al., 2011b). The differential time course of oxidative stress and inflammatory responses to nano particulate matter (nPM) from nose to OB and to other brain regions (Cheng et al., 2016) indicate that slow cumulative transport of inhaled nPM into the brain may contribute to delayed responses of proximal and distal brain re- gions, with potential input from systemic factors (Cheng et al., 2016).
Commercial human microglial, astrocyte and neuron cell lines showed no apparent change in morphology nor viability when exposed to ultra
fine particles (UFP) (Campbell et al., 2014).
12. Astrocyte activation in response to TRAP
Not surprisingly, astrocytic activation usually occurs simultaneously with, or immediately after microglia stimulation and contributes to the release of oxidant species and pro-inflammatory cytokines, that may enhance neurotoxicity of PMs and create a proinflammatory environ- ment in the brain. The extent of literature on TRAP effects in astrocytes is not as extensive as in microglia. Table 4 summarizes studies with evidence of TRAP inducing astrocytic activation.
In order to investigate air pollution effects on astrocytes, different component of TRAP have been studied. Studies have used particles of different sizes collected near highways and ambient nanoparticles col- lected on filters along with commercially available particles. These studies have shown both time- and dose-dependent PM effects in as- trocytes
in vitro(Li et al., 2016; Xu et al., 2016).
In vivostudies used different approaches of particulate delivery, including intranasal in- stillation (Li et al., 2018; Onoda et al., 2014, 2017b; 2017a; Umezawa et al., 2018), intratracheal instillation (Tseng et al., 2019a,b) or in- halation in exposure chambers (Andrade-Oliva et al., 2018; Cheng et al., 2016; Haghani et al., 2020; Liu et al., 2014; Morgan et al., 2011;
Woodward et al., 2017a). In studies describing effects of exposure on astrocytes, the nanoscale subfraction of TRAP (< 250 nm diameter) has been mainly used. Unfortunately, it is impossible to compare the effect
Table 3Oxidative stress and metabolism related factors induced by air pollutant exposure associated with microglial activation.Main findings are presented as arrows: ↑level of factor was increased; ↓level of factor was decreased; ↔ no statistical differences were indicated in the study.
Factor Biological function Findings and references
Metabolic mediators
ATP Adenosine triphosphate, involved in many processes, energy transfer and cell
signaling ↓ (Araújo et al., 2019;Sama et al., 2007) (altered)
PARP1 Poly(ADP-Ribose) Polymerase 1, modifies nuclear proteins ↑ (Chen et al., 2018;Jang et al., 2018)
Insulin Peptide hormone regulating metabolism ↑ (Bolton et al., 2012)
Glycolysis The process of breaking down glucose, associated with inflammatory activation ↑ (Araújo et al., 2019)
NAD Nicotinamide adenine dinucleotide, involved in redox reactions ↓ (Araújo et al., 2019;Jang et al., 2018) (changed) NADPH oxidase Nicotinamide adenine dinucleotide phosphate oxidase, catalyzes the production of a
superoxide free radical ↑ (Block et al., 2004)
MMP Mitochondrial membrane depolarization, Impaired mitochondrial function, a
prerequisite for mitophagy ↑ (Sama et al., 2007;Veronesi et al., 2005)
Products of oxidative stress
TBARs, lipid peroxidation Thiobarbituric acid reactive substances, formed as a byproduct of lipid peroxidation ↑ (Bai et al., 2019;Cole et al., 2016;Zhang et al., 2019) MDA Malondialdehyde,a final product of polyunsaturated fatty acids peroxidation ↑ (Chen et al., 2018;Coburn et al., 2018) ↓(Zanchi et al., 2010)
ROS Reactive oxygen species ↔ (Block et al., 2004;Campbell et al., 2014;Durga et al.,
2015) DCFH-DA Dichloro-dihydro-fluorescein diacetate, used for detection of oxidative species in
cells ↑ (Bai et al., 2019;Chen et al., 2018;Roqué et al., 2016)
4HNE 4-Hydroxynonenal, produced by lipid peroxidation ↑ (Cheng et al., 2016) (only in OB); ↔ (Zhang et al., 2019)
H2O2 Hydrogen peroxide, toxic ROS ↑ (Durga et al., 2015;Levesque et al., 2011b)
Enzymes reducing oxidative stress
Nrf2 Nuclear factor erythroid 2-related factor 2, master regulator of antioxidant pathway ↑ (Guerra-Araiza et al., 2013)
GSH Glutathione, an antioxidant ↓ (Chen et al., 2018;Sama et al., 2007;Zanchi et al., 2010)
SOD1 Superoxide dismutase 1, cytoplasmic and mitochondrial protein, convert superoxide radicals to oxygen and H2O2
