Diesel exhaust activates and primes microglia: air pollution, neuroinflammation, and regulation of dopaminergic neurotoxicity

Abstract

Background: Air pollution is linked to central nervous system disease, but the mechanisms responsible are poorly understood.

Objectives: Here, we sought to address the brain-region-specific effects of diesel exhaust (DE) and key cellular mechanisms underlying DE-induced microglia activation, neuroinflammation, and dopaminergic (DA) neurotoxicity.

Methods: Rats were exposed to DE (2.0, 0.5, and 0 mg/m3) by inhalation over 4 weeks or as a single intratracheal administration of DE particles (DEP; 20 mg/kg). Primary neuron-glia cultures and the HAPI (highly aggressively proliferating immortalized) microglial cell line were used to explore cellular mechanisms.

Results: Rats exposed to DE by inhalation demonstrated elevated levels of whole-brain IL-6 (interleukin-6) protein, nitrated proteins, and IBA-1 (ionized calcium-binding adaptor molecule 1) protein (microglial marker), indicating generalized neuroinflammation. Analysis by brain region revealed that DE increased TNFα (tumor necrosis factor-α), IL-1β, IL-6, MIP-1α (macrophage inflammatory protein-1α) RAGE (receptor for advanced glycation end products), fractalkine, and the IBA-1 microglial marker in most regions tested, with the midbrain showing the greatest DE response. Intratracheal administration of DEP increased microglial IBA-1 staining in the substantia nigra and elevated both serum and whole-brain TNFα at 6 hr posttreatment. Although DEP alone failed to cause the production of cytokines and chemokines, DEP (5 μg/mL) pretreatment followed by lipopolysaccharide (2.5 ng/mL) in vitro synergistically amplified nitric oxide production, TNFα release, and DA neurotoxicity. Pretreatment with fractalkine (50 pg/mL) in vitro ameliorated DEP (50 μg/mL)-induced microglial hydrogen peroxide production and DA neurotoxicity.

Conclusions: Together, these findings reveal complex, interacting mechanisms responsible for how air pollution may cause neuroinflammation and DA neurotoxicity.

Figure 1

Inhalation exposure to DE elevates markers of neuroinflammation. WKY rats were exposed to 0 (air control), 0.5, or 2.0 mg/m³ DEP (n = 3/treatment group). TNFα (A) and MIP‑1α (B) mRNA levels (mean ± SE) were determined in olfactory bulbs using quantitative real-time RT-PCR; values represent the fold increase from control 2^–ΔΔCT normalized with α-tubulin and expressed as a percentage of control. Representative images show changes in IL‑6 (C) and IBA‑1 (D) in whole-brain homogenate assayed by Western blotting; GAPDH was used as a loading control. (E) Whole-brain homogenate was also assayed for nitrosylated protein by ELISA; values shown are mean ± SE. *p < 0.05, compared with controls.

Figure 2

DE elevates microglial markers, cytokines, and chemokines in brain regions. WKY rats were exposed to 0 (air control), 0.5, or 2.0 mg/m³ DEP (n = 4/treatment group). Protein levels of TNFα (A), IL‑1β (B), IL‑6 (C), MIP‑1α (D), RAGE (E), and IBA‑1 (microglial marker; F) from the olfactory bulb (OB), cortex, and midbrain were measured by ELISA. In controls, IBA‑1 was significantly higher in the midbrain compared with other brain regions, indicating higher levels of microglia in the absence of exposure. Notably, the midbrain region also showed the highest levels of cytokines, chemokines, and microglial markers in response to DE. *p < 0.05, compared with the corresponding region in controls. **p < 0.05 for IBA‑1 in midbrain, compared with olfactory bulbs and cortex levels in controls.

Figure 3

DEP cause TNFα production and microglia activation in vivo. Sprague-Dawley rats were treated IT with saline or 20 mg/kg DEP (n = 3/treatment group). Serum (A) and brain (B) TNFα levels were determined 6 hr postexposure, as measured by ELISA. (C) Representative images of immunohistochemically stained substantia nigra sections at 20 hr post-DEP treatment show enhanced microglial IBA‑1 expression, consistent with mild microglial activation. Bar = 100 μm. *p < 0.05, compared with controls.

Figure 4

DEP amplify the microglial proinflammatory response to LPS in vitro. (A and B) HAPI microglia were pretreated for 30 min with DEP followed by LPS treatment; supernatant was collected for analysis at 3 hr for TNFα (A) and at 24 hr for nitrite (B). (C and D) Primary mesencephalic neuron–glia cultures were pretreated for 30 min with low concentrations of DEP (5 μg/mL) followed by low concentrations of LPS (2.5 ng/mL); supernatants were collected for analysis at 3 hr for TNFα (C) and at 24 hr for nitrite (D). *p < 0.05, compared with controls (n = 3). **p < 0.05, compared with LPS alone (n = 3).

Figure 5

Effect of fractalkine on DEP-induced DA neurotoxicity in vitro and in vivo. DEP synergistically enhanced inflammation-mediated loss of DA neuron function. (A) DA neuron function in neuron–glia cultures 7–9 days after DEP exposure was measured with the [³H]DA uptake assay. (B) DE inhalation increased fractalkine levels in olfactory bulb (OB), cortex, and midbrain of rats; WKY rats were exposed to 0 (air control), 0.5, or 2.0 mg/m³ DEP (n = 4/treatment group) for 1 month, and fractalkine levels were measured by ELISA. (C and D) Primary mesencephalic neuron–glia cultures were pretreated for 30 min with fractalkine (100 pg/mL) followed by DEP (50 μg/mL) exposure (n = 3/treatment group). (C) DEP-induced loss of DA neuron function was reduced by fractalkine 7 days after treatment as measured by DA uptake assay. (D) DEP-induced H₂O₂ in microglia was reduced by fractalkine 3 hr after treatment. *p < 0.05, compared with controls. **p < 0.05, compared with LPS alone. ^#p < 0.05, compared with DEP alone.