Final Report: Project 2: The Role of Oxidative Stress in PM-induced Adverse Health Effects

EPA Grant Number: R832413C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R832413
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Southern California Particle Center
Center Director: Froines, John R.
Title: Project 2: The Role of Oxidative Stress in PM-induced Adverse Health Effects
Investigators: Nel, Andre E. , Harkema, Jack , Kleinman, Michael T. , Lusis, Aldons
Institution: University of California - Los Angeles , Michigan State University , University of California - Irvine
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

The primary objective of this application was to determine, using in vitro and in vivo studies, the mechanism(s) by which ambient PM components contribute to asthma and atherogenesis. Our principal hypothesis was that PM-induced oxidative stress initiates a series of biological effects that promote airway inflammation and atherosclerosis. Integral to this hypothesis is the role of antioxidant defense pathways that protect against the pro-inflammatory effects of PM. A failure to respond to oxidative stress by mounting an appropriate antioxidant defense could constitute the basis of PM susceptibility. Progress in studying the adjuvant effect of PM has led to a more in-depth elucidation in the mechanisms by which ambient PM contribute to the pathogenesis of allergic disease such as asthma and allergic rhinitis. Our research on the impact of PM on cardiovascular system has increased our understanding in the mechanisms that are responsible for the proatherogenic effect of PM. We will summarize our conclusions as they relate to the impact on asthma and atherosclerosis.

Summary/Accomplishments (Outputs/Outcomes):

Aim 1: We will use normal and genetically susceptible mice to study the role of oxidative stress in PM-induced exacerbation of asthma and atherosclerosis

1. The adjuvant effect of ambient PM on allergic sensitization is closely reflected by the particles’ oxidant potential

Although studies have suggested that ambient PM can act as an adjuvant to promote sensitization to common environmental allergens, there has been a paucity of direct evidence showing this effect on allergic sensitization. We developed a highly sensitive mouse model, which allows us to demonstrate the adjuvant effect of ambient ultrafine particles (UFP) on ovalbumin (OVA)-induced allergic sensitization in vivo. Analysis of bronchoalveolar lavage (BAL) showed that upon secondary OVA aerosol challenge animals intranasally sensitized by a low dose of endotoxin-free OVA in the presence of UFP developed a significant exacerbation of allergic inflammation in the lung and nose compared to the saline control or OVA alone. Ambient UFP significantly enhanced OVA-induced eosinophil infiltration, airway inflammation, and OVA-specific antibody (OVA-IgE and OVA-IgG1) production. UFP also increased the production of several pro-inflammatory cytokines in the lung including TNFα, IL-5, IL-6, IL-13, KC, MCP-1 and MIP-1α, whereas those in other groups (OVA alone, LPS plus OVA, or F/UF plus OVA) were unaffected. The increase of IL-5 and IL-13 by OVA/UFP is an important indicator that ambient UFP is capable of skewing immune response towards Th2 immunity. Our dose-response studies have shown that a single intranasal exposure to OVA/UFP is sufficient to elicit eosinophilic inflammation in the lung as well as to elevate OVA-IgG1 and OVA-IgE levels in the blood. Using different controls, we showed that neither endotoxin nor ultrafine carbon black particles had an effect on OVA sensitization. Moreover, side-by-side comparison of UFP and fine particles (FP, PM2.5) indicates that this adjuvant effect is specific to the UFP since FP failed to enhance the effect of OVA. This approach has allowed us to compare the contribution of particle size and accompanying differences in the oxidant potential of PM2.5 and UFP in an in vivo model, similar to what we have previously demonstrated in cultured cells.

The results of BAL and OVA-specific antibody analyses were confirmed by histological analysis. Morphometry of the lung showed that the major changes in the lungs of OVA/UFP-treated mice consisted of marked mucous cell metaplasia in the surface epithelium lining the conducting airways (large- and small-diameter bronchioles) and mixed inflammatory cell influx consisting of eosinophils, lymphocytes and plasma cells in the interstitial tissues surrounding these airways.  Airway lesions were most severe in the main axial airways, but were also present to a slightly lesser degree in the small-diameter, terminal bronchioles of the mice exposed to both OVA and UFP.

Along the axial airways, the volume densities of intraepithelial mucosubstances in the proximal and distal generations (5 and 11) were approximately 22 and 24 times greater, respectively, than those measured at the same airway generations in the saline control mice.  Mice exposed only to OVA had epithelial and inflammatory alterations that were similar to those in the OVA/UFP mice, but the severity of these changes in the large-diameter, pre-terminal and small-diameter, terminal bronchioles were less.  The mucosubstances in the airway epithelium lining the proximal axial airways (generation 5) of OVA/UFP-treated mice were approximately twice as abundant as those in animals exposed only to OVA. In the distal axial airway (generation 11), OVA/UFP-exposed mice generated almost five times more intraepithelial mucosubstances compared to those in OVA-alone mice.

