Urban air particulate matter induces mitochondrial dysfunction in human olfactory mucosal cells

Abstract

Background: The adverse effects of air pollutants including particulate matter (PM) on the central nervous system is increasingly reported by epidemiological, animal and post-mortem studies in the last decade. Oxidative stress and inflammation are key consequences of exposure to PM although little is known of the exact mechanism. The association of PM exposure with deteriorating brain health is speculated to be driven by PM entry via the olfactory system. How air pollutants affect this key entry site remains elusive. In this study, we investigated effects of urban size-segregated PM on a novel cellular model: primary human olfactory mucosal (hOM) cells.

Results: Metabolic activity was reduced following 24-h exposure to PM without evident signs of toxicity. Results from cytometric bead array suggested a mild inflammatory response to PM exposure. We observed increased oxidative stress and caspase-3/7 activity as well as perturbed mitochondrial membrane potential in PM-exposed cells. Mitochondrial dysfunction was further verified by a decrease in mitochondria-dependent respiration. Transient suppression of the mitochondria-targeted gene, neuronal pentraxin 1 (NPTX1), was carried out, after being identified to be up-regulated in PM_2.5-1 treated cells via RNA sequencing. Suppression of NPTX1 in cells exposed to PM did not restore mitochondrial defects resulting from PM exposure. In contrast, PM-induced adverse effects were magnified in the absence of NPTX1, indicating a critical role of this protein in protection against PM effects in hOM cells.

Conclusion: Key mitochondrial functions were perturbed by urban PM exposure in a physiologically relevant cellular model via a mechanism involving NPTX1. In addition, inflammatory response and early signs of apoptosis accompanied mitochondrial dysfunction during exposure to PM. Findings from this study contribute to increased understanding of harmful PM effects on human health and may provide information to support mitigation strategies targeted at air pollution.

Keywords: Air pollution; Inflammation; Mitochondria; NPTX1; olfactory system; oxidative stress; particulate matter.

Fig. 1

Morphology and gene expression profile of hOM cultures. a Representative phase-contrast image of hOM cells cultured as a monolayer. Scale bar = 100 μm. b. Comparison of DEGs of a representative hOM cell line cultured in growth medium (GM), neurosphere induction medium (NIM) and neuronal medium (NM). Relative transcript levels of several cell markers including nestin, SOX2 and TP63 are specific to basal stem cells. SOX2, PAX6 and TP63 are specific to cells committed to sustentacular cell fate. TUBB3 and OMP are specific to cells committed to olfactory sensory neuronal cell fate. Transcript levels are normalised to GM with hGAPDH as a reference gene, n = six wells/group

Fig. 2

Effects of PM on cytotoxicity and cellular metabolism in hOM cultures. a hOM cells exposed to PM_1–0.2, PM_2.5–1 or PM_10–2.5 for 24 h, after which cell death was measured by the LDH release assay. Results were normalised by lysed cell control, n = four donors. Metabolic activity was measured by the MTT reduction assay of hOM cultures. Results were normalised to the vehicle, n = four donors. ** p < 0.01, *** p < 0.001. c Caspase-3/7 signal intensity in live single cells of a representative culture exposed to all three PM, n = three experimental replicates

Fig. 3

PM effects on the immune response in hOM cultures. Secreted inflammatory mediators were quantified from a representative cell line after 24-h h exposure by CBA. Following exposure to PM, the levels of of (a) IL-6, (b) IL-8, (c) GM-CSF, (d) RANTES and (e) MCP1 in hOM cultures were measured. After a 24 h co-treatment with IFN-ɣ/TNFα and PM_1–0.2, PM_2.5–1 or PM_10–2.5, the levels of IL-6, IL-8, GM-CSF, RANTES and MCP were determined by CBA. *p < 0.05, ***p < 0.001, n = five wells/group and compared to the vehicle. #p < 0.05, ##p < 0.001, ###p < 0.0001, compared to cytokines