↑ (Gasparotto et al., 2019) (only with HFD);
↓ (Araújo et al., 2019) (decrease after 1month); (Chen et al., 2018;Zanchi et al., 2010)
SOD2 Superoxide dismutase 2, mitochondrial isoform ↑ (Guerra-Araiza et al., 2013)
GST Glutathione S-transferase, detoxification reactions ↑ (Gasparotto et al., 2019); ↓ (Araújo et al., 2019) (decrease after 1month)
CAT Catalase, catalyzes the decomposition of H2O2 to water and oxygen ↑ (Gasparotto et al., 2019); ↓ (Chen et al., 2018) LDH Lactic Dehydrogenase, converts lactate to pyruvate and NAD+ to NADH and back,
outside cell indicates cell death ↑ (Bai et al., 2019;Morgan et al., 2011);
↔ (Campbell et al., 2014)
NQO1 NAD(P)H dehydrogenase 1, a cytoplasmic 2-electron reductase ↑ (Chen et al., 2018)
HO-1 Heme oxygenase 1, catalyzes the degradation of heme ↑ (Chen et al., 2018;Fonken et al., 2011;Guerra-Araiza et al., 2013)
NPSH Non-proteinsulfhydryls or thiols, redox reactions ↓ (Sama et al., 2007)
of particles on astrocytes with regards to particle composition or dia- meter since most of the studies used different experimental designs or assays. However, we have attempted to summarize the main common findings of the studies in Table 4.
Increased glial fibrillary acidic protein (GFAP) levels are considered to indicate astrocyte activation and astrogliosis (Eng et al., 2000). An increase in astrocytic activation after acute and subchronic intranasal and inhalation exposure to PM
2.5(particles with diameters < 2.5 μm) has been documented in the brains of both adult and neonatal rats and mice (Andrade-Oliva et al., 2018; Xu et al., 2016). Moreover,
in uteroambient nano-sized particulate matter (nPM, diameters < 0.20 μm) and early neonatal period PM
2.5-induced glial hyperactivation has been shown to result in depression and autism-like behavior in adult animals (Haghani et al., 2020; Li et al., 2018). This emphasizes the contribution of TRAP exposure to early life immune system disruption that later may manifest as different neuropathological conditions (Noriega and Savelkoul, 2014). Interestingly, there may be sexual dimorphism asso- ciated with TRAP-induced astrocytic activation. Early postnatal ex- posure to ambient UFP (Ultra-fine particles, particle aerodynamic dia- meter < 100–200 nm) has been shown to lead to a decrease in GFAP immunoreactivity in males, whereas in females it leads to increased GFAP expression and is accompanied by other markers of neuroin- flammation (Allen et al., 2014a). These findings point towards the importance of studies that explore glial effects of TRAP and segregate the data by gender. In fact, there may be significant subpopulation-
based differences in the vulnerability to the TRAP exposure. In line with this, epidemiological data has pointed out that female APOE
ε4/4 carriers have the highest risk of cognitive deficits after chronic PM exposure (Calderón-Garcidueñas et al., 2016). At the experimental level, intranasal instillation of PM2.5 to rats subjected to ischemic stroke resulted in increased GFAP expression accompanied by a de- crease in spontaneous locomotion and exploratory activity (Zhang et al., 2016). The authors suggest that air pollution may worsen the neurological and neurobehavioral deficits in stroke patients. This is a relevant finding considering that the prevalence of heart attacks is in- creased while commuting in traffic (Peters et al., 2004).
Similar to studies pointing out the ability of TRAP to induce mi- croglial activation, TRAP-induced activation of astrocytes may also result in overexpression of proinflammatory cytokines.
In vitrostudies using mixed glial cultures (astrocytes and microglia) with nPM treat- ment have shown an increase in the expression of proinflammatory cytokines together with a decrease in the levels of anti-inflammatory cytokines (Cheng et al., 2016; Morgan et al., 2011; Woodward et al., 2017a). It is likely that microglia account for most of the cytokine re- sponses, yet the contribution of astrocytes was also evaluated (Cheng et al., 2016; Xu et al., 2016). Likewise,
in vivo, exposure to PM of dif-ferent diameters leads to astrocyte activation accompanied with an increase in the levels of proinflammatory cytokines and decrease in anti-inflammatory ones (Allen et al., 2014a; Haghani et al., 2020; Li et al., 2018; Liu et al., 2014; Xu et al., 2016; Zhang et al., 2016a,b).
