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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Environ Pollut. 2018 May 22;241:279–288. doi: 10.1016/j.envpol.2018.05.047

In utero exposure to fine particulate matter results in an altered neuroimmune phenotype in adult mice

Joshua A Kulas 1, Jordan V Hettwer 1, Mona Sohrabi 1, Justine E Melvin 1, Gunjan D Manocha 1, Kendra L Puig 1, Matthew W Gorr 2, Vineeta Tanwar 2, Michael P McDonald 3, Loren E Wold 2, Colin K Combs 1
PMCID: PMC6082156  NIHMSID: NIHMS971040  PMID: 29843010

Abstract

Environmental exposure to air pollution has been linked to a number of health problems including organ rejection, lung damage and inflammation. While the deleterious effects of air pollution in adult animals are well documented, the long-term consequences of particulate matter (PM) exposure during animal development are uncertain. In this study we tested the hypothesis that environmental exposure to PM 2.5 microns in diameter in utero promotes long term inflammation and neurodegeneration. We evaluated the behavior of PM exposed animals using several tests and observed deficits in spatial memory without robust changes in anxiety-like behavior. We then examined how affects the brains of adult animals by examining proteins implicated in neurodegeneration, synapse formation and inflammation by western blot, ELISA and immunohistochemistry. These tests revealed significantly increased levels of COX2 protein in PM2.5 exposed animal brains in addition to changes in synaptophysin and Arg1 proteins. Exposure to PM2.5 also increased the immunoreactivity for GFAP, a marker of activated astrocytes. Cytokine concentrations in the brain and spleen were also altered by PM2.5 exposure. These findings indicate that in utero exposure to particulate matter has long term consequences which may affect the development of both the brain and the immune system in addition to promoting inflammatory change in adult animals.

Keywords: Air Pollution, PM2.5, COX2, Neuroinflammation

Graphical abstract

graphic file with name nihms971040u1.jpg

Introduction

Air pollution has become a prominent global health issue, with many cities and industrial areas around the world releasing high levels of toxic pollutants into the environment. In 2013, 87% of the world’s population lived in areas where air particulate concentrations reached greater than 10 μg/m3 (Brauer et al., 2016). The World Health Organization estimated that in 2012 alone, air pollution was responsible for around 3 million deaths, while another report estimated that 3.24 million premature deaths were caused by airborne particulate matter (PM) in 2010. (Apte et al., 2015; WHO, 2016). The widespread and apparently severe effects of air pollution warrant the need for additional studies into the biological consequences of pollution exposure.

Airborne PM has a heterogeneous composition and is commonly classified by its size, with coarse particles ranging from 2.5-10 μm (PM10), fine particles less than 2.5 μm (PM2.5), and ultrafine particles less than 0.1 μm (PM.01) (Brook et al., 2010). PM2.5 may be particularly toxic due to its ability to penetrate the lung tissue and enter the circulatory system (Nemmar et al., 2002; Nemmar et al., 2001; Wold et al., 2006). Numerous reports have demonstrated the toxicity of PM2.5 both in animal models and in humans (Bhatt et al., 2015; Morakinyo et al., 2016; Van Winkle et al., 2015; Wold et al., 2012). The National Ambient Air Quality Standards (NAAQS) currently states that exposure to PM2.5 for healthy individuals should not exceed 15 micrograms per cubic meter annually.

Previous studies indicated that there are wide array of potential health complications from prolonged exposure to PM, including potent effects on the cardiovascular system (Apte et al., 2015; Gorr et al., 2015; Ibald-Mulli et al., 2001; Morakinyo et al., 2016; Sun et al., 2010; Weinmayr et al., 2015; Wold et al., 2012). In addition to these well studied effects, epidemiological reports indicate that air pollution also has the ability to adversely affect the brain (Chen et al., 2017; Chen et al., 2015). PM2.5 promotes brain inflammation and adversely affects cognition (Bhatt et al., 2015; Block and Calderon-Garciduenas, 2009; Calderon-Garciduenas et al., 2008; Hogan et al., 2015; Liu et al., 2015). It has been proposed that inflammation plays an important role in the pathogenesis of Alzheimer’s disease (McGeer and McGeer, 2013). Thus, PM2.5 may promote changes in the brain leading to neurodegeneration. The blood brain barrier itself may also be compromised by PM exposure. Recent work suggests exposure to heterogeneous PM generated by combustion engines leads to compromised blood-brain barrier integrity in adult mice (Suwannasual et al., 2018).

