Diurnal variation in the proinflammatory activity of urban fine particulate matter (PM_2.5) by in vitro assays

Introduction

Particulate matter (PM) with an aerodynamic diameter less than 2.5 µm (fine PM or PM_2.5), is associated with diverse health problems and chronic diseases, including asthma, chronic obstructive pulmonary disease (COPD), lung cancer, and coronary heart disease (Delfino et al., 2005; Delfino et al., 2011; Dockery et al., 1993; Dominici et al., 2006; Kaufman et al., 2016; Kim et al., 2013; Landrigan et al., 2018; Shah et al., 2013). Findings of recent epidemiological studies extend chronic PM_2.5 exposure risk to Alzheimer’s disease and accelerated cognitive decline (Cacciottolo et al., 2017; Chen et al., 2015; Chen et al., 2017). Corresponding rodent models show robust indicators of inflammatory and oxidative stress to PM_2.5 fractions in pathological responses of aorta (Li et al., 2003), brain (Cheng et al., 2016b; Levesque et al., 2011; MohanKumar et al., 2008; Morgan et al., 2011), and lung (Zhang et al., 2012).

In addition to the epidemiological associations with chronic disease, we must also consider diurnal variations in airborne particulate matter chemistry that are not included in most long-term epidemiological studies. Diurnal variation in air pollution toxicity is suggested by diurnal variations in emergency department admissions for dementia (Linares et al., 2017), ischemic stroke (Han et al., 2016), and respiratory conditions (Darrow et al., 2011). Although these admissions were more strongly associated with ozone than with PM_2.5 in all three of these studies, diurnal changes in PM_2.5 chemistry must also be considered as an influencing factor. Freshly emitted primary PM undergoes photochemical oxidation reactions over the course of the day, catalyzed by ultraviolet (UV) sunlight, which results in diverse oxidized organic and inorganic products (secondary PM) (Forstner et al., 1997; Grosjean & Seinfeld, 1989), along with concomitant changes in PM toxicity. These diurnal changes in PM_2.5 composition and associated toxicity are relevant to and may inform future long-term epidemiological studies of primary and secondary particulate matter. While prior studies in the Los Angeles air basin have shown extensive diurnal variations in PM composition and size, the findings of PM oxidative activity have been inconsistent and differ between various assays of oxidative potential (Saffari et al., 2015; Verma et al., 2009; Wang et al., 2013b).

The current study further examined diurnal variations in composition and oxidative potential of PM samples collected at the central Los Angeles site used in the three studies mentioned above. However, unlike these earlier studies, PM samples were collected by a direct aerosol-into-liquid collection method to provide time-integrated aqueous PM_2.5 slurries for both morning and afternoon periods. This technology allows for a more comprehensive analysis than the filterable (i.e. water extracted) particulate samples examined in our prior studies (Morgan et al., 2011; Saffari et al., 2015; Verma et al., 2009; Woodward et al., 2017a).

Microglia were used for in vitro assays of oxidative and inflammatory responses to PM_2.5 exposures because of their increasingly recognized role in environmental neurotoxicology (Krafft, 2015). Air pollution can induce premature microglial activation, as documented in rodent models (Cheng et al., 2016a; Hanamsagar & Bilbo, 2017; Morgan et al., 2011) and as indicated for young adults living in the highly polluted Mexico City (Calderón-Garcidueñas et al., 2008; Calderón-Garcidueñas et al., 2018). Microglia (BV-2) cell cultures were assayed for induction of nitric oxide (NO) and for proinflammatory gene mRNA responses of interleukins 6 and 1β (IL-6 & IL-1β), and monocyte chemoattractant protein 1 (MCP-1), also known as chemokine (C-C motif) ligand 2 (CCL2). These markers were chosen because of their in vivo and in vitro responses to ultrafine PM shown in prior studies (Cheng et al., 2016b; Morgan et al., 2011; Woodward et al., 2017b).

