Diesel exhaust impairs TREM2 to dysregulate neuroinflammation

Abstract

Background: Air pollution has been linked to neurodegenerative diseases, including Alzheimer's disease (AD), and the underlying neuroimmune mechanisms remain poorly understood. TREM2 is a myeloid cell membrane receptor that is a key regulator of disease-associated microglia (DAM) cells, where loss-of-function TREM2 mutations are associated with an increased risk of AD. At present, the basic function of TREM2 in neuroinflammation is a point of controversy. Further, the impact of air pollution on TREM2 and the DAM phenotype is largely unknown. Using diesel exhaust (DE) as a model of urban air pollution exposure, we sought to address its impact on TREM2 expression, the DAM phenotype, the association of microglia with the neurovasculature, and the role of TREM2 in DE-induced neuroinflammation.

Methods: WYK rats were exposed for 4 weeks to DE (0, 50, 150, 500 μg/m³) by inhalation. DE particles (DEP) were administered intratracheally once (600 μg/mouse) or 8 times (100 μg/mouse) across 28 days to male mice (Trem2^+/+, Trem2^-/-, PHOX^+/+, and PHOX^-/-).

Results: Rats exposed to DE exhibited inverted-U patterns of Trem2 mRNA expression in the hippocampus and frontal cortex, while TREM2 protein was globally diminished, indicating impaired TREM2 expression. Analysis of DAM markers Cx3Cr1, Lyz2, and Lpl in the frontal cortex and hippocampus showed inverted-U patterns of expression as well, supporting dysregulation of the DAM phenotype. Further, microglial-vessel association decreased with DE inhalation in a dose-dependent manner. Mechanistically, intratracheal administration of DEP increased Tnf (TNFα), Ncf1 (p47^PHOX), and Ncf2 (p67^PHOX) mRNA expression in only Trem2^+/+ mice, where Il1b (IL-1β) expression was elevated in only Trem2^-/- mice, emphasizing an important role for TREM2 in DEP-induced neuroinflammation.

Conclusions: Collectively, these findings reveal a novel role for TREM2 in how air pollution regulates neuroinflammation and provides much needed insight into the potential mechanisms linking urban air pollution to AD.

Keywords: Air pollution; Alzheimer’s disease; Brain; Disease-associated microglia; Microglia; Neuroinflammation; TREM2.

Fig. 1

Diesel exhaust impairs TREM2 expression during neuroinflammation. WKY rats were exposed to diesel exhaust (0, 50, 150, or 500 μg/m³) by inhalation for 4 weeks. Representative images taken at × 40 of IBA-1 immunofluorescence in the hippocampus are shown (n = 3). The scale bar depicts 50 μm (a). In the hippocampus, the number of microglia with a volume greater than 600 μm (hypertrophic microglia) (b), the average branch length per microglia (c), and the total number of microglia (d) were counted in the slices were assessed. Tnf (TNFα) mRNA levels were measured in the frontal cortex (e) and hippocampus (f). Trem2 mRNA levels were assessed in the hippocampus (g) and frontal cortex (h). mRNA levels were analyzed by qRT-PCR and normalized to Gapdh using the 2^−ΔΔCT method. i Frontal cortex TREM2 protein levels were analyzed by ELISA. Data are reported as the mean ± SEM. *p < 0.05 when compared to control. ^#p < 0.05 compared between exposure groups (n = 7)

Fig. 2

Diesel exhaust dysregulates markers of disease-associated microglia. WKY rats were exposed to diesel exhaust (0, 50, 150, or 500 μg/m³) by inhalation for 4 weeks. Changes in gene expression of the disease-associated microglia markers were analyzed by qRT-PCR. Stage 1 DAM markers Cx3cr1 and Lyz2 were analyzed in the frontal cortex (a, b) and the hippocampus (d, e). The stage 2 DAM marker Lpl was analyzed in the frontal cortex (c) and hippocampus (f). Data are reported as the mean ± SEM. *p < 0.05 when compared to control. ^#p < 0.05 compared between exposure groups (n = 7)

Fig. 3

Diesel exhaust impairs microglial association with the neurovasculature. WKY rats were exposed to diesel exhaust (0, 50, 150, or 500 μg/m³) by inhalation for 4 weeks. The changes in the association of microglia cell bodies with the neurovasculature were assessed with confocal images of microglia (IBA-1, green) and vascular endothelial cells (CD31, red) in the hippocampus (4 slices per brain). Representative maximum intensity projection images are shown. The scale bar depicts 50 μm (top) or (20 μm) bottom (p < 0.05, n = 3) (a). The number of vessel-associated microglia in the CA-1 hippocampus was counted (p < 0.05, n = 3) (b). The total number of IBA-1+ cells in the confocal images was counted (p < 0.05, n = 3) (c). mRNA analysis of Aif1 (IBA-1) expression demonstrates no change in expression of IBA-1 in the hippocampus of WKY rats (p < 0.05, n = 7). Data are reported as the mean ± SEM. *p < 0.05 when compared to control

Fig. 4

TREM2 deficiency selectively augments cortical Il1b gene expression. Trem2^−/− and Trem2^+/+ mice were treated with DEP (100 μg/mouse IT, 2× per week, 4 weeks) or vehicle (IT, 2× per week, 4 weeks). mRNA levels were measured by qRT-PCR for Tnf (TNFα) (a, b) and Il1b (IL-1β) (c, d) in the cortex (a, c) or hippocampus (b, d) of mice. *p < 0.05, compared with controls (n = 3–6)

Fig. 5

TREM2 deficiency augments neutrophilic pulmonary response to diesel exhaust particles. Trem2^+/+ and Trem2^−/− mice were treated with DEP (600 μg/mouse IT, single dose) or vehicle (IT, single dose). Total cell infiltration in bronchoalveolar lavage fluid (BALF) was analyzed by automated cell counter (a). Neutrophils were counted from Wright-Giemsa-stained cytospin preparations (b). Total protein in BALF was analyzed by BCA assay (c). Representative H&E-stained lung sections (d). *p < 0.05, compared with controls. ^#p < 0.05 compared between IT-DEP-exposed groups (n = 5–6)

Fig. 6

TREM2 regulates diesel exhaust particle-induced NADPH oxidase gene expression. Trem2^−/− and Trem2^+/+ mice were treated with DEP (100 μg/mouse IT, 2× per week, 4 weeks) or vehicle (IT, 2× per week, 4 weeks). mRNA levels were measured by qRT-PCR for Cybb (gp91^PHOX) (a, b), Ncf1 (p47^PHOX) (c, d), and Ncf2 (p67^PHOX) in the cortex (a, c, e) or hippocampus (b, d, f). *p < 0.05, compared with controls (n = 3–6)