The effect of PM2.5 exposure on the brain during critical developmental periods remains uncertain. However, recent research indicates that there may be both developmental impairments and long term detriments to brain physiology in mice exposed to concentrated ambient particles. A recent study by Klocke et al. found that exposure to concentrated fine and ultrafine particles during embryonic development affected oligodendrocyte maturation and brain myelination in adulthood (Klocke et al., 2017a). Behavior abnormalities and neuropathological changes in mice exposed to concentrated ultrafine particles during development have also been observed (Klocke et al., 2017b). In this study, we tested the hypothesis that in utero exposure to concentrated PM2.5 promotes long term neurodegeneration and brain inflammation. We examined the effect of in utero exposure to PM2.5 on the brains of adult mice. Pregnant females were exposed daily to either filtered air (FA) or PM2.5 through the entire course of gestation. At four months of age, male offspring were then assessed for changes in memory and exploratory or anxiety-like behavior. In addition to behavioral assessments, the brains and spleens of these animals were evaluated for neurodegenerative or inflammatory changes. Our results demonstrated that PM2.5 exposure in utero produced adverse effects on working memory but not exploratory or anxiety-like behavior in four-month-old mice. Moreover, this correlated with altered protein levels of the presynaptic protein marker, synaptophysin and cyclooxygenase 2 (COX-2). Increased levels of the activated astrocyte marker GFAP were observed by immunohistochemistry, although total levels of GFAP in western blot lysates were not significantly different. Finally, PM2.5 animals demonstrated attenuated levels of multiple cytokines in both brains and spleens. These data suggest that in utero exposure to PM2.5 is sufficient to provide long lived adverse effects on both nervous and immune system function.

Methods

Exposure to Particulate Matter (PM) or Filtered Air (FA)

FVB male and female mice were obtained from The Jackson Laboratories (Bar Harbor, ME) and housed for at least 1 week in the animal facility prior to breeding. Females were paired with males and the presence of a vaginal plug indicated successful mating and was designated gestational day 1. The pregnant dams were randomly divided into two groups for exposure either to filtered air (FA) or particulate matter (PM2.5) for 6 hrs/day, 5 days/week throughout the gestational (in utero) period. No difference in the health of the female dams was observed across FA and PM exposed animals. Following exposure, a maximum of 2 pups from each of 5 separate litters were used in both the FA and PM conditions resulting in 10 total animals in each group. The aerosol concentration system located at the Ohio State University was used for the concentrated PM2.5 exposure from the Columbus, OH region as described previously (Wold et al., 2012). The average daily PM2.5 concentration that the dams were exposed to was 46.70 μg/m3. Since the animals were exposed for only 6 hrs/day (one-quarter of the day), the average 24 hr concentration was equivalent to 11.67 μg/m3 which is below the national air quality standard of 15 μg/m3. The PM filter allowed any particle of size ≤ 2.5 microns which includes the ultra-fine fraction. Mice were whole body exposed and food and water were restricted during exposures. For the FA treatment, an identical system was used, except that a HEPA filter at the inlet to the system was used to remove all ambient particles. In this study we chose to utilize male animals to control for any confounding effects the estrous cycle may have on behavior and inflammation. After birth, male pups from in utero FA and PM2.5-exposed groups were raised in room air until 10 weeks of age before transporting to the University of North Dakota School of Medicine and Health Sciences (UNDSMHS). Although no differences in birth weights of pups were noted, to avoid any confounds of litter differences, as mentioned above, 2 pups from each litter were used for the experiments. While litter affects may indeed be present and could account for some variability in data, we elected to only screen for effects caused by the exposure of PM to our animal cohorts. Males from the same litter were co-housed. Mice transported to the UNDSMHS were allowed to adapt to the new environment for 14 days before behavioral assessment. Ten mice per group were used and housed 3-4 per cage during the 14 day period and provided food and water ad libitum with a 12 hour light dark cycle. Animal use was approved by the Ohio State University and University of North Dakota Institutional Animal Care and Use Committees.