We hypothesized that afternoon PM_2.5 (pm-PM_2.5), with its high proportion of secondary photochemical oxidation products, would have greater oxidative and proinflammatory activity than freshly emitted, primary PM collected during morning hours (am-PM_2.5).

Methods

Results

Nitric Oxide (NO)

A dose-dependent NO response to PM_2.5 treatments relative to control was observed at all timepoints (30 min., 60 min., 24 hr.), which was greater for am-PM_2.5 than pm-PM_2.5 exposures (Figure 1). am-PM_2.5 samples induced consistently higher levels of NO for all concentrations and post-exposure timepoints, with a peak effect, 7-fold greater than control (p = 0.0077), observed at 60 minutes in response to the highest am-PM_2.5 dose of 20 µg/mL (Figure 1A). At 30 minutes post-treatment, there was also a significant 5.3-fold increase of am-PM_2.5 relative to control (p = 0.0020), and a significant difference between the responses to am-PM_2.5 and pm-PM_2.5, with am-PM_2.5 eliciting a 3.1-fold greater NO response than pm-PM_2.5 (p = 0.0094). There was also a significant a significant 2.9-fold increase of am-PM_2.5 relative to control (p = 0.0007) at 24 hours post-treatment. The NO responses to pm-PM_2.5 paralleled the effects of am-PM_2.5 exposures, but were at least 50% smaller (Figure 1B): the 20 µg/mL pm-PM_2.5 treatment induced 1.7-, 3.5-, and 2.0-fold increases in NO concentration relative to control at 30 min., 60 min. and 24 hrs., respectively, but these effects were not significant. The acute effects of PM exposure seen within the first hour of exposure, at 30 and 60 minutes post-treatment, are due to direct NO induction, while the sustained effect still measurable after 24 hours indicates that there has been upregulation of the iNOS enzyme that produces NO. Thus, this overall effect is two-fold, with the increase in NO secretion due to PM_2.5 exposure mediated by two distinct mechanisms, acute and delayed.

Figure 1. Nitric oxide (NO) induction by microglia.

BV-2 microglial responses to PM_2.5 slurries in vitro, assayed in culture media by the Griess reaction (control = 1.0 µM nitrite). A. Morning samples (am-PM_2.5); B. Afternoon samples (pm-PM_2.5). am-PM_2.5 samples induced consistently higher NO responses for all concentrations and post-exposure timepoints. At 30 minutes post-treatment, there was a significant effect of am-PM_2.5, as well as a significant difference between the responses to am-PM_2.5 and pm-PM_2.5 (overall ANOVA: p = 0.0017; am-PM_2.5 20 µg/mL vs. control: 5.3-fold increase, p = 0.0020; am-PM_2.5 20 µg/mL vs. pm-PM_2.5 20 µg/mL: 3.1-fold increase, p = 0.0094). There was also a significant effect of am PM_2.5 at 60 minutes post-treatment (overall ANOVA: p = 0.010; am-PM_2.5 20 µg/mL vs. control: 7.0-fold increase, p = 0.0077). At 24 hours a significant effect of am-PM_2.5 treatment was also observed (overall ANOVA: p = 0.0005; am-PM_2.5 20 µg/mL vs. control: 2.9-fold increase, p = 0.0007). Mean ± SE (n = 3 experiments). 2-way repeated measures ANOVA statistical analysis with Bonferroni post hoc tests: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Inflammatory gene responses