Nasal morphometry showed that mice exposed to OVA plus UFP had airway epithelial and inflammatory changes consistent with an acute allergic rhinitis. The major alterations were mucous cell metaplasia/hyperplasia of airway epithelium accompanied by inflammatory cell influx including eosinophils and mononuclear cells (lymphocytes and plasma cells) in the underlying lamina propria in the nose. These changes were restricted to intranasal regions lined by transitional or respiratory epithelium. There was a markedly greater amount of mucosubstances in the nasal transitional epithelium lining the maxilloturbinates compared to the mice in the control or OVA-alone groups.  These data suggest that UFP exert its adjuvant effect immediately upon contact with the allergen in the nasal turbinate and this may explain the association between air pollution and allergic rhinitis. 

To obtain evidence that the adjuvant effect of UFP was correlated to its redox-active organic chemical contents, we characterized these ambient PM for their chemical composition, redox potential, and the ability to induce intracellular oxidative stress. UFP consistently had higher organic carbon content compared to the FP. The DTT assay, which measures the redox activity of PM based on the interaction between quinones and DTT, showed that UFP had much stronger oxidant potential than the FP. Moreover, UFP also had a greater ability to induce antioxidant enzyme HO-1, a sensitive marker of oxidative stress. Taken together, our findings suggest that the degree of adjuvancy is related to the OC content and oxidant potential of PM. 

In summary, these results suggest that intranasal OVA delivery caused minimal allergic inflammatory responses in the lungs and nose of mice without concomitant intranasal UFP instillation. Thus, intranasal exposure to UFP exerts an adjuvant effect that could not be obtained with intranasal fine PM (1).

2. Inhalation of “real-life” UFP could effectively boost the secondary immune response in previously sensitized animals

After publishing the work on the adjuvant effect of ambient UFP on the primary immune response and how that leads to allergic sensitization (1), we continued our efforts to answer the question whether inhalation of ambient UFP could boost the secondary immune response in previously sensitized animals upon re-exposure to the same allergen. In collaboration with Dr. Sioutas, we conducted aerosolized inhalation exposure in the mobile animal research laboratory located near the 110 Freeway in downtown Los Angeles. Mice were sensitized by two intranasal administrations of OVA (10 µg) together with a low dose of UFP (0.5 µg). After a 2-week interval, animals were given inhalation exposures to either filtered air or concentrated ambient UFP for 4 hrs per day for 5 days. OVA aerosol (1%) challenge was given on the last two days of inhalation exposure.

Our results show that as few as five inhalation exposures to “real-life” UFP were sufficient to boost the secondary immune response during OVA challenge. The UFP-enhanced secondary immune response was characterized by a profound eosinophilic airway inflammation, increased systemic OVA-IgG1 and OVA-IgE production and Th2 and IL-17 cytokine gene expression profiles in the lung.  Real-time PCR analysis confirmed that UFP inhalation significantly increased the expression of eotaxin, IL-5, IL-13, TNFα, KC, IL-10, and Muc5ac genes in the lungs of prior sensitized animals. In addition, UFP inhalation also enhanced IL-17a gene expression in the lung, which was accompanied by increased BAL neutrophil counts. Lung morphometry demonstrated that UFP targeted the centriacinar region of the distal lung during the secondary immune response, which stands in contrast to the adjuvant effect of UFP on the primary immune response that elicits inflammation in the nose and axial airways. That UFP-boosted allergic inflammation was accompanied by increased Ym1 protein expression, a PM-induced oxidative stress marker previously identified by proteome analysis, in the same centriacinar region suggests that the pro-inflammatory effects of UFP during the secondary immune response involve the generation of local oxidative stress (2). 

Taken together, the significance of this work is that our results showed, for the first time, that pro-oxidative UFP inhalation exposure is quite effective in boosting the secondary immune response after only a limited number of exposures. This leads to the exacerbation of allergic airway inflammation characterized by Th2 and Th17 cytokine profiles in already-sensitized animals. Inhaled UFP target the distal lung, including the alveolar duct and alveolar parenchyma, where the generation of oxidative stress play a role in enhancing the secondary immune response. Our study is also important from the perspective of asthma prevention and exacerbation by air pollution particles. Epidemiological studies indicate a close link between surges in ambient PM levels and asthma flares. While a host of atmospheric conditions and pollutant sources may contribute to these exacerbations, it is important to consider the role of freeway proximity in determining exposure to relevant UFP concentrations.

3. Genetic deficiency in Nrf2-mediated antioxidant/detoxification pathways significantly enhances the adjuvant effect of ambient UFP on allergic sensitization

Induction of cellular oxidative stress by organic chemicals or inorganic metals/elements on particle surface has been identified as one of the key mechanisms by which PM exerts its adverse health effects including asthma exacerbation. A crucial pathway that protects cells from oxidant injury is the antioxidant and phase II enzyme defense system mediated by the transcription factor, Nrf2. In addition to protecting cells against oxidative and electrophilic injury, Nrf2 signaling is also involved in attenuating inflammation-mediated respiratory disorders (e.g. asthma and emphysema). The anti-inflammatory affect of Nrf2 is likely the result of a cooperation of Nrf2-targeted genes in regulating the innate immune response, leading to the suppression of pro-inflammatory genes. Thus, deficiency or complete loss of Nrf2 signaling may enhance the susceptibility not only to oxidative and electrophilic stress but also to inflammatory tissue injury, including the impact of environmental pollutants. Using our intranasal sensitization mouse model we tested the hypothesis that Nrf2 deficiency could enhance the adjuvant effect of PM on allergic sensitization. The successful backcross of Nrf2 knockout (Nrf2-/-) mice onto a Balb/C background allowed us to conduct the follow studies:

We sensitized wild-type (Nrf2+/+) and Nrf2-/- mice through intranasal instillation of endotoxin-free OVA (10 µg) with or without a low dose of ambient UFP (0.5 µg) and challenged them with 30-min OVA aerosol (1%) on three consecutive days two weeks later. BAL analysis showed that while OVA/UFP effectively induced an eosinophil influx in the lungs of both Nrf2+/+ and Nrf2-/- mice compared to saline- or OVA-sensitized animals, the number of eosinophils from Nrf2-/- mice was significantly greater than that from Nrf2+/+ mice in the same treatment group. Sensitization by OVA alone increased OVA-IgG1 production in Nrf2+/+ as well as Nrf2-/- mice. While UFP further increased OVA-IgG1 production in both types of animals, the level of this antibody was significantly higher in Nrf2-/- mice compared to their Nrf2+/+ counterpart. OVA sensitization also elevated OVA-IgE level in both Nrf2+/+ and Nrf2-/- mice compared with respective saline controls. However, the enhancing effect of UFP on OVA-IgE production was only observed in Nrf2-/- mice. Moreover, UFP-induced allergic airway inflammation was accompanied by increased levels of IL-13, a signature Th2 cytokine, in the BAL fluid of Nrf2-/- mice.  Consistent with BAL analysis, lung histology showed that only Nrf2-/- mice exposed to OVA/UFP developed a prominent inflammation in the small airways of the lung. These results provide direct evidence indicating that lack of Nrf2 can significantly augment the adjuvant effect of UFP on allergic sensitization.

4. Use of proteome analysis to study the oxidative stress response in a bronchial epithelial cell line and BAL fluid

Since the identification of global protein expression changes in BALF and lung tissue from OVA sensitized mice could provide new insights into the complex molecular mechanisms involved in asthma, we used two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify significantly increased protein expression in a murine asthma model and potential protein targets of the antioxidant, N-acetylcysteine (NAC) in allergic airway inflammation. Six proteins were found to be significantly increased in BAL from OVA-challenged mice compared to a control group, including chitinase 3-like protein 3 (Ym1), chitinase 3-like protein 4 (Ym2), acidic mammalian chitinase (AMCase), pulmonary surfactant-associated protein D (SP-D), resistin-like alpha (FIZZ1), and haptoglobin a-subunit. A total of 11 protein spots on 2D-gels were significantly increased in lung tissue; these include Ym1, Ym2, FIZZ1, and other lung remodeling related proteins.  Western blotting confirmed increased Ym1/Ym2, SP-D, and FIZZ1 expression measured from BAL fluid and lung tissue from OVA-challenged mice. Intra-peritoneal administration of NAC before OVA inhalation inhibited Ym1/Ym2, SP-D, and FIZZ1 expression in the lung. Proteins Ym1/Ym2, FIZZ1 and SP-D identified in this study could be associated with the pathogenesis of asthma and suggest a link between oxidative stress-induced inflammation and asthma (3).

We also used this technology to identify new proteins that may be useful biomarkers for the allergic airway inflammation related to PM exposure. Using our intranasal sensitization mouse model (1), we sought to assess whether the adjuvant effect of ambient PM leads to an altered proteome profile in BAL fluid. We hypothesized that the mechanistic link between the adjuvant effect of PM and changes observed in the proteome profile can be used (i) to develop biomarkers for allergic inflammatory responses caused by air pollutants, (ii) confirm the hierarchical oxidative stress model, and (iii) elucidate the mechanism of the adjuvant effect. BALF proteins from control and sensitized mice were resolved by 2D-PAGE, and identified by mass spectrometry. A total of 30 protein spots from 16 proteins were significantly changed in OVA-sensitized mice and even more significantly changed in mice exposed to an OVA plus UFP combination.  The 7 proteins showing the highest protein expression level changes were confirmed by western blotting and RT-PCR.  These proteins include surfactant protein-D, polymeric immunoglobulin receptor (PIGR), complement C3, neutrophil gelatinase-associated lipocalin (NGAL), and a family of chitinases, including chitinase-3-like protein 3 (Ym1), chitinase-3-like protein 4 (Ym2), and acidic mammalian chitinase. Among them, Ym1, Ym2, acidic mammalian chitinase, PIGR, complement C3, and NGAL demonstrated significantly enhanced up-regulation by UFP with a polycyclic aromatic hydrocarbon (PAH) content and a higher oxidant potential. The proteins identified in this study may be the important specific elements targeted by PM, through the ability to generate reactive oxygen species in the immune system, and may be involved in allergen sensitization and asthma pathogenesis (4).