Behavioral Analysis

Each mouse was tested once in each behavioral test in this study. A cross maze apparatus was used to compare working memory in FA and PM2.5 mice. Mice explored the cross-shaped maze at their own discretion without explicit stressors or motivators, such as lights, sound or food deprivation. The maze consisted of four arms in a cross configuration, 30 cm long and 5 cm wide with a 5 cm2 central area. Each arm had a 15 cm high wall along each side and at the end. The central area was open for the duration of the session. All mice were placed in the same arm of the maze and allowed to explore and choose additional arms. A choice was defined as having all four feet within an arm. Mice were allowed to explore the maze for 12 min. and arm entries were recorded. The number of alternations were counted (defined as 4 consecutive entries into 4 different arms), and % alternation for each mouse was calculated as follows: # alternations/(total entries-3). After exiting an arm, immediate re-entry into the same arm was not counted as an arm choice; thus the chance alternation rate (poor memory) for this task is 22.2%. Mice were returned to their home cages for 30 minutes to await further assessments.

A light-dark box was used to assess anxiety-like behavior in mice exposed to FA or PM2.5 in utero. A 40 cm × 40 cm × 35 cm container split into two sides, one light and one dark, was used to allow the mouse to move and explore freely. All mice were placed in the same corner of the light side of the box and the movements were recorded for 5 min. Blind assessment of movement and time spent in light vs dark areas were quantified from video captured using Anymaze (Stoelting Co., Wood Dale, IL) software. Mice were again returned to their home cages for 30 minutes before further assessment.

In order to assess any general locomotion changes associated with the treatment, mice were placed in an open field 30 min. after completion of the light-dark test. All mice were placed in the same quadrant of a 40 × 40 × 35-cm arena and allowed to move and explore freely. After 30 s of habituation, mice were allowed to explore for 10 min. Time mobile and immobile, as well as distance traveled, time spent in the center, or in the field corners (quadrants), were scored by blind raters from video captured using Anymaze software. Time spent in the center was considered an adjunct measure of anxiety-like behavior.

Tissue Collection

Following the behavior testing, the animals were returned to their home cages for one day prior to tissue collection. Animals were approximately 3.5 months old at the time of collection. No obvious differences in overall health and fitness between FA and PM exposed animals were observed at the time of collection. To collect tissue, animals were sacrificed and perfused with PBS containing Ca++. One hemisphere of the brain was post-fixed in 4% paraformaldehyde for immunohistochemistry while the other hemisphere was flash frozen in liquid nitrogen for western blot and ELISA analyses. Spleens were collected and flash frozen in liquid nitrogen.

Antibodies

Antibodies were utilized for western blot (WB) and/or immunohistochemistry (IHC). The COX1 antibody (160110) was purchased from Cayman Chemicals. The COX2 (15191) and APP (32136) antibodies were purchased from Abcam. The Iba1 antibody (019-19741) was purchased from Wako Chemicals. The GFAP (D1F4Q), BACE (D10E5), PSD95 (2507S) and Arg1 (D4E3M) antibodies were purchased from Cell Signaling. The synaptophysin antibody (mab5258) was purchased from Millipore. The iNOS (N20) and GAPDH (6C5) antibodies were purchased from Santa Cruz Biotechnology.

Western Blots

Brain temporal cortices were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Sigma product P8340) and centrifuged. The soluble fraction was collected and protein content assessed using the Bradford method (Bradford, 1976). 10 μg of protein was resolved by 10% SDS-PAGE and proteins were transferred to PVDF membranes for western blotting. Membranes were blocked for 1 hour in 5% bovine serum albumin (BSA) in tris-buffered saline solution (TBST). Following blocking, primary antibodies were diluted 1:1000 in 5% BSA-TBST solution and applied to membranes overnight at 4 °C. Chemiluminescent visualization was utilized to detect proteins of interest using horseradish peroxidase conjugated secondary antibodies. Exposures were captured using an Aplegen Omega Lum G imaging system. Optical densities were quantified using Adobe Photoshop 12.0 software. Optical density values were normalized to the loading control GAPDH optical density values from the same membrane, and then compared using a Student’s t-test.