BV-2 cells were treated with 10 μg/mL of am-PM_2.5 and pm-PM_2.5 and analyzed for mRNA responses by qPCR after 24 hours incubation. The 10 μg/mL dose was chosen as below threshold for metabolic impairment based on prior studies from our group (e.g. Cheng et al., 2016b; Morgan et al., 2011; Woodward et al., 2017b). Induction of all three cytokines was increased by both morning and afternoon PM_2.5 samples, with more modest responses to pm-PM_2.5 (Figure 2). As shown in Figure 2A, treatment with am-PM_2.5 induced a significant 4.8-fold increase in IL-1β expression relative to control (p = 0.0070). Both am-PM_2.5 and pm-PM_2.5 induced significant increases in IL-6 mRNA production relative to control, with am-PM_2.5 exposure resulting in a 5.1-fold increase (p < 0.0001) and pm-PM_2.5 resulting in a 3.5-fold increase (p = 0.0046) (Figure 2B). Treatment with am-PM_2.5 also induced a significant 2.0-fold increase in MCP-1 mRNA production (p = 0.0022), while pm-PM_2.5 had a 33% smaller effect (Figure 2C). This difference in MCP-1 mRNA production induced by am-PM_2.5 as compared to pm-PM_2.5 was marginally significant (p = 0.0527).

Figure 2. Inflammatory gene mRNA induction in microglia.

After exposing BV-2 cells to 10µg/mL of morning (am-PM_2.5) and afternoon (pm-PM_2.5) slurries, cellular mRNA production was assessed by qPCR. Relative to control, both am-PM_2.5 and pm-PM_2.5 exposures increased mRNA levels of A. Interleukin 1β (IL-1β), B. Interleukin 6 (IL-6), and C. monocyte chemoattractant protein 1 (MCP-1). Treatment with am-PM_2.5 induced a significant 4.8-fold increase in IL-1β expression relative to control (overall ANOVA: p = 0.0090; am-PM_2.5: 4.8-fold increase, p = 0.0070). Both am-PM_2.5 and pm-PM_2.5 induced significant increases in IL-6 mRNA production (overall ANOVA: p < 0.0001; am-PM_2.5: 5.1-fold increase, p < 0.0001; pm-PM_2.5: 3.5-fold increase, p = 0.0046). Treatment with am-PM_2.5 also induced a significant increase in MCP-1 mRNA production, while pm-PM_2.5 had an effect 33% smaller than am-PM_2.5 (overall ANOVA: p = 0.0028; am-PM_2.5: 2.0-fold increase, p = 0.0022; am-PM_2.5 vs. pm-PM_2.5: p = 0.0527). Mean ± SE (n = 12). 2-way repeated measures ANOVA statistical analysis with Bonferroni post hoc tests: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Chemical composition of PM_2.5 slurry samples

The am-PM_2.5 and pm-PM_2.5 time-integrated aqueous slurry samples were analyzed for chemical composition, including total carbon (TC), inorganic ions, and total metals and trace elements, and are presented as PM_2.5 mass fractions in Figures 3A, 3B, and 3C, respectively. PM_2.5 TC content decreased by 40% from morning (0.50 μg/μg-PM) to afternoon (0.31 μg/μg-PM) (Figure 3A). Mass concentrations of inorganic secondary ions (NO₃^-, SO₄^2-, NH₄⁺, Na⁺) were approximately 5-fold higher in the afternoon as compared to morning slurries (Figure 3B). For the sixteen metals and trace elements analyzed, the am-PM_2.5 slurry contained higher mass concentrations of several measured elements as compared to the pm-PM_2.5 slurry (Figure 3C, note log scale; Table S1, Supplementary File 1). Arsenic, chromium, and manganese showed the largest diurnal decline, represented as am-PM_2.5:pm-PM_2.5 ratios: arsenic (11.6), chromium (7.9), and manganese (6.0).

Figure 3. Chemical analyses.

Time-integrated PM_2.5 slurries collected during morning (6–9am) and afternoon (12–4pm) periods analyzed for A. Total Carbon (TC), B. Inorganic ions (ion chromatography), C. Total metals and trace elements (ICP-MS). Mean values presented are based on triplicate analysis of one sample aliquot. Error bars represent laboratory uncertainty values based on contributions of analytical error (standard deviation) and blank subtraction (standard deviation of at least three method blanks).

Dataset 1.The following raw data sets are provided as comma separated values (.csv) files.

