Exacerbation of Nanoparticle-Induced Acute Pulmonary Inflammation in a Mouse Model of Metabolic Syndrome

Abstract

Nanotechnology has the capacity to revolutionize numerous fields and processes, however, exposure-induced health effects are of concern. The majority of nanoparticle (NP) safety evaluations have been performed utilizing healthy models and have demonstrated the potential for pulmonary toxicity. A growing proportion of individuals suffer diseases that may enhance their susceptibility to exposures. Specifically, metabolic syndrome (MetS) is increasingly prevalent and is a risk factor for the development of chronic diseases including type-2 diabetes, cardiovascular disease, and cancer. MetS is a combination of conditions which includes dyslipidemia, obesity, hypertension, and insulin resistance. Due to the role of lipids in inflammatory signaling, we hypothesize that MetS-associated dyslipidemia may modulate NP-induced immune responses. To examine this hypothesis, mice were fed either a control diet or a high-fat western diet (HFWD) for 14-weeks. A subset of mice were treated with atorvastatin for the final 7-weeks to modulate lipids. Mice were exposed to silver NPs (AgNPs) via oropharyngeal aspiration and acute toxicity endpoints were evaluated 24-h post-exposure. Mice on the HFWD demonstrated MetS-associated alterations such as increased body weight and cholesterol compared to control-diet mice. Cytometry analysis of bronchoalveolar lavage fluid (BALF) demonstrated exacerbation of AgNP-induced neutrophilic influx in MetS mice compared to healthy. Additionally, enhanced proinflammatory mRNA expression and protein levels of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and interleukin-6 were observed in MetS mice compared to healthy following exposure. AgNP exposure reduced mRNA expression of enzymes involved in lipid metabolism, such as arachidonate 5-lipoxygenase and arachidonate 15-lipoxygenase in both mouse models. Exposure to AgNPs decreased inducible nitric oxide synthase gene expression in MetS mice. An exploratory lipidomic profiling approach was utilized to screen lipid mediators involved in pulmonary inflammation. This assessment indicates the potential for reduced levels of lipids mediators of inflammatory resolution (LMIR) in the MetS model compared to healthy mice following AgNP exposure. Statin treatment inhibited enhanced inflammatory responses as well as alterations in LMIR observed in the MetS model due to AgNP exposure. Taken together our data suggests that MetS exacerbates the acute toxicity induced by AgNPs exposure possibly via a disruption of LMIR leading to enhanced pulmonary inflammation.

Keywords: acute inflammation; lipid mediators of inflammatory resolution; nanotoxicology; silver nanoparticles; statin; susceptibility.

FIGURE 1

Characterization of body weight and serum lipid levels in healthy and MetS mouse models following 14 weeks on either a control or high-fat western diet (HFWD). Healthy and MetS mice without or with statin treatment were characterized by examination of (A) body weight, (B) serum total cholesterol, (C) serum high-density lipoprotein (HDL), (D) serum low-density lipoprotein (LDL), and (E) serum triglyceride levels following oropharyngeal aspiration of saline (control) or AgNPs (50 μg). Values are expressed as mean ± standard error of mean (SEM, n = 5–6). $ denotes significant differences between healthy and MetS, (p < 0.05).

FIGURE 2

Effect of AgNP exposure on BALF (A) total protein, (B) albumin, (C) total cells, (D) macrophages and (E) neutrophils from healthy and MetS mice and the influence of statin treatment. Values are expressed as mean ± SEM (n = 5–6). * denotes significant differences due to AgNP exposure, $ denotes significant differences between healthy and MetS mouse models, # denotes significant differences due to statin treatment (p < 0.05).

FIGURE 3

Effect of AgNP exposure on pro-inflammatory gene expression in lung tissue from healthy or MetS mice and the influence of statin treatment. AgNP-induced alterations in the gene expression of (A) macrophage inflammatory protein-2 (MIP-2), (B) interleukin-6 (IL-6), (C) monocyte chemoattractant protein-1 (MCP-1), (D) interleukin-1β (IL-1β), and (E) chemokine 1 (CXCL1) were evaluated in lung tissue. Values are expressed as mean ± SEM (n = 5–6). * denotes significant differences due to AgNP exposure, $ denotes significant differences between healthy and MetS mouse models, # denotes significant differences due to statin treatment (p < 0.05).