Immunohistochemistry

Immunohistochemistry (IHC) was performed on serial brain sections collected from individual animals (n=10 FA and n=10 PM). Following collection, brains were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) overnight. After PFA fixation, brains were cryoprotected in 15% sucrose-PBS overnight and then 30% sucrose-PBS in PBS overnight. Brains were embedded into a block of 10% gelatin and cut into 40 μm sections using a freezing microtome. Immunohistochemistry was performed on free floating brain sections embedded in gelatin. At least five gelatin sections for both FA and PM (n=10) were stained for each protein of interest. We chose sections containing both hippocampus and TC to assess regions of interest. For Arg1 and COX2 immunostaining, antigen retrieval was required and performed using sodium citrate Antigen Unmasking Solution (Vector Labs product H-3300) at 60 degrees centigrade for 40 minutes prior to blocking. Brain slices were rinsed in PBS and then blocked for one hour in PBS containing 0.5% BSA, 0.1% Triton X-100, 5% horse serum and 0.02% sodium azide. Primary antibodies were diluted in additional blocking solution and incubated with tissue at 4 °C overnight. Biotinylated secondary antibodies were used followed by Vector Laboratory ABC kits addition of streptavidin/peroxidase. The visible light chromogen Vector VIP (Vector Labs) was utilized for visualization. The tissue was washed in PBS, mounted on slides overnight, dehydrated, and plated on coverslips. Quantitation of immunohistochemistry was performed from 5 serial sections per brain using Adobe Photoshop CS6 software. Temporal cortex images were converted to greyscale and the mean optical density for each image was calculated. Values obtained from densitometry were normalized to the mean staining intensity value of the FA group. Normalized values from FA and PM conditions were then compared by Students t-test.

Enzyme Linked Immunosorbent Assay (ELISA)

Pre-weighed and flash frozen temporal cortex from individual animals (n=10, FA and n=10, PM) were lysed on ice with RayBiotech 1x Lysis Buffer (Raybiotech, Inc., Norcross GA). Supernatants were collected and ELISAs performed as per the manufacturer’s protocol. Cytokine levels were normalized to the protein content of each homogenized sample, averaged, and plotted ± SD.

Statistics

ELISA and densitometric western blot data were analyzed using Student’s t-test with Sigmaplot 12.0 software (Systat Software, Inc., San Jose, CA), with significance set at p < 0.05. Data are represented as mean values ± SD. Behavior data were analyzed using Mann-Whitney U-test with Sigmaplot 12.0 software with significant differences set at p<0.05 and the data are presented as mean values ± SEM.

Results

Mice exposed to PM2.5 had impaired memory but no difference in exploratory or anxiety-like behavior compared to FA exposed mice

We began assessing the adult consequences of in utero PM2.5 exposure on brain health by first conducting behavioral tests on mice exposed to PM2.5 in utero and then housed under standard conditions until 4 months of age. We first tested the working memory of mice using a cross maze. The cross maze is a “+” shaped apparatus and is similar in principal to the Y maze. The cross maze test challenges the memory of mice by measuring their ability to alternate to unexplored arms of the maze over repeated trials (Jawhar et al., 2012; Wang et al., 2017). There was no significant difference in the total number of arm choices, but we observed a significant reduction in the percent alternations made by PM2.5-exposed mice (Figure 1A). These findings indicate that PM2.5 exposure in utero may have a deleterious effect on a specific form of hippocampal-based memory.

Figure 1.

Figure 1

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Mice exposed to in utero PM2.5 demonstrated a decrease in working memory without robust effects on anxiety-like behavior or general locomotion when compared to FA exposed control mice. (A) Working memory was assessed with cross maze exploratory behavior to quantify numbers of arm entries and percent alternations. Mouse anxiety-like and exploratory behavior were assessed mice by (B) light dark box and (C) open field testing to quantify time spent in quadrants (Q), time in the center, time in the dark, latency to enter the dark, latency to exit the dark, along with distance traveled. Animals were tested at 4 months of age (n=10), * p<0.05. Error bars represent mean ±SEM of the data graphed.

