Peripheral HMGB1 is linked to O3 pathology of disease-associated astrocytes and amyloid

Abstract

Introduction: Ozone (O₃) is an air pollutant associated with Alzheimer's disease (AD) risk. The lung-brain axis is implicated in O₃-associated glial and amyloid pathobiology; however, the role of disease-associated astrocytes (DAAs) in this process remains unknown.

Methods: The O₃-induced astrocyte phenotype was characterized in 5xFAD mice by spatial transcriptomics and proteomics. Hmgb1^fl/fl LysM-Cre⁺ mice were used to assess the role of peripheral myeloid cell high mobility group box 1 (HMGB1).

Results: O₃ increased astrocyte and plaque numbers, impeded the astrocyte proteomic response to plaque deposition, augmented the DAA transcriptional fingerprint, increased astrocyte-microglia contact, and reduced bronchoalveolar lavage immune cell HMGB1 expression in 5xFAD mice. O₃-exposed Hmgb1^fl/fl LysM-Cre⁺ mice exhibited dysregulated DAA mRNA markers.

Discussion: Astrocytes and peripheral myeloid cells are critical lung-brain axis interactors. HMGB1 loss in peripheral myeloid cells regulates the O₃-induced DAA phenotype. These findings demonstrate a mechanism and potential intervention target for air pollution-induced AD pathobiology.

Highlights: Astrocytes are part of the lung-brain axis, regulating how air pollution affects plaque pathology. Ozone (O₃) astrocyte effects are associated with increased plaques and modified by plaque localization. O₃ uniquely disrupts the astrocyte transcriptomic and proteomic disease-associated astrocyte (DAA) phenotype in plaque associated astrocytes (PAA). O₃ changes the PAA cell contact with microglia and cell-cell communication gene expression. Peripheral myeloid cell high mobility group box 1 regulates O₃-induced transcriptomic changes in the DAA phenotype.

Keywords: O3; amyloid plaques; disease‐associated astrocytes; lung–brain axis; peripheral HMGB1.

FIGURE 1

O₃ exposure increases astrocyte density in the cortex of 5xFAD mice. Male 5xFAD mice (10–11 weeks old) were exposed to either FA or 1.0 ppm O₃ for 3 consecutive days each week for 4 hours/day for 13 weeks. A, Representative 10× images depicting cortical astrocyte density (GFAP, red) in FA‐ and O₃‐exposed mice. Scale bar: 1000 or 100 µm. B, Quantification of the number of GFAP‐positive areas and GFAP‐positive cells in the entire cortex (layers I–VI). C, Representative maximum intensity projection images taken at 40× in the cortex, staining for plaques (Methoxy‐X34, gray) and astrocytes (GFAP, green). Scale bar: 50 µm. D, Quantification of plaque number in the 40× confocal images. E, Quantification of plaque‐associated astrocytes normalized to plaque number. Correlation of the number of plaques with the number of (F) periplaque and (G) non‐plaque astrocytes in the cortex. Astrocytes were considered periplaque if their cell bodies or branches reached within the circular periplaque region of interest drawn at 50 µm diameter around the plaque center; if not, they were considered nonplaque. Data are represented as the mean ± SEM, n = 8–9 mice/exposure group. ^* = P < 0.05; Welch t test. FA, filtered air; GFAP, glial fibrillary acidic protein; O₃, ozone; SEM, standard error of the mean

FIGURE 2

O₃ altered the astrocytic protein expression pattern, dependent on spatial localization with plaques. Male 5xFAD mice (10–11 weeks old) were exposed to FA or 1.0 ppm O₃ for 3 consecutive days each week for 4 hours/day for 13 weeks. A, Representative images from the NanoString GeoMX DSP platform illustrating periplaque (left) and non‐plaque astrocytes (right), as defined by plaque staining (Aβ, magenta), astrocyte (GFAP, green) staining, and the area from which samples were collected for analysis (blue). Scale bar: 10 µm. B, Venn diagram showing the number of plaque environment‐induced changes in the astrocyte proteomic profile in FA and O₃ groups, indicating protein changes shared between the two groups. Volcano plots representing differentially expressed proteins on periplaque versus plaque‐distant astrocytes in the (C) FA and (D) O₃ groups. n = 68–72 ROIs/region per exposure group (n = 4 mice/exposure group). P < 0.05. Aβ, amyloid beta; DSP, Digital Spatial Profiling; FA, filtered air; GFAP, glial fibrillary acidic protein; O₃, ozone; ROI, region of interest

