Ambient air pollution contains ubiquitous PM that vary in size and composition. Each PM type has a unique toxicological signature which may determine its interactions with molecular cascades in impact organs. We exposed rats separately to PM 2.5–10 , PM <2.5 and UFPM from Riverside, California. We show that PM 2.5–10 and UFPM exposures lead to cerebral metal accumulation. However, only the PM 2.5–10 exposures triggered upregulation of inflammation and cancer-related genes. Based on known toxicological profiles of ambient PM from Southern California, we conclude that combinatorial exposures to certain metals and toxins in PM 2.5–10 are necessary and responsible for the expression of inflammation and cancer-related biomarkers. This hypothesis is summarized in Fig. 5 and discussed in more detail below.

Figure 5 Working hypothesis to explain gene upregulation observed following PM 2.5–10 exposures. PM 2.5–10 contains both nickel (see Table 1) and LPS (see text) and triggers upregulation of several genes. PM <2.5 contain nickel (see Table 1) and LPS (see text) but presumably deposit poorly in the nasal and endotracheal airways (see text); hence no Ni accumulation was observed after PM <2.5 exposures. UFPM contain nickel (Table 1) but are not always associated with LPS, and do not trigger gene upregulation. Thus, the combination of Ni accumulation and LPS appears to be required to trigger gene upregulation; additional toxins and metals that are not mentioned here are likely contributing factors. Full size image

Combinatorial toxicity of metals and other toxins in PM 2.5–10

The PM 2.5–10 that we used in exposure experiments contained Ni (see Table 1) and led to cerebral Ni accumulation (Fig. 2A). A previous analysis of PM 2.5–10 from Southern California concluded that endotoxin concentrations are elevated in ambient PM from Riverside28. Endotoxins, or bacterial lipopolysaccharides (LPS), are potent triggers of inflammatory responses in pulmonary, cardiovascular, and brain tissue22,36,37,38,39. We thus hypothesize that the combined exposure to metals (i.e., Ni) and LPS contained in ambient PM 2.5–10 from Riverside triggered upregulation of EGR2, IL-16, IL-13Rα1 and RAC-1 and putatively an inflammatory response in rat brains (Fig. 5A).

No gene upregulation was observed following intermediate-length exposures to PM <2.5 . PM <2.5 contain metals (see Table 1) and LPS28. However, PM <2.5 deposit poorly in rat airways. Tissue deposition of PM is governed by inertia and Brownian diffusion, which favor the deposition of micrometer-sized coarse PM and nanometer-sized UFPM, respectively. Fine PM <2.5 is too light for deposition by inertia and too large for absorption via Brownian diffusion, and thus accumulates only marginally in nasal40 and endotracheal/pulmonary airways41,42. Ultimately, this limits the interaction between PM <2.5 and biological tissues, and hence, the transfer of toxic metals and LPS to impacted organs. This would explain why we failed to observe cerebral Ni accumulation (Fig. 2) and gene upregulation (Fig. 3B) following exposures to PM <2.5 (Fig. 5B).

Finally, UFPM contain toxic metals (Table 1) and UFPM exposures lead to cerebral Ni accumulation (Fig. 2A). However, UFPM in Southern California may not always contain LPS27, and do not correlate in ambient concentration28 or distribution with endotoxins43, suggesting that endotoxins present themselves independently of UFPM. Indeed, we observed that UFPM do not trigger the expression of EGR2, IL-16, IL-13Rα1 and RAC-1 in rat brains (Fig. 5C) and we therefore conclude that (1) adequate PM deposition, (2) Ni (or other metal) accumulation and (3) LPS (or other toxin) exposures were necessary to induce the upregulation of the gene repertoire studied in this experiment.