Table 4
Studies supporting an astrocytic role in TRAP-mediated effects on the brain.
Model Major outcomes References
Primary mixed rat glial cultures 24 h incubation with ambient nano-sized particulate matter (nPM; 10 μg/ml and 12 μg/ml) mediated induction of proinflammatory cytokines. Activation of MyD88-dependent TLR4 and NF-κB pathways.
(Cheng et al., 2016;Morgan et al., 2011;Woodward et al., 2017a)
Primary neuronal cultures Inhibition of neurite outgrowth and reduction of neurite number after
incubation with conditioned medium from nPM-treated glia. (Cheng et al., 2016;Morgan et al., 2011) C57BL/6J mice Glial activation and proinflammatory cytokines induction in cerebral cortex
and hypothalamus via NF-κB pathway activation after PM exposure.
Chronic PM exposure induced hippocampal RNA changes for TLR4 pathway components and the TNFα pathway in hippocampus and cerebral cortex.
Inhibition of NF-κB and up regulation of Nrf2 pathway protects against astrocyte-stimulated PM-induced neuroinflammation.
(Allen et al., 2014b,2014a;Allen et al., 2017a,b;
Haghani et al., 2020;Morgan et al., 2011;Woodward et al., 2017a;Xu et al., 2016)
Micein uteroexposure β-sheet-rich waste protein accumulation and swelling of astrocytic end-foot in perivascular regions of the cerebral cortex in the TRAP-exposed offspring.
Dose-dependent GFAP increase in astrocytes surrounding blood vessels in the cerebral cortex and hippocampus.
Alterations in the gene expression profile of the frontal and temporal cortex, impaired spatial memory.
(Haghani et al., 2020;Kulas et al., 2018a;Onoda et al., 2018,2017b;2017a,2014;Umezawa et al., 2018)
Male Sprague-Dawley rats Early postnatal intranasal PM2.5exposure resulted in glial activation and proinflammatory cytokine induction in prefrontal cortex and hippocampus accompanied by autism-like phenotype. Acute and subchronic inhalation exposure resulted in a significant increase in GFAP immunoreactivity in the striatum.
(Andrade-Oliva et al., 2018;Li et al., 2018)
C6 rat glioma cells PM-induced overexpression of iNOS and IL-1β release in a dose- and time- dependent manner via activation of p38/JNK/ERK MAPKs and JAK2/STAT3 pathway in astrocytes.
Li et al. (2016)
Primary murine Nrf2+/+ and
Nrf2−/− astrocytes Inhibition of NF-κB and up regulation of Nrf2 pathway protects against PM2.5-
mediated neuroinflammation (24 h, dose-dependent manner). Xu et al. (2016) Ex vivohippocampal tissues of 3xTg-
AD mice 24 h fine PM (< 4 μm; 30 μg/ml)-treatment increased Aβ levels and activated glial cells. Pharmacological inhibition of PARP-1 ameliorated PM-induced effects.
Jang et al. (2018)
KKay mice, a genetically susceptible
model of Type II diabetes Whole body chronic PM2.5-inhalation resulted in increased hypothalamic proinflammatory cytokines and microglial/astrocyte reactivity via NF-κB pathway. Hyperglycemia and insulin resistance were observed in exposed group.
Liu et al. (2014)
Although dissecting out the exact contribution of microglia versus as- trocytes is difficult in mixed cultures or
in vivostudies, overall, the findings support the hypothesis that adverse effects of TRAP exposure are as a result of glial activation and that astrocytes are expected to play a role in the observed effects.