PM_Diurnal_Variation_NO_Fig1_DATAPM_Diurnal_Variation_qPCR_Fig2_DATAPM_Diurnal_Variation_TC_Fig3A_DATAPM_Diurnal_Variation_Ions_Fig3B_DATAPM_Diurnal_Variation_Metals_Fig3C_DATA

Discussion

Diurnal variations in urban PM_2.5 oxidative and proinflammatory activity showed consistent decreases from morning to afternoon sampling periods in two independent in vitro assays using the BV-2 microglia cell line. The collection of total PM_2.5 as an aqueous slurry was enabled by direct aerosol-into-liquid sampling that more efficiently captures water-insoluble components of ambient PM_2.5 than traditional filter-based sampling methods used in several prior studies (e.g. Saffari et al., 2015; Verma et al., 2009). These slurry samples are more representative of the full range of ambient PM components and their toxicities than filter-trapped and water eluted PM. Additionally, the results of the NO assay and the qPCR assay for inflammatory gene responses extend findings from the widely used dithiothreitol (DTT) and alveolar macrophage (dichlorodihydrofluorescein, DCFH) assays for oxidative potential, which can be confounded by oxidative recycling from transition metals (Forman & Finch, 2018). Our findings, that primary PM_2.5 results in a greater oxidative and proinflammatory response than secondary PM_2.5, are contrary to expectations based on prior reports that secondary, photo-oxidized PM exhibits greater oxidative activity than primary PM.

While there may be a concern that the concentrations of PM_2.5 treatments used in the in vitro assays do not reflect the exact concentrations of PM_2.5 reaching microglia in the brain following ambient exposures, these assays do serve as a useful model for how brain cells in living organisms would respond to PM_2.5 at the given concentrations (i.e. 1, 5, 10, and 20 μg/mL). The focus of the paper was to investigate the differences between morning (primary-dominated) and afternoon (secondary-dominated) PM_2.5, rather than to quantify actual exposure concentrations, and subsequent CNS concentrations, that would be considered harmful. We modeled these interactions between PM_2.5 and microglia using concentrations that are below the threshold for cell death, as evaluated by the MTT assay. While there is evidence that particles can directly enter the brain through the olfactory tract (Oberdörster et al., 2004), and thus perhaps maintain higher concentrations than PM_2.5 passing through the periphery, the concentration of particles interacting directly with brain cells via this route has not been quantified, and thus comparisons to these results could not be made.

Previous studies of diurnal variations in PM composition and oxidative activity have not been consistent and were limited in using only simple assays of oxidative potential (i.e. DTT and DCFH) on filter-captured PM. Relying solely on oxidative potential measures such as the DTT and DCFH assays provides us with only an imprecise measure of cellular oxidative and proinflammatory activity that lacks specificity. The current study improves on the experimental design of past studies by utilizing direct measures of acute oxidative stress and inflammation, including free radical production induced by PM as nitric oxide (NO) and cellular proinflammatory mRNA responses. Additionally, by using the direct aerosol-into-liquid method to collect aqueous slurries in our study, water-insoluble PM species were more efficiently captured, providing samples more representative of the full range of ambient PM components and their toxicities.

Further insight into the sources of particulate toxicity may be gleaned by the apportionment of redox properties to its water soluble and insoluble chemical components, including water-soluble and water-insoluble organic carbon (WSOC and WIOC, respectively). WSOC species are generally defined as hydrophilic, while WIOC are hydrophobic (Turpin & Lim, 2001). Wang et al. (2013b) collected aqueous PM_2.5 slurries by a similar aerosol-into-liquid sampling method, and found that increased WIOC content in PM_2.5, relative to WSOC content, was highly correlated with redox activity on a per mass basis, indicating a greater intrinsic toxicity of WIOC as compared to WSOC. While this study was limited by its use of the DCFH assay, the greater oxidative potential associated with increased WIOC mass concentrations was attributed to organic compounds such as PAHs, as well as iron and other transition metals.