5. ApoE knockout mice exposed to CAPs on the freeway shows increased atherogenic potential of ambient ultrafine particles

Epidemiological studies demonstrating that exposure to ambient particulate matter (PM) increases cardiovascular morbidity and mortality. Both epidemiological and animal-based studies suggest that the exacerbation of atherosclerosis by PM plays an important role in these outcomes. We hypothesized that PM synergizes with known pro-atherogenic stimuli and mediators in their ability to elicit oxidative stress and promote atherosclerosis, and that most of the pro-inflammatory potential resides in UFP that are highly enriched for redox cycling chemicals.

Two experimental protocols were used to test our hypothesis. In the first protocol (chow protocol), 6-week-old male C57BL/6J apoE null mice were placed on a chow diet and exposed to concentrated ambient particles (CAPs) over a 40-day period, while in the second study (high fat diet, HFD protocol), 2-month-old male apoE null mice were fed a HFD over a 56-day period. Food and water were administered ad libitum.  Mice destined for CAPs exposure were transported to the mobile research laboratory in downtown of Los Angeles, close (~300 m) to the I-110 freeway. The animals were housed in a Hazelton Chamber ventilated with air from which 99.9% of the incident particles were removed using a HEPA filter (chow protocol) or in top-filter cages (HFD protocol).  There were three exposure groups (17-18 mice/group), namely filtered air (FA), particles < 2.5 μm (FP) and particles < 0.18 μm (UFP).  Whole body exposures were performed simultaneously for five hours per day (exposure session), three days per week, for a combined total of 75 and 125 hours in the chow-fed and HFD-fed protocols, respectively. Animals were euthanized 24-48 hours after completion of the last CAPs exposure, and aortas and various organs harvested.

Our data showed that Chow-fed apoE null mice exposed to concentrated UFP developed significantly (p<0.05) larger early aortic atherosclerotic lesions. Exposure to UFP resulted in an inhibition of the anti-inflammatory capacity of plasma high density lipoproteins (HDL) and increased systemic oxidative stress markers as evidenced by a significant: (i) increase in hepatic malondialdehyde levels and (ii) upregulation of Nrf2-related phase-2 response genes (e.g., catalase, superoxide dismutase) when compared to FA or NE mice. While HFD-fed apoE null mice did not exhibit differences in their aortic atherosclerosis, they still showed evidence of increased systemic oxidative stress as demonstrated by a significant upregulation of Nrf2 and Nrf2-regulated genes (5).

Aim 2: We will use in vitro toxicology studies to assess the effects of variation in ambient PM composition on the induction of oxidative stress and inflammation in tissue culture macrophages, airway epithelial cells, and endothelial cells. 

1. Pro-oxidative DEP chemicals could perturb the function of dendritic cells (DCs), which may explain the adjuvant effect of PM in asthma

DCs play a key role in antigen presentation in the immune system. There is growing evidence that the redox equilibrium of DCs influence their ability to induce T-cell activation and regulate the polarity of immune response. To fully understand how PM promotes the pathogenesis of allergic airway inflammation, systematic dissection of the molecular pathways involved in the adjuvant effect of PM is of great general interest and it is also a key priority in the research of asthma and allergy. We tested the hypothesis that altered cellular redox equilibrium by PM and adsorbed redox cycling organic chemicals leads to the perturbation of DC function and favors Th2 skewing of the immune response. We investigated how DEP-induced oxidative stress interferes with DC function including maturation, antigen uptake and presentation, expression of costimulatory molecules, cytokine/chemokine production, and T-cell activation.

Exposure of bone marrow-derived DCs (BMDC) to organic DEP diesel exhaust particle (DEP) extract (DEPext) resulted in a dose-dependent glutathione depletion and the induction of heme oxygenase 1 (HO-1). Although DEP chemicals per se did not change the expression of DC surface functional molecules (I-Ad, CD54, and CD86), DEPext was able to suppress LPS-induced their expression in a dose-dependent fashion. Similarly, while DEPext alone failed to exert an effect on IL-12p40 and IL-12p70 production by BMDC, it suppressed the LPS-induced production of these cytokine. The inhibitory effects of DEPext on LPS-induced CD86 expression and IL-12 production could be neutralized by thiol antioxidant, N-acetyl cysteine, indicating the involvement of oxidative stress. Using CD4+ T cells from a T-cell receptor transgenic mouse strain (DO11.10) that recognizes OVA323-339 in the context of the BALB/c MHC class II (I-Ad), we have demonstrated that simultaneous exposure of DCs to DEPext and LPS induced a significant decrease in IFN- production compared with that in the cells treated with LPS only. Furthermore, exposure of DCs to DEPext alone, before antigen pulsing, significantly increased IL-10, a Th2 cytokine, production in the co-cultured T cells. In addition to inhibiting LPS effect (TLR4), organic DEPext also suppressed CD86 expression induced by TLR2, TLR3, and TLR9 agonists suggesting that TLRs may be targets of DEP chemicals. Using bone BMDC from Nrf2-deficient mice, we have shown that Nrf2 is required for the suppression of LPS effects by DEP chemicals and DEPext inhibits LPS effects on DC by interfering with NFkB signaling pathway. Our findings demonstrate that organic DEP chemicals indeed alter the redox equilibrium in DCs and that oxidative stress does interfere with several DC responses, leading to the suppression of Th1 response.  Our studies also demonstrated that Nrf2-mediated phase II response and NF-kB signaling pathway play a keys role in modulating DC function under conditions of PM-induced oxidative stress (6). 

