Early pulmonary response is critical for extra-pulmonary carbon nanoparticle mediated effects: comparison of inhalation versus intra-arterial infusion exposures in mice

Abstract

Background: The death toll associated with inhaled ambient particulate matter (PM) is attributed mainly to cardio-vascular rather than pulmonary effects. However, it is unclear whether the key event for cardiovascular impairment is particle translocation from lung to circulation (direct effect) or indirect effects due to pulmonary particle-cell interactions. In this work, we addressed this issue by exposing healthy mice via inhalation and intra-arterial infusion (IAI) to carbon nanoparticles (CNP) as surrogate for soot, a major constituent of (ultrafine) urban PM.

Methods: Equivalent surface area CNP doses in the blood (30mm² per animal) were applied by IAI or inhalation (lung-deposited dose 10,000mm²; accounting for 0.3% of lung-to-blood CNP translocation). Mice were analyzed for changes in hematology and molecular markers of endothelial/epithelial dysfunction, pro-inflammatory reactions, oxidative stress, and coagulation in lungs and extra-pulmonary organs after CNP inhalation (4 h and 24 h) and CNP infusion (4 h). For methodological reasons, we used two different CNP types (spark-discharge and Printex90), with very similar physicochemical properties [≥98 and ≥95% elemental carbon; 10 and 14 nm primary particle diameter; and 800 and 300 m²/g specific surface area] for inhalation and IAI respectively.

Results: Mild pulmonary inflammatory responses and significant systemic effects were observed following 4 h and 24 h CNP inhalation. Increased retention of activated leukocytes, secondary thrombocytosis, and pro-inflammatory responses in secondary organs were detected following 4 h and 24 h of CNP inhalation only. Interestingly, among the investigated extra-pulmonary tissues (i.e. aorta, heart, and liver); aorta revealed as the most susceptible extra-pulmonary target following inhalation exposure. Bypassing the lungs by IAI however did not induce any extra-pulmonary effects at 4 h as compared to inhalation.

Conclusions: Our findings indicate that extra-pulmonary effects due to CNP inhalation are dominated by indirect effects (particle-cell interactions in the lung) rather than direct effects (translocated CNPs) within the first hours after exposure. Hence, CNP translocation may not be the key event inducing early cardiovascular impairment following air pollution episodes. The considerable response detected in the aorta after CNP inhalation warrants more emphasis on this tissue in future studies.

Keywords: Aorta; Cardiovascular; Heart; Inflammation; Inhaled soot; Lung; Particle translocation; Ultrafine particulate matter.

Fig. 1

Analysis of bronchoalveolar lavage (BAL) cell differentials following carbon nanoparticle (CNP) inhalation exposure in BALB/cJ mice compared to control. a. Total BAL cell counts do not exhibit any significant changes. BAL cell analysis following CNP inhalation exposed mice revealed increased macrophages (b) [Control: 5.5 ± 0.8 × 10⁵ versus 6.7 ± 0.8 × 10⁵] at 4 h post exposure followed by a strong granulocyte influx (c) [Control: 2.6 ± 0.8 × 10³ versus 10.0 ± 2.8 × 10³] at 24 h post exposure. Data are shown as Mean ± SEM; n = 8 and asterisks (*) denote p < 0.05. White bars: Clean air exposed; Black bars: CNP exposed

Fig. 2

Heatmap representation of lung transcript and protein panel assays following inhalation and intra-arterial infusion exposure of carbon nanoparticles (CNP). a. Transcript expression levels of 59 genes representing epithelial/endothelial activation, inflammatory cell markers, inflammation mediators and oxidative stress are shown. b. Protein expression levels of 34 markers representing epithelial/endothelial activation, inflammatory cell markers, inflammation mediators are shown. Samples were pooled from 4 animals/experimental group for transcript and protein analysis. Expression values are provided as percentage relative to time matched control. Fold changes below 1.5 were considered insignificant and are indicated in black color. IHA: inhalation; IAI: intra-arterial infusion

