The bidirectional lung brain-axis of amyloid-β pathology: ozone dysregulates the peri-plaque microenvironment

Abstract

The mechanisms underlying how urban air pollution affects Alzheimer's disease (AD) are largely unknown. Ozone (O3) is a reactive gas component of air pollution linked to increased AD risk, but is confined to the respiratory tract after inhalation, implicating the peripheral immune response to air pollution in AD neuropathology. Here, we demonstrate that O3 exposure impaired the ability of microglia, the brain's parenchymal immune cells, to associate with and form a protective barrier around Aβ plaques, leading to augmented dystrophic neurites and increased Aβ plaque load. Spatial proteomic profiling analysis of peri-plaque proteins revealed a microenvironment-specific signature of dysregulated disease-associated microglia protein expression and increased pathogenic molecule levels with O3 exposure. Unexpectedly, 5xFAD mice exhibited an augmented pulmonary cell and humoral immune response to O3, supporting that ongoing neuropathology may regulate the peripheral O3 response. Circulating HMGB1 was one factor upregulated in only 5xFAD mice, and peripheral HMGB1 was separately shown to regulate brain Trem2 mRNA expression. These findings demonstrate a bidirectional lung-brain axis regulating the central and peripheral AD immune response and highlight this interaction as a potential novel therapeutic target in AD.

Keywords: HMGB1; TREM2; air pollution; amyloid plaque; lung-brain axis; microglia.

Figure 1

Ozone impairs microglial-plaque association and TREM2 on plaque-associated microglia. 5xFAD mice (10–11 weeks old) were exposed to O₃ (0.3 or 1.0 ppm) or FA by inhalation for 13 weeks. (A) Representative confocal images of TREM2 (red), IBA1 (green) and 6e10 (white) in the cortex of 5xFAD mice exposed to O₃ or FA control. Scale bars = 100 μm; Inset = 25 μm. (B and C) Number of (B) plaque-associated microglia and (C) total microglia per image. (D and E) Quantification of (D) microglial TREM2 relative to plaque area and (E) the ratio microglial TREM2 to IBA-1. (F) RT-qPCR of the pro-inflammatory genes Nlrp3 and Il1b as well as Trem2 in the cortex of O₃ exposed mice. (G) Correlation of %ThioS in the cortex and Trem2 mRNA. Data are represented as mean ± SEM, n = 5–7 mice/exposure group. *P < 0.05; one-way ANOVA, Bonferroni post hoc or Welch’s t-test.

Figure 2

Ozone exposure exacerbates amyloid pathology in 5xFAD mice. 5xFAD mice (10–11 weeks old) were exposed to O₃ (0.3 or 1.0 ppm) or FA by inhalation for 13 weeks. (A) Representative images of ThioS+ plaques in the cortex and hippocampus. Scale bars = 1000 μm; Inset = 100 μm. (B) Quantification of the number of ThioS+ plaques per mm2 in the hippocampus and cortex. (C) Representative images of 6e10+ Aβ in the cortex and hippocampus. Scale bars = 1000 μm; Inset = 100 μm. (D) Quantification of the number of 6e10+ plaques per mm² in the hippocampus and cortex. (E) Quantification of the average size of 6e10+ plaques in the hippocampus and cortex. (F) The ratio of DEA soluble Aβ₄₂ to Aβ₄₀ in the hippocampus and cortex. Data are represented as mean ± SEM, n = 8–10 mice/exposure group. *P < 0.05, one-way ANOVA, Bonferroni post hoc.

Figure 3

Ozone exposure exacerbates neuritic dystrophy and dysregulates acetylcholinergic gene expression. 5xFAD mice (10–11 weeks old) were exposed to O₃ (0.3 or 1.0 ppm) or FA by inhalation for 13 weeks. (A) Representative confocal images of ThioS (green) and LAMP1 (magenta) taken at ×20 (top) or ×63 (bottom) in the M1/M2 cortex of 5xFAD mice exposed to O₃. Scale bars: top = 100 µm; bottom = 25 µm. (B) Quantification of LAMP1 per cent area. (C) mRNA expression of Slc18a3 (VAChT), Chat and Ache in the cortex of O₃ exposed 5xFAD and WT mice. n = 8–10 mice/exposure group. *P < 0.05; one-way ANOVA, Bonferroni post hoc or Welch’s t-test.