We next subjected mice to light-dark box testing as a measure of anxiety-like behavior (Dhanushkodi and McDonald, 2011; Kulesskaya and Voikar, 2014; Miller et al., 2011). We did not observe any significant differences in total time spent in the light or dark areas, latency to enter the dark, or time spent in the center of the lighted area of the light-dark box. We did, however, observe a significant increase in the latency of PM2.5 exposed mice in re-emerging from the dark (Figure 1B) suggesting PM2.5 exposure may promote a very subtle shift towards anxiety-like behavior. Finally, mice were compared via open field testing, which is used to assess primarily exploratory behavior in mice with some anxiety-like behavior associated with time spent in the center of the field (Crawley, 1985, 1999). No differences were detected in distance traveled or time spent in the center or corner quadrants of the open field, indicating that PM2.5 exposure did not affect general exploratory or anxiety-like behavior in these mice (Figure 1C). These findings further supported the idea that in utero PM2.5 exposure did not significantly alter anxiety-like behavior of the mice.

PM2.5-exposed mouse brains show changes in synaptic and immune markers

Based upon the behavioral differences, we next determined whether in utero exposure to PM2.5 altered levels of proteins involved in neuroinflammation, neurodegeneration, or synapse formation. Temporal cortices of FA vs PM2.5 in utero exposed mice were compared via western blots. The COX enzymes catalyze the formation of prostaglandins (Poorani et al., 2016) and COX2 is known to be upregulated in the brain during inflammatory change (Nakayama et al., 1998; Yan et al., 2015). Westerns blots for COX2 revealed a significant increase in COX2 but not COX1 protein levels in in utero PM2.5 exposed compared to FA mice (Figure 2).

Figure 2.

Figure 2

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COX2, Arg1, and synaptophysin protein levels were increased in in utero PM2.5 exposed temporal cortex compared to FA control mice (n=10). Temporal cortices from FA and in utero PM2.5 exposed mice (n=10) were lysed and proteins resolved by 10%SDS-PAGE and western blotted. Representative blots from 5 brains per group are shown. Densitometry was performed and each sample was normalized to GAPDH as a loading control. Data were graphed as mean fold change of FA (± SD) by dividing each normalized value by the average of the FA group, * p<0.05.

After observing the robust increase in COX2 protein levels, we next examined Iba1 and GFAP protein levels as these proteins have been shown to be elevated in reactive microglia and astrocytes, respectively, during brain inflammation (Sumbria et al., 2016). We did not detect significant differences in the levels of microglial IBA1, nor did we observe a significant increase in total GFAP levels (Figure 2). We further examined brain tissue for arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS) content, the levels of which have been shown to change during inflammation and tissue damage (Murphy and Gibson, 2007). While iNOS levels were unchanged, we observed a significant increase in Arg1 levels in in utero PM2.5 exposed temporal cortices (Figure 2).

It has been suggested by several groups, including our own, that exposure to air pollution increases the risk of dementia and Alzheimer’s disease (Ailshire and Crimmins, 2014; Calderon-Garciduenas, 2016; Jung et al., 2015; Oudin et al., 2016). Indeed, our prior work demonstrated increased amyloid precursor protein (APP) and β secretase (BACE) protein levels in brains of mice exposed to PM2.5 from 2 to 9 months of age (Bhatt et al., 2015). To determine whether a similar increase in AD-related proteins resulted from in utero exposure to PM2.5, we quantified both APP and BACE protein levels due to their central role in formation of amyloid plaques during Alzheimer’s disease. However, we observed no differences in the levels of these proteins in in utero PM2.5 exposed mice compared to FA exposed controls (Figure 2).

In order to determine a correlation with the observed changes in working memory, we elected to quantify both the presynaptic and postsynaptic compartments via examining synaptophysin and PSD95 protein levels, respectively. Surprisingly, in utero PM2.5 exposed mice showed elevated levels of synaptophysin although PSD95 was unchanged (Figure 2). This demonstrated that in utero PM2.5 exposure resulted in a pervasive change in preferentially the presynaptic compartment that correlated with impaired working memory.