FIGURE 3

O₃ increased astrocyte–microglia cell contact in the plaque microenvironment. Male 5xFAD mice (10–11 weeks old) were exposed to either FA or 1.0 ppm O₃ for 3 consecutive days each week for 4 hours/day for 13 weeks. A,= Representative image showing colocalized areas (yellow) of cell‒cell contact, as indicated by white arrows in a single image from a set of confocal Z‐stack images taken at 40×. Scale bar: 10 µm. Quantification of astrocyte–microglia colocalization in the (B) periplaque and (C) non‐plaque (right) space from confocal Z‐stacks taken at 40× in the cortex. Data are represented as the mean ± SEM, n = 8–9 mice/exposure group. ^* = P < 0.05; Welch t test. D, Representative maximum intensity images taken at 60× showing plaques (Methoxy‐X34, gray), astrocytes (GFAP, green), and microglia (IBA1, red) in the O₃ and FA groups. Scale bar: 10 µm. FA, filtered air; GFAP, glial fibrillary acidic protein; IBA1, ionized calcium‐binding adapter molecule 1; O₃, ozone; SEM, standard error of the mean

FIGURE 4

O₃ alters the astrocytic transcriptional profile in the plaque microenvironment. Male 5xFAD mice (10–11 weeks old) were exposed to either FA or 1.0 ppm O₃ for 3 consecutive days each week for 4 hours/day for 13 weeks. A, Representative image showing the digital spatial profiler scan for regions of interest containing astrocytes localized in the periplaque space, as defined by plaque staining (6e10, magenta), astrocyte staining (GFAP, green), and the area from which GFAP‐positive cells were collected for mRNA analysis (blue). Scale bar, 50 µm. B, Volcano plot showing O₃‐induced gene expression in plaque‐associated astrocytes compared to that in the FA group. Highlighted genes represent a subset of significantly changed genes (red dots) after FDR correction (Benjamini‒Hochberg, P < 0.05). Pathway analysis of O₃ versus FA cortical astrocyte gene expression in plaque‐associated astrocytes depicting significantly (C) increased and (D) decreased pathways of interest (Benjamini–Hochberg, P < 0.05. n = 96 ROIs/region per exposure group (n = 3 mice/exposure group). FA, filtered air; FDR, false discovery rate; GFAP, glial fibrillary acidic protein; O₃, ozone; ROI, region of interest

FIGURE 5

Peripheral HMGB1 modulates O₃‐induced astrocytic dysregulation. A, Subchronic (13‐week) O₃ (1 ppm) exposure reduced HMGB1 mRNA expression in the BAL cells of 5xFAD mice. Data are represented as the mean ± SEM, n = 6 mice/exposure group. Welch t test. B, Hmgb1 ^fl/fl _. LysM‐Cre⁺ mice have Hmgb1 genetically ablated in peripheral myeloid cells (comprising a substantial component of BAL fluid cells) but not microglia. Hmgb1 ^fl/fl _. LysM‐Cre⁻ and Hmgb1 ^fl/fl _. LysM‐Cre⁺ mice were exposed to O₃ (1.0 ppm) or FA once for 4 hours. Cell counts of eosinophils, neutrophils, and lymphocytes infiltrating the BAL are shown. Data are represented as the mean ± SEM, n = 3–5 mice/group. ^* = P < 0.05, ^**  = P < 0.01, ^***  = P < 0.001; Welch t test. C, Hmgb1 ^fl/fl _. LysM‐Cre⁻ and Hmgb1 ^fl/fl _. LysM‐Cre⁺ mice were exposed to O₃ (2.0 ppm) or FA once for 4 hours. Serpina3n mRNA levels in the midbrain after a single 2 ppm O₃ exposure are shown. Data are represented as the mean ± SEM, n = 9–10 mice/exposure group. ^* = P < 0.05, ^†† = P < 0.01. D, Gfap, C3, and Aqpn4 mRNA levels were assessed 3 hours after a tail vein injection of rHMGB1 (32.5 µg). Data are represented as the mean ± SEM, n = 6–8 mice/exposure group. ^* = P < 0.05; Welch t test. BAL, bronchoalveolar lavage; FA, filtered air; HMGB1, high mobility group box 1; O₃, ozone; SEM, standard error of the mean

FIGURE 6

Plaque‐associated astrocytes and peripheral myeloid cells interact in the O₃‐dysregulated lung–brain axis: Implications for Alzheimer's disease. O₃, a reactive gas component of urban air pollution that cannot reach the brain, increased astrocyte density in the 5xFAD mouse cortex, concomitant with decreased bronchoalveolar lavage fluid cell (predominantly myeloid) HMGB1 expression and an exacerbated plaque burden. O₃‐induced astrocyte effects (transcriptomic and proteomic) were dependent on the localization of astrocytes relative to plaques, indicating that this air pollution exposure selectively and qualitatively changes astrocytes in the plaque microenvironment, accelerates the astrocyte transcriptomic shift to a disease‐associated astrocyte phenotype, and increases astrocyte contact with microglia but not plaques. Mechanistically, O₃‐exposed mice with HMGB1 deleted from the peripheral myeloid cells but not microglia exhibited a perturbed pulmonary immune response to O₃ and disrupted DAA markers in the brain, indicating that peripheral myeloid cells and HMGB1 regulate the astrocyte DAA response to O₃. These findings provide much‐needed insight into how urban air pollution may dysregulate the lung–brain axis, disrupt astrocytic function, and increase the amyloid burden. DAA, disease‐associated astrocytes; HMGB1, high mobility group box 1; O₃, ozone