Fig. 4

Elevated cellular ROS in hOM cultures after PM exposure. a Measurement of mt-ND4 content in hOM cultures after 24 h exposure to PM_1–0.2, PM_2.5–1 and PM_10–2.5 . Data are normalized the vehicle treated cells and using the reference gene hGAPDH. b Cytoplasmic reactive oxygen intermediates were also detected via H2DCFHA (DCF) signal intensities examined in cells after 4 h and 24 h exposure in a representative cell line. c Cellular ROS levels quantified by CellROX reveals a slight increase in average CellROX-positive cells exposed to PM_2.5–1 and PM_10–2.5 . n = four donors

Fig. 5

Exposure to PM increased mitochondrial ROS and altered key mitochondrial functions. a Mitochondrial ROS levels were quantified by MitoSOX labelling after 4 h exposure revealed a significant increase in cells exposed to PM_2.5–1 and PM_10–2.5 . b Significant loss of mitochondrial membrane potential was observed in all PM size classes. c Line plot displaying oxygen consumption rate (OCR) of hOM cultures exposed to PM and quantified in pmol/minute/μg after the addition of oligomycin (Olig), FCCP and Rotenone/Antimycin (Rot+Ant). d Basal respiration in cells exposed to PM_1–0.2 (5.17 ± 0.59), PM_2.5–1 (2.92 ± 1.03) and PM_10–2.5 (2.32 ± 1.29), compared to the vehicle (4.44 ± 0.28). e Maximal respiration after exposure to PM_1–0.2 (10.74 ± 0.26), PM_2.5–1 (5.14 ± 0.86) and PM_10–2.5 (4.96 ± 2.3), compared to the vehicle (11.01 ± 1.08). f Non-mitochondrial respiration after exposure to PM_1–0.2 (1.38 ± 0.35), PM_2.5–1 (1.86 ± 0.12) or PM_10–2.5 (1.65 ± 0.25), compared to the vehicle (3.35 ± 0.16). g ATP production in hOM cells exposed for 24 h to PM_1–0.2 (4.17 ± 0.72), PM_2.5–1 (1.81 ± 0.01) or PM_10–2.5 (1.1 ± 0.58), compared to the vehicle (3.47 ± 0.19). h Intracellular ATP levels reflected little variation in hOM cultures exposed to PM_1–0.2 (0.28 μM ± 0.02 μM), PM_2.5–1 (0.2 μM ± 0.04 μM) or PM_10–2.5 (0.28 μM ± 0.13 μM) from vehicle control (0.31 μM ± 0.04 μM). The OCR was normalised by protein levels. * p < 0.05, **p < 0.01. n = four donors/group

Fig. 6

Transcriptomic analyses reveal NPTX1 as a novel target of PM in hOM cells. a Volcano plot of DEGs in the transcriptome of hOM cells exposed to PM_2.5–1 reveals 377 DEGs, of which 114 are altered by two-fold or more. b Heatmap comparing 114 DEGs in 4 cell lines above the FDR cut-off of 0.05 and log fold change ≥1 (≥2x). c List of most up- and down-regulated transcripts in cells exposed to PM_2.5–1d GO terms enriched with relation to significantly up- and down-regulated transcripts. N = four donors/group

Fig. 7

NPTX1-knockdown did not ameliorate mitochondrial function via ROS signalling. a Experimental timeline. b Validation of NPTX1 expression shows a significant increase in hOM cells exposed to PM_2.5–1 and significant suppression of NPTX1 following RNAi in a representative cell line. NPTX1 transcripts levels are presented as log fold change (logFC) and normalized by the reference gene, GAPDH and to the scrambled control (NC1). c MTT assay results of 24 h exposure to PM after 48 h treatment with siRNA. d LDH release assay results of 24 h exposure to PM after 48 h treatment with siRNA. e Proportion of mitoSOX-positive cells increased in NPTX1-kd cells exposed to PM. f Measurement of OCR with SeahorseFX technology revealed that suppression of NPTX1 (in green lines) did not further increase the failure of cellular respiratory activity after PM exposure. g Intracellular ATP levels were not altered by NPTX1-kd. (H) STRING interaction network of NPTX1 in human. *p < 0.05 **p < 0.01 ***p < 0.0001. N = 3–6 wells/group