In utero PM2.5 exposure increased neuronal COX2 and Arg1 immunoreactivity with minimal evidence of glial activation

It is well established that the astrocytes and microglia of the central nervous system have prominent roles in various brain diseases and immune responses (Ransohoff, 2016). Since increased protein levels of COX2 and Arg1 were observed in brains from in utero PM2.5 exposed mice, we elected to verify whether these changes were occurring in glia. We performed immunohistochemical analyses of brain sections FA or in utero PM2.5 exposed mice to assess localization and immunoreactivity for COX2 and Arg1 but also the glial markers, Iba1 (microglia) and GFAP (astrocytes). Interestingly, we observed widespread increased cellular staining for COX2 in the neurons, rather than glia, of in utero PM2.5-exposed mice consistent with the western blot findings (Figure 3). Similarly, consistent with the western blot findings, APP and Iba1 immunoreactivities were not dramatically different between the two groups. In contrast to our western blot findings, we found that astrocyte GFAP levels to be significantly increased (Figure 3) by immunohistochemistry. Surprisingly, Arg1 immunoreactivity localized to neuronal populations rather than the expected microglia and was robustly increased in the in utero PM2.5 exposed mice compared to FA exposed controls (Figure 3).

Figure 3.

Figure 3

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Brains of in utero exposed PM2.5 mice demonstrated increased neuronal COX2 and Arg1 immunoreactivity. Mouse brain hemispheres were sectioned and immunostained using primary antibodies against COX2, GFAP, Iba1, APP and Arg1. Vector VIP was utilized as the chromogen. Representative images from the temporal cortex are shown from n=10 mice per group. Immunostaining was quantified by densitometry and values were normalized to the mean of the FA exposed group. Values obtained were compared by students t-test with * p<0.05. Data is graphed as mean ±SD.

Cytokine levels were attenuated in brains and spleens from in utero PM2.5-exposed mice compared to FA-exposed controls

Based upon the increased neuronal Arg1 immunoreactivity and its known role in driving immunosuppressive responses in the brain, we next quantified cytokines from the brains and spleens of the in utero PM2.5 exposed and FA control mice (Colton et al., 2006; Kan et al., 2015).

Using ELISAs, we quantified temporal cortex levels of several cytokines and normalized to tissue weight. As expected, the levels of a number of cytokines were significantly reduced in the in utero PM2.5 exposed tissue. These included the cytokines IL-1α, IL-2, IL-4 IL-6, IL-10, IFN-γ, GM-CSF and TNF-α. (Figure 4)

Figure 4.

Figure 4

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Levels of multiple cytokines were reduced in in utero PM2.5 exposed temporal cortex and spleens compared to FA exposed control mice. Isolated temporal cortex and spleens (n=10) were lysed then proteins levels of 12 different cytokines were quantified by ELISA. Cytokine values were normalized to tissue wet weight to obtain pg/mL/mg, * p<0.05. Error bars represent mean ±SD of the data graphed.

To determine if the immunosuppressive effect of in utero PM2.5 exposure on cytokine levels was confined to the brain or extended to peripheral organs, we also quantified spleen cytokines from FA and in utero PM2.5 exposed mice. Similar to the effects observed in the brain, several spleen cytokines were reduced in the PM2.5 exposed mice including IL-2, IL-6, IL-10 and TNF-α. These data indicate that in utero PM2.5 exposure leads to long-term inhibitory effects on the immune system in general.

Discussion

In this study we report that sustained in utero exposure to high levels of PM2.5 results in changes in working memory and synaptic compartments at adulthood as well as robust immunosuppression in both the brain and the periphery. Our results suggest that in utero PM2.5 exposure has developmental consequences affecting not only the nervous system but also the immune system.

Male mice in this study were exposed to concentrated PM2.5 from the Colombus, Ohio region using the “Ohio Air Pollution Exposure System for Interrogation of Systemic Effects 1” or OASIS-1 which concentrates ambient PM and exposes the whole body of the animal to PM2.5.

This type exposure system has been described in detail and used for a variety of exposure studies (Maciejczyk et al., 2005; Sun et al., 2005; Sun et al., 2008; Wold et al., 2012). The composition of the PM2.5 generated by the OASIS-1 has been characterized. It has been found to contain relatively high amounts of elemental sulfur as well as a range of metals including lead, zinc and iron (Xu et al., 2010; Ying et al., 2009). PM were not visualized in the study and it is important to note that the PM filter utilized in this study allows any particle of size ≤ 2.5 microns which includes the ultra-fine fraction. The average daily concentration of PM2.5 to which mice were exposed in this study was 46.70 μg/m3. Animals were exposed for 6 hours every day, thus the average 24 hour concentration was equivalent to 11.67 μg/m3. This dose of PM2.5 exposure is both below the NAAQS recommended limits and well within the range of PM2.5 concentrations observed in cities and industrial areas observed worldwide (worldwide PM2.5 levels can be viewed at: http://aqicn.org/map/world). For example, in Lima, Peru, the mean PM2.5 concentration was recently measured at 26 μg/m3 (Silva et al., 2017). Additionally, PM2.5 levels have been observed exceeding 100 μg/m3 in populated areas around the world (Costa et al., 2017; Nagar et al., 2017; Xiong et al., 2016).