Fig. 3

Hematological analysis of blood samples following inhalation and intra-arterial infusion of carbon nanoparticle (CNP) in mice compared to control. CNP exposure related changes are shown as percentage relative to control. a. Granulocyte and monocyte numbers were increased in the blood following 24 h CNP inhalation exposure. [Granulocytes: 0.46 ± 0.09 versus 0.75 ± 0.05 × 10E3; Monocytes: 0.04 ± 0.01 versus 0.07 ± 0.01 × 10E3 cells/ml blood]. Platelet counts, particularly that of large platelets, were significantly increased both after 4 h and 24 h post exposure periods. [Platelet (4 h post exposure): 810 ± 87 versus 1203 ± 55; Platelet (24 h post exposure): 869 ± 97 versus 1104 ± 18 and Large platelet (4 h post exposure): 1.9 ± 0.4 versus 17.6 ± 2.7; Large platelet (24 h post exposure): 4.3 ± 1.3 versus 9.0 ± 1.3 x10E3 cells/ml blood]. b. Intra-arterial infusion of CNP resulted in increased lymphocyte counts after 4 h [Lymphocyte: 1.52 ± 0.07 versus 1.92 ± 0.13 x10E3 cells/ml blood] but the platelet count remained unaltered. Data are shown as Mean ± SEM; inhalation: n = 8, infusion: n = 6 and asterisks (*) denote p < 0.05. WBC: White blood cell; Neutro: neutrophilic granulocytes; Mono: Monocytes; Lympho: Lymphocytes; RBC: Red blood cells; PLT: Platelets; Large PLT: Large platelets

Fig. 4

Characterization of peripheral blood monocytes and leucocytes for surface expression of adhesion molecules following inhalation and intra-arterial infusion of carbon nanoparticle (CNP) in mice compared to control using fluorescent automated cell sorting (FACS). a. Reduced expression of CD49d and CD11b was noted in granulocytes after 24 h of CNP inhalation exposure whereas CD18 expression was reduced in granulocytes at both 4 h and 24 h post inhalation exposure. b. Reduced expression of CD49d in monocytes was observed after 24 h of CNP inhalation exposure, while reduced CD18 expression in monocytes was noted following both 4 h and 24 h post CNP inhalation exposure. c. No altered expression of CD49d, CD11b and CD18 was noted in granulocytes following 4 h of intra-arterial infusion of CNP. d. No altered expression of CD49d, CD11b and CD18 was noted in monocytes following 4 h of intra-arterial infusion of CNP. Data are shown as Mean ± SEM; inhalation: n = 8, infusion: n = 6; and asterisks (*) denote p < 0.05. White bars: Clean air exposed; Gray bars: CNP exposed

Fig. 5

Expression profiling of acute phase reactants and systemic inflammation markers in the plasma of carbon nanoparticle (CNP) exposed mice compared to control following inhalation and intra-arterial infusion. a. CNP exposure related changes of protein concentrations are shown as percentage relative to control following 4 h and 24 h post inhalation is shown. b. CNP exposure related changes of protein concentrations are shown as percentage relative to control following 4 h post intra-arterial infusion. Data are presented as Mean ± SEM; inhalation: n = 8, infusion: n = 6; and asterisks (*) denote p < 0.05

Fig. 6

Heatmap representation of the transcript (59 genes) and protein (34 analytes) panel profiling from heart, aorta and liver following carbon nanoparticle (CNP) inhalation (4 h and 24 h) and intra-arterial infusion (4 h) comparing exposed versus control mice. a. Transcript expression levels of 59 genes representing epithelial/endothelial activation, inflammatory cell markers, inflammation mediators and oxidative stress of heart, aorta and liver are shown. b. Protein expression levels of 34 markers representing epithelial/endothelial activation, inflammatory cell markers, inflammation mediators of heart and liver are shown. Samples were pooled from 4 animals/experimental group for transcript and protein analysis. Expression values are provided as percentage relative to time matched control. Fold changes below 1.5 were considered insignificant and are indicated in black color. Ox. stress: Oxidative Stress

Fig. 7

Schematic representation of the experimental strategy. The figure outlines the rationale and corresponding experimental strategy to compare and contrast the pulmonary and extra-pulmonary effects of carbon nanoparticle (CNP) exposure following inhalation and intra-arterial infusion in BALB/cJ mice. BAL: bronchoalveolar lavage; BET: Brunauer–Emmett–Teller; CNP: carbon nanoparticle; FACS: fluorescence automated cell sorting