2. Nrf2 deficiency in DC enhances the adjuvant effect of ambient UFP on allergic sensitization

Following the demonstration that (i) Nrf2-/- mice were more susceptible to the adjuvant effect of intra-nasally administered ambient UFP on OVA sensitization, and (ii) that DEP chemicals could induce oxidative stress that suppressed the LPS-induced Th1 response in DC, we tested the hypothesis that the adjuvant effect of UFP on allergic sensitization took place at DC level and that the lack of Nrf2 expression in DCs could further strengthen this effect.

To assess the role of Nrf2 in mediating DC response to UFP, we investigated the direct impact of these particles using BMDC obtained from Nrf2+/+ and Nrf2-/- mice. BMDC were stimulated with UFP (10 µg/ml) for 16 hrs before being analyzed for the expression of MHC class II as a surrogate maturation marker and co-stimulatory molecules (CD80, CD86 and CD40). Flow cytometric analysis revealed that UFP increased the expression of MHCII on the surface of CD11c+ BMDC from both Nrf2+/+ and Nrf2-/- mice, indicating that these particles were capable of inducing DC maturation. Exposure to UFP also led to increased expression of co-stimulatory molecules CD80 and CD86 on Nrf2+/+ and Nrf2-/- BMDC surface, whereas it had no effect on the expression of CD40. However, the extent of UFP-induced surface marker expression was similar between Nrf2+/+ and Nrf2-/- DC. 

Since we did not observe any difference in the UFP effect on surface molecule expression between Nrf2+/+ and Nrf2-/- DC, we asked whether UFP could induce different cytokine profiles between Nrf2+/+ and Nrf2-/- DC. To answer this question, the media from the cell cultures were analyzed for the levels of cytokines (IL-12 p70, IL-6 and IL-1) that were relevant to allergic airway inflammation. Our results showed that under resting condition there was a significant difference in the level of IL-12p70 between Nrf2+/+ and Nrf2-/- DC. While untreated Nrf2+/+ DC maintain a basal level of IL-12p70, Nrf2-/- DC failed to produce this cytokine. Although UFP stimulation significantly increased IL-12p70 production by Nrf2-/- DC, the level of this cytokine remained significantly lower as compared to their Nrf2+/+ counterparts. In contrast, un-stimulated Nrf2-/- DC produced greater levels of IL-6 than Nrf2+/+ DC. Although UFP stimulation increased IL-6 production by both Nrf2+/+ and Nrf2-/- DC, the level of IL-6 produced by Nrf2-/- DC was significantly higher than that from Nrf2+/+ DC. UFP also induced IL-1 production, but there was no significant difference between Nrf2+/+ and Nrf2-/- DC. Among many cytokines involved in allergic inflammation, IL-12 p70 and IL-6 play important roles in mediating T cell respons. Thus, our data suggest that Nrf2 deficiency alone is capable of creating a unique cytokine environment that could promote an altered T cell response e.g. favoring Th2 immunity.

To determine whether above differences between Nrf2+/+ and Nrf2-/- DC were reflected in the adjuvant activity of UFP in vivo, we performed intra-tracheal adoptive transfer studies using Nrf2+/+ and Nrf2-/- BMDC that had been prior exposed to saline, OVA or OVA/UFP to sensitize wild-type Balb/C recipient mice. BMDC from Nrf2+/+ and Nrf2-/- mice were stimulated with endotoxin-free OVA (50 µg/ml) with or without UFP (10 µg/ml) for 16 hrs. Cells in the control group received equal volume of vehicle alone. Recipient mice were sensitized by intratracheal instillation of Nrf2+/+ or Nrf2-/- DC (106 cells) and challenged with OVA aerosol two weeks later.

Our results demonstrated that animals sensitized by OVA/UFP-primed Nrf2-/- DC developed a more significant allergic inflammatory response in their lungs as compared to those in all other treatment groups. BAL differential cell counts revealed that sensitization by OVA- or OVA/UFP-treated Nrf2+/+ DC increased the numbers of eosinophils in the lungs of recipient mice compared to the animals sensitized by control DC, but we found no statistically significant difference between these two groups. By contrast, while sensitization by OVA- and OVA/UFP-treated Nrf2-/- DC also increased BAL eosinophil number in recipient mice, OVA/UFP-treated Nrf2-/- DC caused a further increase in eosinophil counts. Adoptive transfer of OVA/UFP-treated Nrf2+/+ and Nrf2-/- DC also increased BAL lymphocyte count compared with respective control and OVA-treated groups; this effect was markedly significant in the mice that had received Nrf2-/- DC. Consistent with the BAL cellular data, OVA-IgG1 levels in the blood were also significantly higher in mice sensitized by OVA/UFP-exposed Nrf2-/- DC, whereas it remained unchanged in animals sensitized by Nrf2+/+ DC. While sensitization by Nrf2+/+ DC exposed to either OVA alone or OVA/UFP had little impact on IL-13 production in the lungs of recipient animals, OVA-treated Nrf2-/- DC significantly increased IL-13 production in the lung, which was further increased in the presence of UFP. Histological analysis showed that sensitization by OVA/UFP-treated Nrf2-/- DC led to a significant inflammation in the lung with multiple inflammatory foci along the conducting airways, whereas mice that received Nrf2+/+ DC in the same treatment group only had a minimal response. 