It is important to consider the potential contribution of the various components of PM2.5 in contributing to neurodegeneration. As stated earlier, PM2.5 has a heterogeneous composition and is known to contain or be associated with metals, polycyclic aromatic hydrocarbons (PAHs) and other trace elements. PAHs, which are produced during the combustion of organic matter, are known to associate with PM2.5 (Li et al., 2017; Liu et al., 2017) and it has been shown that stimulation of the aryl hydrocarbon receptor (AHR) to which PAHs bind can affect brain development in zebrafish (Aluru et al., 2017). It has been suggested that, in humans, exposure to PAHs negatively affects brain development and cognition, possibly by changing BDNF levels (Perera et al., 2015). Developmental toxicity of PM2.5 may also be linked, in part, to the metal content of the particles. It has recently been reported that PM2.5 exposure leads to deposition of metals within numerous tissues including brain (Ku et al., 2017). This contributes to the idea that the lungs are not the sole site of PM2.5 toxicity but rather the particles and/or their components have some ability to enter the circulation and effect other tissues depending on their physical characteristics and the overall length of exposure (Kreyling et al., 2013). It has long been known that metals have neurotoxic effects on development (Clarkson, 1987; Karri et al., 2016) and growing evidence suggests that metal exposure may also result in neuroinflammation (Chibowska et al., 2016).

Previous research, including work from our group, has shown that adult animals or humans exposed to PM2.5 have significantly increased COX2 protein levels in the brain. (Bhatt et al., 2015; Bos et al., 2012; Calderon-Garciduenas et al., 2004; Calderon-Garciduenas et al., 2013). Interestingly, our data indicate that in utero exposure to air pollution also elevates COX2 levels in the CNS, suggesting some common mechanism independent of the exposure paradigm. It is interesting to speculate what the consequence of elevated neuronal COX2 protein levels indicates. Elevated COX2 protein levels have been shown to correlate with decreased protein levels of synaptophysin in conditions such as schizophrenia (Rao et al., 2013) and Alzheimer’s disease (Rao et al., 2011). In addition, prior work in vitro has shown an inverse relationship between COX2 and synaptophysin expression, in which decreasing COX2 expression results in increased synaptophysin levels (Narayanan et al., 2006). These observations are consistent with the idea that elevated COX2 production of prostaglandins potentiates degenerative neuroinflammatory changes in the brain. However, COX2 has been shown to localize to synaptophysin-positive presynaptic terminals (Lin et al., 2014) as well as the postsynaptic compartment (Sang et al., 2005), and has a well-characterized role in generating prostaglandins to act presynaptically to modulate neurotransmission and even long-term potentiation (Chen et al., 2002; Jadhav et al., 2009; Koch et al., 2010; Sang et al., 2005; Slanina and Schweitzer, 2005). Therefore, it is not unreasonable to expect that elevated neuronal COX2 expression leads to altered synaptic compartment function and neurotransmission to contribute to the memory deficit observed.

Although total Iba1 protein levels or immunoreactivity were not robustly different across the treatment groups, we did observe increased immunoreactivity for GFAP in the temporal cortex of PM exposed mice. GFAP is known to be upregulated in activated astrocytes suggesting that PM2.5 exposure promotes a pro-inflammatory-like response in these cells (Balasingam et al., 1994). Additionally, attenuated levels of multiple cytokines were observed in the temporal cortex of in utero PM2.5 exposed mice. Interestingly, in agreement with these findings, a study of induced in utero inflammation in rabbits using bacterial toxins also showed similar changes in impaired microglial function and morphology in cultured brain slices (Zhang et al., 2016). This suggests that air pollution exposure during critical developmental periods may fundamentally alter the immune system. In agreement with these data, several recent reports indicate that maternal immune activation or exposure to environmental toxicants during pregnancy can alter development of both the nervous and immune systems and modulate cytokine levels (Crum et al., 2016; Fischer et al., 2016; Prahl et al., 2016; Zhang et al., 2016). In addition, glia are known to play an important role in synapse pruning and this function may be impaired by PM2.5 induced inflammation contributing to, for example, the increased presynaptic protein synaptophsyin in the in utero PM2.5 exposed brains (Bialas and Stevens, 2013; Chung et al., 2015; Schafer et al., 2012).