Together with a description of the changes of cytokine levels, some studies have attempted to pinpoint exact molecular pathways that were activated by TRAP in astrocytes. Experiments conducted on rat C6 glioma cells revealed that JAK2/STAT3 and p38/JNK/ERK MAPKs pathways are activated in PM-exposed astrocytes and are involved in the induction of iNOS and IL-1β production (Li et al., 2016). It is not surprising that many of the studies have shown NF-
κB to be responsible for these effects. Increased expression of inhibitor of nuclear factor kappa-B (IKKβ) has been reported together with elevated proin- flammatory cytokine expression after TRAP exposure in mice. This subunit plays a key role in the induction of NF-κB activity (Li et al., 1999). In the same study design, the administration of a pharmacolo- gical inhibitor of IKKβ reversed PM-induced abnormalities, normalized energy metabolism and reduced IL-6 expression (Liu et al., 2014), further strengthening the notion that this pathway is important in TRAP-exposed glia. Furthermore, an Affymetrix whole genome micro- array study on nPM and LPS induced glia showed extensive overlap in inflammatory responses between PM and LPS (Woodward et al., 2017a). Astrocytes do not express receptors for LPS, but share the common downstream pathways with microglia. In contrast to LPS, nPM activated the NF-κB inflammatory response through MyD88-dependent signaling in glia (Woodward et al., 2017a). These findings emphasize that astrocyte activation by TRAP is usually accompanied by a proin- flammatory NF-κB pathway activation which could be considered as a target for future therapeutic approaches. The various molecules shown to be altered in astrocytes upon TRAP exposure are summarized in Table 5.
13. TRAP induced oxidative stress in astrocytes
Airborne particles contain different chemicals and metals, and their translocation to the body leads to the generation of ROS and in- flammation (Xia et al., 2006). The most sensitive cellular response to oxidative stress is the activation of antioxidant phase II enzymes. The expression of these antioxidant enzymes is regulated by the Nrf2 tran- scription factor (Itoh et al., 1999). Nrf2 pathway activation and its in- teraction with NF-κB signaling plays a critical role in cell protection and survival (Buelna-Chontal and Zazueta, 2013). Xu et al. showed that Nrf2 deficient mice show increased astrocytic activation in response to concentrated PM
2.5inhalation exposure. Chronic PM
2.5exposure in- duced both inflammatory NF-κB mediated gene expression as well as Nrf2 transportation to the nucleus and overexpression of phase II en- zymes (Xu et al., 2016). The data suggest that PM, in addition to to- gether activating the inflammatory pathway, also induces cellular anti- oxidative responses to detoxify the extensive oxidative stress caused by exposure.
14. Air pollution and glial priming
The onset of neurodegenerative diseases is often related to predis- posing factors, such as environmental factors, either prenatally or postnatally inducing priming of CNS cells (Chin-Chan et al., 2015). The word “priming” can also be used in context with microglia and astro- cytes. Priming refers to a cell fate that is induced upon two subsequent inflammatory stimuli and is related to the capacity of the cell to re- member these stimuli, also known as immunological memory (Perry and Holmes, 2014). When glia show exaggerated responses upon a secondary inflammatory stimulus, we refer to them as being primed. A primed microglia profile is determined by higher expression of in- flammatory markers or mediators; a lower threshold for the change to a pro-inflammatory stage and an immune activation after the second
inflammatory stimuli (Norden et al., 2015; Perry and Holmes, 2014).
Many of the neuropsychiatric disorders are thought to be linked to glial priming effects, the so called two-hit hypothesis, in which the second hit initiates the disease progression (Feigenson et al., 2014). Indeed, prenatal and early life glia-priming exposures often lead to neurode- velopmental abnormalities and behavioral deficits.
There are a variety of stimuli that prime microglia or astrocytes both prenatally and postnatally. Prenatal stimulus, such as maternal immune activation (MIA) or hyperglycemia, predispose the offspring to neuro- developmental disorders, such as schizophrenia or autism, and altered brain development (Hanamsagar and Bilbo, 2017). At the cellular level, these stimuli lead to decreased neurogenesis, microsomia and down- regulation of apoptosis regulatory proteins (Piazza et al., 2019). After MIA, microglial reactivity increases evidenced by increased cytokine release (Antonson et al., 2019). Maternal viral infections have been shown to lead to increased IL-6 in placenta and amniotic fluid, to ab- normal development (Hanamsagar and Bilbo, 2017) and astrogliosis (O'Loughlin et al., 2017). Similarly, LPS, when infused during a post- natal period, induces microglia priming and even chronic inflammation (Bellesi et al., 2017; Chin-Chan et al., 2015). Early-life exposure to stress (Hanamsagar and Bilbo, 2017) and disturbances in physiological processes like sleep, can also prime both astrocytes and microglia.