Taken together, these data showed that allergic sensitization by adoptive transfer of OVA/UFP-treated DC could yield an in vivo outcome similar to that in the intranasal sensitization model, including increased eosinophil count and IL-13 level in the BAL and significantly enhanced OVA-IgG1 production. This provided direct evidence that lack of functional Nrf2 in DC could markedly strengthen the adjuvant effect of ambient UFP on allergic sensitization. A manuscript describing the findings from these studies along with those in the section of “Genetic deficiency in Nrf2-mediated antioxidant/detoxification pathways significantly enhances the adjuvant effect of ambient UFP on allergic sensitization” is in preparation and will be submitted shortly.

3. Genome-wide analysis screening in endothelial cells revealed synergistic cellular stress responses during exposure to organic DEP extracts and oxidized LDL components.

We used human microvascular endothelial cells (HMEC) to test the hypothesis that pollutant particles synergize with known pro-atherogenic stimuli and mediators in their ability to elicit oxidative stress and promote atherosclerosis. We studied the combined effects of a model air pollutant, diesel exhaust particles (DEP), and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on genome-wide gene expression. After HMEC were treated with organic DEP extract (5 µg/ml), ox-PAPC (10, 20 and 40 µg/ml) or combination of both compounds for 4 hours gene expression profiles were assessed by Illumina microarray technology. Our data showed that ox-PAPC as well as DEP regulated a large number of genes (up- and down-regulated) in a dose-dependent fashion. More important, a marked degree of co-regulation was present where the combined action of DEP and ox-PAPC resulted in a different effect than DEP or ox-PAPC alone. All together, 1555 genes were significantly upregulated (> 1.5 fold, p < 0.05) by the three DEP and ox-PAPC combinatory treatments. Notably, some genes were uniquely regulated by ox-PAPC and not by DEP, and vice versa, some genes were regulated by DEP but not by ox-PAPC (7).

Using weighted gene co-expression network analysis (WGCNA), we identified 12 modules of densely interconnected genes. We found three modules that were most highly enriched in genes that were differentially regulated by the stimuli, all of which exhibited patterns of additive/synergistic interaction where the combined action of DEP and ox-PAPC led to a greater effect than each compound alone. Based on this study we developed a novel synergistic index that allows us to differentiate in between additive effects and synergistic effects. Conceptually, we defined synergy as a response to DEP plus ox-PAPC that was greater than the effects induced by either compound alone and greater than the summation of those individual effects. Interestingly, the above three modules concentrated 83% of the synergistically expressed genes identified in the gene network. These three modules were also enriched in synergistically co-regulated genes and pathways relevant to vascular inflammation (7).

These gene expression data were further validated by quantitative PCR (qPCR) in the same set of samples analyzed by microarray analysis and in a set of samples from an independent experiment. Representative genes from various pathways were selected including ARE-regulated genes [e.g. HO-1, selenoprotein S (SELS)], inflammatory response genes [e.g. Interleukin 8 (IL-8), chemokine (C-X-C motif) ligand 1 (CXCL1)], immune response genes [e.g. Interleukin 11 (IL-11)], UPR genes [e.g. ATF 4, heat shock 70kDa protein 8 (HSPA8), X-box binding protein 1  (XBP1)], oxygen and reactive oxygen species metabolism genes [e.g. dual specificity phosphatase 1 (DUSP1), PDZ and LIM domain 1 (PDLIM1)]. qPCR confirmed 91 % of the synergistic effects that were revealed by microarray technology. All considered, the synergistic response profile represents a combination of hierarchical oxidative stress as well as protein unfolding response genes (9).

We also validated this synergy on selected genes in vivo by demonstrating that liver gene expression of hypercholesterolemic mice (HFD protocol from section 1) exposed to ambient UFP exhibited significant up-regulation of the module genes (5). Indeed, liver tissue was assayed for mRNA expression of HO-1, as well as two key UPR transcription factors, XBP1 and ATF4. UFP-exposed animals exhibited a significant up-regulation (p < 0.05) of all three genes in comparison with FP, FA and NE mice. These results indicate that the synergistic effects predicted by our in vitro studies have important in vivo outcomes, in which pro-oxidative PM chemicals may gain access to the systemic circulation from the lung and may then be able to synergize with circulating ox-LDL. 

4. Identification of human bronchial epithelial cell response to DEP by DIGE technology

In collaboration with Dr. Joseph Loo from the Keck Proteomics facility at UCLA, we used a 2D-difference gel electrophoresis (DIGE) technology to demonstrate that organic redox cycling DEP chemicals induce a tiered cellular oxidative stress that can further lead to a pro-inflammatory response in human bronchial epithelial cells (8). The DIGE method uncovered induction of an unfolding protein response (UPR) that is characterized by increased IL-6 and IL-8 production in parallel with increased expression of Hsp70, HSF-1, and ATF4 (9). The UPR pathway is activated under stress conditions in order to govern the protein processing and/or folding in the ER.  These results corroborate the demonstration of a protein unfolding response by the gene clustering analysis that was performed in endothelial cells as well as the gene response profile in the livers of apoE null mice exposed to concentrated air pollution particles as reported below.