Work by others has clearly demonstrated that increased Arg1 expression and activity in the brain of Alzheimer’s disease mice increases arginine metabolism with the resultant amino acid deficiency leading to an immunosuppressive phenotype of glial cells as well as memory loss (Kan et al., 2015). These observations are entirely consistent with our findings of elevated Arg1 protein levels, attenuated cytokine levels, and memory dysfunction in the in utero PM2.5-exposed mice. It is interesting to speculate that a prolonged deficiency of arginine may lead to cellular death as well as contribute to progression of age-associated cognitive dysfunction.

The mechanism by which developing embryos may be affected by PM2.5 remains to be determined. Pregnancy is a biologically complex situation in which the immune system and cytokines of the mother play an important role (PrabhuDas et al., 2015). As stated above, previous research indicates that PM2.5 may directly enter the circulation where it exerts toxic effects and activates a robust inflammatory response. Interestingly, PM2.5 may directly damage the placenta. Pregnant rats exposed to PM2.5 show markers of systemic inflammation in addition to pathological changes in the placenta as well as placental inflammation (Liu et al., 2016). Another study has shown that in humans, gene expression, including the BDNF pathway, is altered in PM2.5 exposed placental tissue (Saenen et al., 2015). It is plausible that PM2.5 adversely affects mouse development, in part, through these effects. Our study is most similar to recently published work by Klocke et al. (Klocke et al., 2017a; Klocke et al., 2017b) which demonstrated that exposure to concentrated ambient particles alters both mouse behavior and impairs normal oligodendrocyte maturation and function. Interestingly, their data suggests female mouse brain development may be impaired to a greater extent by PM2.5 exposure than male brains. Taken together with our study, it appears that the brain, and in particular the glial cells, may be compromised by PM2.5 exposure during developmental windows.

Epidemiological evidence indicates that humans exposed to air pollution in utero experience long term immunomodulation sequelae. For example, work from others suggests that high PM2.5 or air pollution exposure during gestation increases the chances of developing asthma (Bharadwaj et al., 2016; Leon Hsu et al., 2015). While the implications of our findings may be somewhat limited as this study utilized male animals, our results contribute to a growing body of literature demonstrating the toxic effects of prolonged exposure to PM and suggest in utero exposure to PM2.5 promotes long term changes in mouse behavior, brain inflammation and immune activation. Although several significant changes were observed in the mice exposed in utero to PM2.5, we noted considerable variability in the data. This may be partly due to differential responses to PM2.5, litter effects, or differential responses to PM2.5 by the pregnant mothers. A larger number of animals for each condition may be necessary to reduce the variance in subsequent study of the neuroimmune consequences of exposure. Indeed, future research will attempt to determine what effect, if any, PM2.5 may have in transgenic neurodegenerative models as well as induced models of neurodegeneration.

Capsule.

Our data indicate that in utero exposure to particulate matter has long term consequences in the adult brain including behavioral alterations, increased COX2 expression and broad changes in cytokine levels.

Highlights.

  • Exposure to PM2.5 in utero impaired spatial memory in adult animals with minimal effects on anxiety-like behavior

  • Brains harvested from animals exposed to PM2.5 in utero had increased protein levels of COX2, Arg1 and synaptophysin.

  • Modest morphological changes in microglia and astrocytes were observed by immunohistochemistry in PM2.5 exposed animals.

  • Exposure to PM2.5 altered the levels of numerous cytokines in both the brain and spleen.

Acknowledgments

This publication was supported by NIH 5R01AG042819 and NIH 5R01ES019923.

Footnotes

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Declaration: The authors declare that they have no competing financial interests.

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