Acute sleep deprivation and chronic sleep loss have been shown to lead to an increased glial phagocytosis. This is associated with increased expression of MERTK and lipid peroxidation (Bellesi et al., 2017) and deficits in spatial learning and memory (Chin-Chan et al., 2015; Piazza et al., 2019). Finally aging, an unavoidable physiological process, is known to be a microglia primer (Hou et al., 2019a; Raj et al., 2014).
Indeed, in aged animals, microglial processes are reduced and glia- neuron signaling processes are impaired (Hefendehl et al., 2014).
In addition to the above-mentioned stimuli, air pollution is one of the most important environmental factors that has been shown to prime microglia and astrocytes, which can eventually lead to neurodevelop- mental deficits. There are many types of air pollutants, but most of the studies showing priming of the glial cells were focused on PM, DEP, nanoparticles, UFP and TRAP, which may all have a specific outcome for the priming effect. Here we shortly summarize the evidence of TRAP effects on neurodevelopmental deficits linked with abnormal glial ac- tivation.
15. Maternal exposure effects
To study effects of maternal exposure on offspring brain develop- ment Tseng et al. (Chao et al., 2017; Tseng et al., 2019a,b), studied pregnant rats exposed to intratracheal instillation of PM
2.5and mea- sured gene expression in the fetal cerebral cortex and hippocampus.
They showed increased inflammatory responses in pregnant rats, and
increased cytokines and free radicals in the amniotic fluid. Although
fetuses appeared normal, PM
2.5exposure changed the expression levels
of different microRNAs in both fetal cortex and hippocampus. Later
studies extended these findings and showed that
in uteroPM-inhalation
exposure led to increased GFAP immunoreactivity and altered expres-
sion of inflammatory genes that were accompanied with impaired
spatial memory and depression in adulthood. Interestingly, the micro-
glia activation marker IBA1 was not changed in exposed animals
(Haghani et al., 2020; Kulas et al., 2018a). A study with maternal in-
tranasal and inhalation exposure of ultrafine carbon black on gesta-
tional days 5 and 9 demonstrated astrogliosis surrounding blood vessels
in the cerebral cortex and hippocampus of the mouse offspring (Onoda
et al., 2017c, 2014; Umezawa et al., 2018). Also, β-sheet-rich protein
accumulation has been detected in the regions with perivascular ab-
normalities induced by maternal TRAP exposure. These results suggest
that β-sheet-rich waste proteins, which are denatured by maternal ex-
posure, were accumulated in the perivascular space in the brain leading
to astrocyte activation and macrophage denaturation (Onoda et al.,
2017a). In addition, the maternal exposure study with an intranasal
carbon black nanoparticle administration showed up-regulation of genes related to astrocyte activation and development, including
Gfap, Drd1and
Tspan2(Onoda et al., 2018).
To study the glial mediated nPM effects on neurite outgrowth, Morgan and coworkers treated mixed glial cultures from the cerebral cortex of neonatal rats with nPM for 24 h after which the conditioned media was transferred to neuron cultures (Cheng et al., 2016; Morgan et al., 2011). They found increased TNFα mRNA levels in mixed glia cultures and reductions of neurite outgrowth and neurite number after the incubation. TNFα has previously been reported to be a part of the neuroinflammatory glial response to air pollution and its induction was shown both in rodent (Campbell et al., 2005; Levesque et al., 2011b, 2011a; Xu et al., 2016) and human models (Calderón-Garcidueñas et al., 2008c; Campbell et al., 2014). Amongst a plethora of other ac- tions, TNFα regulates neurite outgrowth (Neumann et al., 2002). These
studies pinpoint that activated glia may underlie the reduced neurite outgrowth induced by PM and support the hypothesis that
in uteroex- posure to PM promotes neuroinflammation that may provoke neuro- pathological changes, impair neurodevelopment and result in memory deficits in adulthood (nicely reviewed in (Brockmeyer and D'Angiulli, 2016)). Indeed, TRAP exposure, shown to initiate glial cell activation (Bolton et al., 2012), phagocytic activity (Bolton et al., 2017) or even microglial cytotoxicity and cell death (Bai et al., 2019) can lead to al- terations in the structure of the CNS; decreasing the expression Myelin Binding Protein (MBP) and inducing atrophy in CA1 neurites (Babadjouni et al., 2018; Cheng et al., 2016; Woodward et al., 2017b).
