Microglial priming through the lung-brain axis: the role of air pollution-induced circulating factors

Abstract

Air pollution is implicated in neurodegenerative disease risk and progression and in microglial activation, but the mechanisms are unknown. In this study, microglia remained activated 24 h after ozone (O3) exposure in rats, suggesting a persistent signal from lung to brain. Ex vivo analysis of serum from O3-treated rats revealed an augmented microglial proinflammatory response and β-amyloid 42 (Aβ42) neurotoxicity independent of traditional circulating cytokines, where macrophage-1 antigen-mediated microglia proinflammatory priming. Aged mice exhibited reduced pulmonary immune profiles and the most pronounced neuroinflammation and microglial activation in response to mixed vehicle emissions. Consistent with this premise, cluster of differentiation 36 (CD36)(-/-) mice exhibited impaired pulmonary immune responses concurrent with augmented neuroinflammation and microglial activation in response to O3 Further, aging glia were more sensitive to the proinflammatory effects of O3 serum. Together, these findings outline the lung-brain axis, where air pollutant exposures result in circulating, cytokine-independent signals present in serum that elevate the brain proinflammatory milieu, which is linked to the pulmonary response and is further augmented with age.-Mumaw, C. L., Levesque, S., McGraw, C., Robertson, S., Lucas, S., Stafflinger, J. E., Campen, M. J., Hall, P., Norenberg, J. P., Anderson, T., Lund, A. K., McDonald, J. D., Ottens, A. K., Block, M. L. Microglial priming through the lung-brain axis: the role of air pollution-induced circulating factors.

Keywords: glia; inhaled pollutants; neuroinflammation.

Figure 1.

Changes in microglia morphology persist 24 hours after short-term O₃ exposure. Young adult male rats were exposed to FA or O₃ (1 ppm) for 4 h, and changes in microglia morphology were assessed 24 h later. Three coronal sections (40 µm) per animal were stained with the IBA1 antibody. Red arrows: activated microglia morphology (n = 3). Magnification, ×40; scale bar, 50 μm.

Figure 2.

O₃ serum primes the microglial proinflammatory response to LPS ex vivo. Young adult male rats were exposed to FA or O₃ (1 ppm) for 4 h, and serum was collected 24 h later, during the time microglia had shown activated morphology. Ex vivo serum bioactivity was assessed using cultures treated with 2% rat serum acquired from FA- or O₃-exposed rats. A, B) Although O₃ serum did not initiate TNFα production, O₃ serum enhanced LPS-induced TNFα production in the 3 h supernatant of the HAPI rat microglial cell line (A) and rat primary microglia cultures (B). C) O₃ serum caused a slight but insignificant elevation of H₂O₂ at 3 h after treatment and amplified LPS-induced H₂O₂ in primary rat microglia cultures. D) O₃ serum failed to cause any microglia toxicity alone at 3 h after treatment in HAPI rat microglia cell lines and significantly reduced LPS-induced toxicity, as measured by MTT (n = 3–6). Values are reported as the mean or the mean percentage of control ± sem. *P < 0.05 vs. control; ^†P < 0.05, O₃ serum vs. FA serum.

Figure 3.

O₃ serum primes the microglial ROS response and neurotoxicity to Aβ42 ex vivo. Young adult male rats were exposed to FA or O₃ (1 ppm) for 4 h, and serum was collected 24 h later, during the time microglia had shown activated morphology. Ex vivo serum bioactivity was assessed in cultures treated with 2% rat serum acquired from FA- or O₃-exposed rats. A) O₃ serum caused a slight but insignificant elevation of H₂O₂ and amplified Aβ42-induced H₂O₂ in primary microglia cultures. B) O₃ serum amplified Aβ42-induced neurotoxicity in rat cortical neuron–glia cultures, showing that the serum may augment AD-like neuropathology (n = 3). Values are reported as mean percentage of control ± sem. *P < 0.05 vs. control; ^†P < 0.05, O₃ serum vs. FA serum.

Figure 4.