Aim 3: We will use serum samples collected from indoor exposed elderly human subjects with ischemic heart disease in Project 4 to determine how oxidation of HDL affects the anti-inflammatory and anti-oxidative properties of this lipoprotein fraction

Based on the results obtained on the preferential upregulation of hepatic antioxidant and unfolded protein response genes by UFP as compared with FP or filtered air, we performed genomic expression analysis on lung tissue from apoE KO mice exposed to CAPs of different sizes (UFP and FP) vs. filtered air (FA), in collaboration with Dr. Araujo and Lusis. Two different CAPs inhalation exposures, designated as short-term and long-term exposures, were conducted in our mobile laboratory (AIRCARE 1) located in downtown Los Angeles. In the short-term exposure, 9~10-weeks old male apoE null mice were exposed to CAPs vs. FA for 5 hours/day for 4 days while in the long-term exposure, similar mice of 6 weeks of age were exposed to ambient particles for 5 hours/day, 3 days/week for 5 weeks. Lungs were harvested upon euthanasia, mRNA was prepared and Illumina microarrays performed.

In the short-term exposures, analysis of genomic expression profiles unveiled that there were 804 differentially expressed genes in the FP and UFP groups as compared with the FA controls (Beadstudio detection score > 0.95,  student T-test, p<0.05, FDR<0.5%). Both FP and UFP mice exhibited similar number of differentially expressed genes, ~ 27% of which were shared. Approximately a third of the regulated genes displayed levels that were over 20% of FA controls. Interestingly, UFP exposures regulated as many as three times more genes than FP exposure. Network analysis allowed us to identify gene cluster that were preferentially regulated by FP and UFP exposures. Ingenuity pathway analysis revealed inflammatory pathways centered on NF-kB, both stimulatory and inhibitory. Although we could not detect evidence of NF-KB activation at pathway level, increased expression of CXCL12 denotes activation of a NF-kB-driven pathway. Long-term exposures resulted in a much greater number of regulated genes with a greater degree of differential expression. Pathway analysis confirmed involvement of similar pathways as in the short-term exposures. In the long-term exposures, we have determined that both FP and UFP exposures promote the formation of dysfunctional HDL, as determined by a monocyte chemotactic assay developed at UCLA. In collaboration with Drs. Ralph Delfino, Jesus Araujo, Diana Shih and Jake Lusis, we evaluated whether exposure to PM, as encountered at ambient levels, correlates with the induction of HDL dysfunction in human subjects in Dr. Delfino’s CHAPS study.

In response to an invitation by the Particle, Fibre and Toxicology journal, we have published an article on the role of particle size and chemical composition in the development of atherosclerosis. In this paper, we reviewed the epidemiological, clinical and experimental animal evidence that support the association of PM with atherogenesis. We also discussed the possible pathogenic mechanisms involved, the physicochemical variables of importance in the enhanced toxicity of small particles, interaction with genes and other pro-atherogenic factors as well as important elements to consider in the design of future mechanistic studies (10).