16. Maternal exposure and blood–brain barrier integrity
Astrocytes extend their end-feet to neighboring blood vessels and
Table 5Astrocyte-related molecules deregulated as a result of air pollutant exposureMain findings are presented as arrows: ↑-increased expression of RNA or protein level were identified in the study; ↓-level of molecule was decreased; ↔-no statistical differences were indicated in the study.
Molecule Biological function Findings and references
Morphological changes
GFAP Glial fibrillary acidic protein, marker of activated astrocytes ↑(Allen et al., 2014b(females),Allen et al., 2014a(cortex females);Allen et al., 2017a,b(males);Andrade-Oliva et al., 2018(striatum);Jang et al., 2018;Kulas et al., 2018a; Li et al., 2018a;Liu et al., 2014;Morgan et al., 2011;Onoda et al., 2014,2017b,2017a;Umezawa et al., 2018;Xu et al., 2016)
↔(Allen et al., 2014b;Andrade-Oliva et al., 2018) (cortex);
↓(Allen et al., 2014a) (males) Cell-surface receptors
Aqp4 Aquaporin-4, water channel protein ↑ (Onoda et al., 2017b,2017a)
TNFR1 Tumor necrosis factor receptor 1, membrane receptor that binds
TNFα ↔ (Woodward et al., 2017a)
Proteins indicating the inflammatory NF-κB pathway activation
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells,
controller of an inflammatory signaling pathway (Xu et al., 2016) (↑ in a nucleus and ↓ in cytoplasm),
↑ (Woodward et al., 2017a)
NF-κBia and IκBkb NF-κB pathway related factors ↑ (Xu et al., 2016)
IKKa, IKKβ IκB kinases involved in NF-κB signalling pathway ↑ (Liu et al., 2014) p-IKKβ, p-IκBα and p-
NF-κB Phosphorylated forms of NF-κB signaling pathway kinases ↑ (Xu et al., 2016) iNOS Inducible nitric oxide synthase, produces NO, reacts to
proinflammatory stimulation ↑ (Li et al., 2016;Xu et al., 2016)
(Haghani et al., 2020) (↑in vitroand ↓in vivo) COX2 Cyclooxygenase 2, promotes inflammation, catalyzes the formation of
inflammatory prostaglandins ↑ (Kulas et al., 2018a;Xu et al., 2016)
↓ (Haghani et al., 2020) Cytokines
IL-1α Interleukin 1α, cytokine, mediates proinflammatory events ↑ (R.Chen et al., 2017;Morgan et al., 2011)
↓ (Kulas et al., 2018a) IL-1β Proinflammatory cytokine, key mediator of the inflammatory
response ↑ (Allen et al., 2014a;Cheng et al., 2016;Haghani et al., 2020;Li et al., 2018,
2016;Morgan et al., 2011;Woodward et al., 2017a;Xu et al., 2016)
↓ (Allen et al., 2014b) (males)
IL-2 Anti-inflammatory cytokine ↓ (Kulas et al., 2018a)
IL-4 Immunoregulatory cytokine, decreases production of IFN-g and IL-12 ↓ (Kulas et al., 2018a)
IL-6 Proinflammatory cytokine ↑ (Allen et al., 2014b;Cheng et al., 2016;Li et al., 2018;Liu et al., 2014;Morgan
et al., 2011;Woodward et al., 2017a;Xu et al., 2016)
↓ (Allen et al., 2014b;Kulas et al., 2018a)
↔ (Haghani et al., 2020)
IL-9 Anti-apoptic cytokine ↓ (Cole et al., 2016)
IL-10 Anti-inflammatory cytokine, inhibits induction of proinflammatory
cytokines ↓ (Kulas et al., 2018a)
IL-13 Anti-inflammatory cytokine ↓ (Woodward et al., 2018)
GM-CSF Granulocyte macrophage-colony stimulating factor, recruiter of
macrophages ↓ (Kulas et al., 2018a)
IFN- γ Interferon-γ, proinflammatory cytokine, activator of macrophages ↑ (Haghani et al., 2020)
↔ (Morgan et al., 2011); ↓ (Kulas et al., 2018a)
TNFα Tumor necrosis factor-α, proinflammatory cytokine, induce apoptosis ↑ (Allen et al., 2014b;Cheng et al., 2016;Li et al., 2018;Morgan et al., 2011;
Woodward et al., 2017a;Xu et al., 2016)
↓ (Allen et al., 2014b(males);Kulas et al., 2018a)
↔ (Haghani et al., 2020;Li et al., 2016)