MAC1 mediates O₃ serum priming in microglia ex vivo. Young adult male rats were exposed to FA or O₃ (1 ppm) for 4 h and serum was collected 24 h later. The ability of O₃ serum to prime microglia and augment LPS-induced TNFα production in the presence of the control IgG antibody or the MAC1 blocking antibody was determined. Microglia cells were pretreated with the MAC1 inhibitor antibody (20 μg/ml) or mouse IgG control antibody (20 μg/ml) for 30 min followed by LPS (10 ng/ml) treatment. Supernatant was collected 3 h later, and TNFα protein was assessed by ELISA. Values are reported as means ± sem. *P < 0.05, difference in LPS-induced TNFα response from IgG antibody control; ^†P < 0.05, O₃ serum augmentation of LPS-induced TNFα response is only significant in the IgG antibody control (n = 3).

Figure 5.

The aging lung. Aging attenuates the pulmonary immune response to mixed vehicle exhaust and augments thoracic inflammation. Young adult (2 mo) and aged (18 mo) male C57BL/6 mice were exposed to 300 μg PM/m³ MVE or FA 6 h/d for a month. A) Total cells in the bronchoalveolar lavage fluid were not significantly altered by exposure in either group. B) Two-month-old mice displayed a significant increase in BAL fluid neutrophils, and 18-mo-old mice exhibited no neutrophilic response to MVE. *P < 0.05. C) LFA-1 SPECT/CT images showed consistent increases in inflammatory patterns in young mice in response to MVE exposure, with no significant response in the 18-mo-old mice. D) Representative SPECT/CT images of [¹¹¹In]-NorBIRT radioligand for LFA-1 (n = 3). Values are reported as means ± SEM. * P < 0.05, MVE exposure vs. FA.

Figure 6.

The aging brain. Aging augments neuroinflammation in response to air pollutants. Young adult (2 mo) and aged (18 mo) male C57BL/6 mice were exposed to either 300 μg PM/m³ MVE or FA 6 h/d for 50 d. A) MVE-induced neuroinflammation is highest in aged mice. Cortical tissue was collected, and TNFα mRNA levels were assessed with quantitative RT-PCR (n = 3). Expression was normalized to GAPDH by using the 2^−ΔΔCt method and is reported as the mean ± sem. *P < 0.05, MVE vs. FA. B) MVE-induced activated microglia morphology is more pronounced in aged mice. Three coronal sections (40 µm) per animal were stained with the IBA1 antibody. Magnification, ×40; scale bar, 50 μm. Red arrows depict activated microglia morphology (n = 3). C) The aging brain is more sensitive to O₃ serum The ability of O₃ serum collected 24 h after a 4 h O₃ exposure to prime microglia and augment LPS-induced TNFα production in mixed glia cultures from young adult (2 mo) and aged (10 mo) rats was determined. Supernatant was collected 3 h after LPS treatment, and TNFα protein was assessed by ELISA. Data are reported as the percentage increase from mean filtered LPS values ± sem (n = 3). ^†P < 0.05, O₃ serum demonstrated significantly greater augmentation of the LPS-induced TNFα response in aged cultures.

Figure 7.

CD36^−/− mice have augmented O₃-induced neuroinflammation and microglia activation. Young adult female CD36^+/+ and CD36^−/− mice were exposed to FA or O₃ (1 ppm) for 4 h, and brain tissue was collected 24 h later. A, B) TNFα (A) and IL-1β (B) mRNA levels were assessed with quantitative RT-PCR. Values were normalized to GAPDH by the 2^−ΔΔCt method and are reported as means ± sem. *P < 0.05 vs. FA control. C) O₃-induced activated microglia morphology is more pronounced in the cortex of CD36^−/− mice. Three coronal sections (40 µm) per animal were stained with the IBA1 antibody (n = 3). Magnification, ×40; scale bar, 50 μm.

Figure 8.

The lung–brain axis. Circulating factors in response to air pollutants prime microglia. Increasing evidence supports the conclusions that pulmonary damage through disease and exposure to air pollutants may regulate the proinflammatory milieu in the brain and potentially influence CNS disease and damage through the lung–brain axis. Outlining the lung–brain axis, the findings from the current study support the conclusions that after exposure to air pollutants such as ground-level O₃, there is a pulmonary immune response, where impaired function (due to genetic modification or aging) is pathology associated with augmented neuroinflammation and microglia responses (A); brain regulating serum factors independent of circulating cytokines are present in circulation (B); and these circulating factors signal and are detected by microglia in the brain to result in a primed proinflammatory phenotype measured by enhanced sensitivity to additional proinflammatory stimuli (C). These findings have significant implications for how air pollution may affect the brain to augment CNS disease and how aging may confer vulnerability to the CNS effects of air pollution.