Our results indicate that morning PM_2.5, which contains a greater proportion of water-insoluble species, may be intrinsically more toxic and induce greater cellular oxidative stress, than afternoon PM_2.5 samples that contain a larger mass fraction of oxidized, water-soluble species that are products of photochemical reactions in the atmosphere (Seinfeld & Pandis, 2016), including the inorganic secondary ions NO₃^-, SO₄^2-, NH₄⁺, and Na⁺. The mechanisms underlying the greater toxicity of primary, morning PM_2.5 may involve non-polar WIOC components, such as PAHs, being able to more easily permeate the hydrophobic lipid-bilayer of cell membranes to trigger the formation of intracellular oxidative species and induce proinflammatory cytokine formation via an acute oxidative stress response.

Primary, traffic-derived PM_2.5 also consists of greater concentrations of redox active and other toxic metals, as compared to the bulk of secondary PM_2.5, which consists largely of hydrophilic products of photochemical oxidation. The metals and trace elements we found to be more prevalent in the morning slurry sample included the heavy metals vanadium, chromium, nickel, and arsenic, which are emitted by vehicles both as fuel combustion products as well as remnants of motor oil degradation (Geller et al., 2006), copper, which is associated with vehicular brake wear (Garg et al., 2000; Sanders et al., 2003; Sternbeck et al., 2002), and zinc, which is primarily a product of tire deterioration (Singh et al., 2002). Elevated levels of these metals in both collection periods correspond to vehicular emissions as the major source of primary particles in close proximity to the I-110 freeway. We believe the higher proportions of these metals and WIOC components in primary PM_2.5 dominant in the morning hours, as compared to photo-oxidized secondary PM_2.5 prevalent in the afternoon, are responsible for the diurnal variation in acute oxidative stress observed in the current study.

Summary and conclusions

The data presented in this study demonstrate that urban PM_2.5 collected during the morning rush hour (6–9am), when primary, traffic-derived PM emissions are dominant, induces greater oxidative and proinflammatory responses in cells as compared to PM_2.5 collected in the afternoon (12–4pm), which contains a higher proportion of photo-oxidized, secondary PM products. Two in vitro assays of the cellular inflammatory response consistently demonstrated greater oxidative and proinflammatory activity due to primary (morning) PM_2.5 exposure. We attribute this effect to the greater transition metal and water-insoluble organic carbon (WIOC) content of primary PM_2.5, two classes of PM components that increase toxicity (Cho et al., 2005; Hu et al., 2008; Li et al., 2009; Shirmohammadi et al., 2015; Tao et al., 2003; Zhang et al., 2008). Our study also improves upon previous research of diurnal variations in PM-induced oxidative stress by utilizing a unique aerosol-into-liquid PM collection system that more efficiently captures water insoluble components, thus providing complete aqueous PM samples more representative of ambient PM.

This research will ultimately help us gain a more complete understanding of the complex nature of particulate matter and how its composition and proinflammatory effects change over time due to photochemical aging in the atmosphere. The Southern California climate of Los Angeles with abundant sunshine, compounded with dense vehicular traffic, generates ubiquitous primary and secondary PM throughout the year. Identifying the health effects of these pollutants is critical as we strive to understand the underlying mechanisms of PM-induced oxidative stress, neuroinflammation and associated morbidity. Our findings may help in further elucidating the role of PM in the etiology, onset and development of widespread, chronic diseases that plague urban populations, including cancer, cardiac and respiratory distress, and neurodegenerative disorders such as Alzheimer’s disease.

Data availability

Dataset 1: The following raw data sets are provided as comma separated values (.csv) files: 10.5256/f1000research.14836.d203329 (Lovett et al., 2018)

PM_Diurnal_Variation_NO_Fig1_DATA

PM_Diurnal_Variation_qPCR_Fig2_DATA

PM_Diurnal_Variation_TC_Fig3A_DATA

PM_Diurnal_Variation_Ions_Fig3B_DATA

PM_Diurnal_Variation_Metals_Fig3C_DATA