Chronic fine particulate matter exposure induces systemic vascular dysfunction via NADPH oxidase and TLR4 pathways

Abstract

Rationale: Chronic exposure to ambient air-borne particulate matter of < 2.5 μm (PM₂.₅) increases cardiovascular risk. The mechanisms by which inhaled ambient particles are sensed and how these effects are systemically transduced remain elusive.

Objective: To investigate the molecular mechanisms by which PM₂.₅ mediates inflammatory responses in a mouse model of chronic exposure.

Methods and results: Here, we show that chronic exposure to ambient PM₂.₅ promotes Ly6C(high) inflammatory monocyte egress from bone-marrow and mediates their entry into tissue niches where they generate reactive oxygen species via NADPH oxidase. Toll-like receptor (TLR)4 and Nox2 (gp91(phox)) deficiency prevented monocyte NADPH oxidase activation in response to PM₂.₅ and was associated with restoration of systemic vascular dysfunction. TLR4 activation appeared to be a prerequisite for NAPDH oxidase activation as evidenced by reduced p47(phox) phosphorylation in TLR4 deficient animals. PM₂.₅ exposure markedly increased oxidized phospholipid derivatives of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in bronchioalveolar lavage fluid. Correspondingly, exposure of bone marrow-derived macrophages to oxPAPC but not PAPC recapitulated effects of chronic PM₂.₅ exposure, whereas TLR4 deficiency attenuated this response.

Conclusions: Taken together, our findings suggest that PM₂.₅ triggers an increase in oxidized phospholipids in lungs that then mediates a systemic cellular inflammatory response through TLR4/NADPH oxidase-dependent mechanisms.

Figures

Figure 1

PM2.5 exposure promotes inflammatory monocyte egress from bone marrow to blood via TLR4 pathways. Inflammatory monocyte population of TLR4wt and TLR4d mice (A) in spleen (B) in peripheral blood, (C) in bone marrow. Data are mean ± SD. (n=4–6/group; *p<0.05). Exposure duration of 20 weeks.

Figure 2

PM2.5 exposure increases NADPH oxidase derived O2·− production in monocytes, aortic tissue and perivascular fat in wildtype mice. (A) O2·− production in response to PM2.5 exposure in bone marrow derived F4/80+ cells, aortic and perivascular tissue from TLRwt and TLR4d mice. (B) O2·− production in response to PM2.5 exposure in F4/80+ cells, aortic and perivascular tissue from Nox2wt and Nox2−/− mice after 20 weeks of exposure. Data are mean ± SD. (n=5, *p<0.05).

Figure 3

PM2.5 impairs macrovascular tonal responses with chronic exposure to PM2.5 through TLR4 pathways (20 weeks of exposure). (A) Constriction of aortic rings without perivascular fat in response to increasing dosages of phenylephrine. (B) Constriction of aortic rings with perivascular fat in response to increasing dosages of phenylephrine. (C) Relaxation of aortic rings without perivascular fat in response to increasing dosages of acetylcholine. (D) Relaxation of aortic rings with perivascular fat in response to increasing dosages of acetylcholine. (n=8–10/group; *p<0.01 vs. TLR4wt FA; #p<0.05 vs. TLR4d FA; †logEC50 vs. same group FA).

Figure 4

Chronic PM2.5 exposure increases Toll-like receptor dependent gene expression in perivascular tissue in TLR4wt mice. 8 of 82 analyzed genes found to have a significant change vs. the TLR4wt FA control group are depicted. (n=4/group).

Figure 5

Chronic PM2.5 exposure over 20 weeks increases monocyte adherence within microvasculature and tissue niches in c-fmsYFP mice (FVB/N background). (A) Representative images and quantification of adherent YFP cells in the cremasteric venular endothelium (open arrow heads adherent monocytes; arrow heads with tail, rolling monocytes). (Original magnification 400×, n=5). (B) Representative images and quantification of adherent YFP cells in the mesenteric adipose tissue. (Original magnification 200×, n=5). (C) Immunohistochemical staining for YFP positive monocyte infiltration into perivascular fat tissue in c-fmsYFP mice exposed to FA or PM2.5. Perivascular fat tissue from mice that express a yellow fluorescent protein (c-fmsYFP, yellow) was stained with DAPI (blue) and isolectin (red) and visualized by confocal microscopy. (L=lumen; P=perivascular fat). (Original magnification 400×, n=3). Quantification of YFP positive cells in BAL fluid (D), in epidydimal fat (E) and in lung tissue (F) (n=4–6). Exposure time was about 20 weeks. Data are mean ± SD. (*p<0.05).

Figure 6

TLR4 deficiency normalizes inflammatory cytokine release and prevents p47phox phosphorylation in response to PM2.5 exposure over 20 weeks. (A) Cytokine analysis of lung homogenates (500 µg/ml protein) in TLR4wt and TLR4d mice measured by a cytokine bead array. (n=8–10/group). (B) Cytokine analysis of plasma samples in TLR4wt and TLR4d mice measured by a cytokine bead array. (n=8–10/group) (C) Immunoblots demonstrating increased p47phox expression in response to PM2.5 exposure compared to FA in TLR4wt and normalization of p47phox phosphorylation in TLR4d mice. Lung homogenates from TLR4wt and TLR4d mice were immunoblotted for p47phox and phospho-p47phox (left). The figure on the right represents the photodensitometric quantification of the blots. (n=5/group). Data are mean ± SD. (*p<0.05).

Figure 7

Airborne particulate matter causes increased levels of two oxidized PAPC derivatives in BAL fluid of PM2.5 exposed mice. Lipid extracts from BAL fluid of TLR4wt and TLR4d mice exposed for 20 weeks to FA or PM2.5 were analyzed by HPLC with positive electrospray ionization mass-spectrometry operating in multiple reaction monitoring mode. Parent PAPC and oxidized derivatives (POVPC and PGPC) ion pairs were monitored by their characteristic retention time and daughter ions. Corresponding chromatograms were post-processed by extraction of POVPC and PGPC ions for quantitative analysis. Representative LC-MS chromatograms are shown for (A) TLR4wt FA, (B) TLR4wt PM2.5, (C) TLR4d FA, (D) TLR4d PM2.5. (E) Chemical structures of monitored phospholipids. Quantitative analysis of levels of (F) POVPC and (G) PGPC against PAPC with an exaggerated level of oxidation in the PM2.5 exposed mice over 20 weeks. In-vitro incubation of PAPC in the presence of PM2.5 or with PBS was performed in time-dependent manner followed by quantification of levels of (H) POVPC and (I) PGPC by LC/MS-MS. BAL fluid of 5 mice per group were pooled for these experiments with extraction of the lipid content. The amount of oxidized phospholipid is set in ratio to non-oxidized phospholipid in order to compare the different groups.

Figure 8

TLR4 triggers inflammatory cytokine release and promotes IRAK modulated p47phox phosphorylation in response to oxidized phospholipid treatment in BMDM derived from TLR4wt and TLR4d mice. (A) BMDM were treated with PAPC and oxPAPC and inflammatory cytokine levels in the supernatant were determined. (n=3/group; *p<0.05) (B) These blots show p47phox expression and phosphorylation in response to PAPC and oxidized PAPC treatment. Lysates from bone marrow derived monocytes isolated from TLR4wt and TLR4d mice were immunoblotted for p47phox and phospho-p47phox (left). A subset of experiments was performed in presence of an IRAK inhibitor. The figure on the right represents the photodensitometric quantification of the blots. (n=3) Data are mean ± SD. (*p<0.05).