Our findings are consistent with prior reports of synergistic toxicological effects of multiple PM components. Dong et al. (1996) demonstrated that upregulation of inflammatory cytokines in alveolar macrophages occurred in response to urban air and diesel exhaust, but only if air and exhaust particles contained LPS44. A similar result was recently published by45. Farina et al. (2013) reported greater toxicity and pathological effects of Milano-sourced PM 10 during the summer months, when concentrations of toxic LPS are elevated23. More recently, Woodward et al.22 demonstrated that LPS and nano-sized PM (<0.2 μm) trigger glial inflammatory responses via activation of the toll-like 4 receptor. Twenty-five percent of the genes that were upregulated in response to either LPS or PM were shared, suggesting a synergistic interplay between PM, LPS and molecular signaling pathways22. Finally, Ni is one of the most frequently identified metals in airborne PM46,47,48 and multiple studies show that Ni is correlated with cardiovascular abnormalities49,50. However, Ying et al. (2013) demonstrated that Ni exposures alone are not effective in inducing cardiovascular tissue responses, and that these occur only in tissue exposed to Ni and LPS48. Combinatorial interactions of metals with other PM components and toxins are therefore required to induce molecular signaling pathways that induce tissue inflammation.

How do PM 2.5–10 affect the brain?

We showed that exposures to PM 2.5–10 triggered the expression of genes related to inflammation and oncogenesis in rat brains. How do exposures to coarse ambient PM result in brain inflammation? Two routes may be considered. First, coarse PM deposit efficiently in superficial airways and to some degree in the lung41,42. Trace metals, endotoxins and other soluble compounds that are present on the coarse PM can leach into the fluids lining the airways, interact with tissue, and trigger the production of reactive oxygen species via Fenton or Fenton-like reactions (i.e., metals)21, or activate molecular signaling cascades that initiate tissue inflammation (i.e., LPS)22. Pro-inflammatory and inflammatory factors may then be released into the bloodstream to interact with- and disrupt the blood brain barrier (BBB). Additional cytokines, monocytes and macrophages can enter the brain via the disrupted BBB to trigger local inflammatory responses3,23. This may explain why brains from individuals in highly polluted cities have increased monocyte infiltration, activated microglia, increased interleukin activity, BBB damage and localized brain lesions8,9,11.

A second route for coarse PM to induce brain inflammation exists via the olfactory system. Coarse PM deposit efficiently in the rat olfactory epithelium40 and leach soluble metals and toxins into the nasal mucosa. Toxic metals translocate directly into olfactory sensory neurons and are transported along their axons into the olfactory bulbs51,52,53. Once inside the olfactory bulbs, metals and other UFPM may trigger local immune responses, or infiltrate upstream brain regions16,54 to trigger additional inflammatory responses. This presumably occurs via synergistic interactions with other toxins (e.g., LPS), cytokines and macrophages that have gained access to the brain via the same route, or via a compromised BBB, as described before. Coarse PM thus pose significant challenges to the normal function and health of the brain and it will be instructive to study the interplay between this type of pollution with molecular mechanisms of inflammation and disease in more detail.

Inflammation and cancer-related genes as proxies for neurological disorders

Gene expression analysis of rat brains revealed changes in several genes related to inflammation following exposures to PM 2.5–10 . A previous study by our group identified changes in gene expression in rat brains after exposure to coarse PM via by RNA-seq analysis26. Based on these data, we investigated the expression of eight genes (Table 2) that could be affected by pollution exposures and may play important roles in the molecular changes that precede brain pathology.

We found a transient upregulation of IL16 and IL-13Rα1. Tissue expression of IL16 recruits and attracts a variety of immune response cells, including monocytes and dendritic cells32. The IL-13Rα1 gene, on the other hand, encodes a receptor subunit of IL13 and IL4 receptors; cytokine receptors of this variant are implicated in diverse regulatory and pathological processes, including alternative macrophage activation55 and neurodegenerative disorder56. EGR2 is an important modulator of immune responses, and acts primarily to suppress them. EGR2 has been linked to autoimmunity and various immune responses related to cancer29,30. Thus, the collective upregulation of IL16, IL-13Rα and EGR2 genes is a strong indication of an ongoing inflammatory process.