Figure 4

O₃ alters the peri-plaque microenvironment and dysregulates key DAM proteins. 5xFAD mice (10–11 weeks old) were exposed to O₃ (0.3 or 1.0 ppm) or FA by inhalation for 13 weeks. (A) Representative images from NanoString GeoMX DSP platform of IBA-1 (green) and Aβ (magenta) in the cortex of 5xFAD mice. (B) Plot comparing changes from peri-plaque to non-plaque areas in FA and O₃. (C) Radial heat map of protein changes from non-plaque (outer circle) to peri-plaque (inner circle) regions in O₃ exposed 5xFAD mice. (D) Volcano plots comparing peri-plaque versus non-plaque areas for FA and O₃. n = 4 mice/group (34–36 peri-plaque and 36 plaque-distant ROIs per mouse).

Figure 5

Ongoing AD pathology augments pulmonary and peripheral immune responses to O₃. 5xFAD mice (10–11 weeks old) were exposed to O₃ (0.3 or 1.0 ppm) or FA by inhalation for 13 weeks. (A) Cellular differentials from bronchoalveolar lavage (BAL) from O₃-exposed 5xFAD and control littermates. (B) Total protein in BAL fluid. (C) Volcano plots from NanoString analysis of BAL cells from O₃ WT versus FA and O₃ 5xFAD versus FA. Highlighted genes represent a subset of significantly changed genes after FDR correction (Benjamani-Hochberg, P < 0.01). (D) Undirected pathway analysis of 5xFAD O₃ versus WT O₃ showing the top 20 significantly changed pathways (P < 0.05). (E) Analysis of gene changes between 5xFAD and WT mice exposed to O₃.

Figure 6

Peripheral HMGB1 regulates O₃-induced CNS changes. 5xFAD mice (10–11 weeks old) were exposed to O₃ (0.3 or 1.0 ppm) or FA by inhalation for 13 weeks. (A) Serum analysis of HMGB1, IL-9, and VEGF in 5xFAD mice exposed to O₃. C57Bl/6 mice were injected with rHMGB1 (32.5 μg) or vehicle (Veh.) by tail and samples were collected 3 h after injection (B) mRNA analysis of acetylcholinergic genes in mice injected with rHMGB1. (C) mRNA analysis of Trem2 in midbrain and cortex of mice injected with rHMGB1. Hmgb1^fl/flLysM-cre⁻ and Hmgb1^fl/flLysM-cre⁺were exposed to O₃ (1.0 ppm) or FA for 3 days, 4 h/day. (D and E) mRNA expression of (D) Trem2 and (E) Nlrp3 in cortex and midbrain of Hmgb1^fl/flLysM-cre⁻ and Hmgb1^fl/flLysM-cre⁺exposed to O₃. n = 5–10 mice/exposure group for 13 wk O₃ exposure, n = 6–7 mice/treatment group for rHMGB1, n = 16–18 mice/exposure group for short term O₃. *P < 0.05; Welch’s t-test, or one- or two-way ANOVA, Bonferroni post hoc.

Figure 7

O₃ regulates the amyloid plaque micro-environment through the bidirectional lung-brain axis. Exposure to O₃, a reactive air pollutant unable to translocate to the brain to exert CNS effects, caused: reduced microglial-plaque association, impaired CNS Trem2 upregulation in response to plaques, exacerbated amyloid and neuronal pathology, and dysregulated the peri-plaque microenvironment in 5xFAD mice, effects commonly associated with loss of TREM2 function. 5xFAD mice also exhibited enhanced circulating factors (HMGB1), elevated pulmonary immune cell trafficking, and an augmented pulmonary immune cell pro-inflammatory transcriptome in response to O₃, when compared to control mice, supporting a role for ongoing Aβ neuropathology in the pulmonary response to air pollution, revealing a bi-directional lung-brain axis governing central and peripheral immune responses. Mechanistically, iv injection of recombinant HMGB1 in control mice impaired brain Trem2 expression and genetic ablation of HMGB1 in peripheral myeloid cells abolished O_3-induced reduction of TREM2, revealing a role for peripheral HMGB1 in the regulation of CNS Trem2 expression. These findings implicate urban air pollution, the periphery, and the lung-brain axis in amyloid pathology and highlight the potential role of TREM2 and HMGB1 in this process, identifying novel potential therapeutic targets for the regulation of CNS Trem2 expression and Alzheimer’s disease.