Journal Articles on this Report : 10 Displayed | Download in RIS Format

Other subproject views: All 34 publications 23 publications in selected types All 23 journal articles
Other center views: All 241 publications 157 publications in selected types All 157 journal articles
Type Citation Sub Project Document Sources
Journal Article Araujo JA, Barajas B, Kleinman M, Wang X, Bennett BJ, Gong KW, Navab M, Harkema J, Sioutas C, Lusis AJ, Nel AE. Ambient particulate pollutants in the ultrafine range promote early atherosclerosis and systemic oxidative stress. Circulation Research 2008;102(5):589-596. R832413 (2008)
R832413 (2009)
R832413 (2010)
R832413 (Final)
R832413C001 (2008)
R832413C001 (Final)
R832413C002 (2007)
R832413C002 (2008)
R832413C002 (2009)
R832413C002 (Final)
R832413C003 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: CirculationResearch-Full Text HTML
    Exit
  • Abstract: CirculationResearch-Abstract
    Exit
  • Other: CirculationResearch-Full Text PDF
    Exit
  • Journal Article Chan RC-F, Wang M, Li N, Yanagawa Y, Onoe K, Lee JJ, Nel AE. Pro-oxidative diesel exhaust particle chemicals inhibit LPS-induced dendritic cell responses involved in T-helper differentiation. Journal of Allergy and Clinical Immunology 2006;118(2):455-465. R832413 (2008)
    R832413C002 (2007)
    R832413C002 (2008)
    R832413C002 (Final)
    R827352 (Final)
    R827352C002 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • Journal Article Gong KW, Zhao W, Li N, Barajas B, Kleinman M, Sioutas C, Horvath S, Lusis AJ, Nel A, Araujo JA. Air-pollutant chemicals and oxidized lipids exhibit genome-wide synergistic effects on endothelial cells. Genome Biology 2007;8(7):R149 (13 pp.). R832413 (2008)
    R832413 (2009)
    R832413 (Final)
    R832413C001 (2008)
    R832413C001 (Final)
    R832413C002 (2007)
    R832413C002 (2008)
    R832413C002 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: GenomeBiology-Full Text HTML
    Exit
  • Abstract: GenomeBiology-Abstract
    Exit
  • Other: GenomeBiology-Full Text PDF
    Exit
  • Journal Article Jung EJ, Avliyakulov NK, Boontheung P, Loo JA, Nel AE. Pro-oxidative DEP chemicals induce heat shock proteins and an unfolding protein response in a bronchial epithelial cell line as determined by DIGE analysis. Proteomics 2007;7(21):3906-3918. R832413 (Final)
    R832413C002 (Final)
  • Abstract from PubMed
  • Abstract: Wiley-Abstract
    Exit
  • Journal Article Kang X, Li N, Wang M, Boontheung P, Sioutas C, Harkema JR, Bramble LA, Nel AE, Loo JA. Adjuvant effects of ambient particulate matter monitored by proteomics of bronchoalveolar lavage fluid. Proteomics 2010;10(3):520-531. R832413 (Final)
    R832413C002 (2010)
    R832413C002 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Abstract: Wiley-Abstract
    Exit
  • Journal Article Li N, Wang M, Bramble LA, Schmitz DA, Schauer JJ, Sioutas C, Harkema JR, Nel AE. The adjuvant effect of ambient particulate matter is closely reflected by the particulate oxidant potential. Environmental Health Perspectives 2009;117(7):1116-1123. R832413 (2009)
    R832413 (2010)
    R832413 (Final)
    R832413C001 (2009)
    R832413C001 (Final)
    R832413C002 (2009)
    R832413C002 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: EHP-Full Text PDF
  • Abstract: EHP-Abstract and Full Text HTML
  • Journal Article Li N, Harkema JR, Lewandowski RP, Wang M, Bramble LA, Gookin GR, Ning Z, Kleinman MT, Sioutas C, Nel AE. Ambient ultrafine particles provide a strong adjuvant effect in the secondary immune response:implication for traffic-related asthma flares. American Journal of Physiology 2010;299(3):L374-L383. R832413 (Final)
    R832413C001 (2010)
    R832413C001 (Final)
    R832413C002 (2010)
    R832413C002 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: AJP-Full Text HTML
    Exit
  • Abstract: AJP-Abstract
    Exit
  • Other: AJP-Full Text PDF
    Exit
  • Journal Article Xiao GG, Wang M, Li N, Loo JHA, Nel AE. Use of proteomics to demonstrate a heirarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line. Journal of Biological Chemistry 2003;278(50):50781-50790. R832413C002 (Final)
  • Abstract from PubMed
  • Full-text: Journal of Biological Chemistry-Full Text HTML
    Exit
  • Abstract: Journal of Biological Chemistry-Abstract
    Exit
  • Other: Journal of Biological Chemistry-PDF
    Exit
  • Journal Article Zhang L, Wang M, Kang X, Boontheung P, Li N, Nel AE, Loo JA. Oxidative stress and asthma:proteome analysis of chitinase-like proteins and FIZZ1 in lung tissue and bronchoalveolar lavage fluid. Journal of Proteome Research 2009;8(4):1631-1638. R832413 (2008)
    R832413 (Final)
    R832413C002 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: ACS-Full Text HTML
    Exit
  • Abstract: ACS-Abstract
    Exit
  • Other: ACS-Full Text PDF
    Exit
  • Journal Article Araujo JA, Nel AE. Particulate matter and atherosclerosis:role of particle size, composition and oxidative stress. Particle and Fibre Toxicology 2009;6:24. R832413 (Final)
    R832413C002 (2010)
    R832413C002 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: PFT-Full Text HTML
    Exit
  • Abstract: PFT-Abstract
    Exit
  • Other: PFT-Full Text PDF
    Exit
  • Supplemental Keywords:

    RFA, Health, Scientific Discipline, Air, particulate matter, Toxicology, Health Risk Assessment, Risk Assessments, Biochemistry, Ecology and Ecosystems, atmospheric particulate matter, particulates, human health effects, PM 2.5, animal model, airway disease, airborne particulate matter, cardiovascular vulnerability, air pollution, human exposure, vascular dysfunction, cardiovascular disease, human health risk

    Progress and Final Reports:

    Original Abstract
  • 2006 Progress Report
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009 Progress Report
  • 2010 Progress Report
  • 2011

  • Main Center Abstract and Reports:

    R832413    Southern California Particle Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832413C001 Contribution of Primary and Secondary PM Sources to Exposure & Evaluation of Their Relative Toxicity
    R832413C002 Project 2: The Role of Oxidative Stress in PM-induced Adverse Health Effects
    R832413C003 The Chemical Properties of PM and their Toxicological Implications
    R832413C004 Oxidative Stress Responses to PM Exposure in Elderly Individuals With Coronary Heart Disease
    R832413C005 Ultrafine Particles on and Near Freeways