FIGURE 4

Alterations in genes involved in lipid metabolism and signaling following AgNP exposure in lung tissue from healthy and MetS mice and the influence of statins. AgNP-induced alterations in the gene expression of (A) arachidonate-5 lipoxygenase (ALOX-5), (B) arachidonate 15-lipoxygenase (ALOX-15), and (C) inducible nitric oxide synthase (iNOS) were evaluated in lung tissue. Values are expressed as mean ± SEM (n = 5–6). * denotes significant differences due to AgNP exposure, and # denotes significant differences due to statin treatment (p < 0.05).

FIGURE 5

Alterations in BALF cytokine/chemokines proteins due to AgNP exposure in healthy and MetS mice and the effect of statin treatment. Proteins levels of (A) MIP-2, (B) MCP-1, (C) IL-6, and (D) CXCL1 protein levels were examined in BALF to determine disease-related differences in AgNP-induced inflammatory response. Values are expressed as mean ± SEM (n = 6–8). * denotes significant differences due to AgNP exposure, $ denotes significant differences between healthy and MetS mouse models, # denotes significant differences due to statin treatment (p < 0.05).

FIGURE 6

Alterations in lipid mediators of inflammation resolution (LMIR) in the lungs of healthy and MetS mice following AgNP exposure and the effect of statins. Exploratory lipid profiling suggests variations in lipids involved in the synthesis of (A) eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA). * denotes lipids significantly reduced only in MetS in response to AgNP exposure, while $ denotes lipids significantly reduced in AgNP exposed MetS compared to AgNP exposed healthy mice (p < 0.05). (B) Representative lipids within the EPA and DHA pathway altered due to AgNP in healthy and MetS mice. * denotes lipids significantly reduced only in MetS in response to AgNP exposure, while $ denotes lipids significantly reduced in exposed MetS compared to exposed healthy mice, and # denotes significant differences due to statin treatment (p < 0.05). Graphs of other components of the pathway can be found in Supplementary Figure S2.

FIGURE 7

Assessment of eicosapentaenoic acid (EPA)-derived lipid mediators of inflammatory resolution (LMIR) in the lungs of healthy and MetS mice following AgNP exposure and the effect of statins. (A) EPA is metabolized to resolvin E1 (RvE1) and E2 (RvE2) via the intermediate 18-HEPE. * denotes lipids significantly reduced only in MetS in response to AgNP exposure, while $ denotes lipids significantly reduced in exposed MetS compared to exposed healthy mice (p < 0.05). (B) Quantitative differences in EPA-derived LMIR due to AgNP exposure in MetS and healthy mice and the influence of statin treatment. * denotes lipids significantly reduced only in MetS in response to AgNP exposure, while $ denotes lipids significantly reduced in exposed MetS compared to exposed healthy mice, and # denotes significant differences due to statin treatment (p < 0.05).

FIGURE 8

Assessment of docosahexaenoic acid (DHA)-derived LMIR in the lung tissue of healthy and MetS mice following AgNP exposure and the effect of statins. (A) DHA is metabolized to protectin D1 (PD1) and the resolvin D series (RvD1, RvD2, RvD5, and RvD6) via intermediates 17-HpDHA and 17-HDHA. Additionally, DHA can be metabolized to maresin-1 via 14-HDHA. * denotes lipids significantly reduced in MetS in response to AgNP exposure, ** denotes lipids significantly reduced in both healthy and MetS mice in response to AgNP exposure, $ denotes lipids significantly reduced in exposed MetS mice compared to exposed healthy mice (p < 0.05). (B) Quantitative differences in representative DHA-derived LMIR due to AgNP exposure in MetS and healthy mice and the influence of statin treatment. * denotes lipids significantly reduced in MetS in response to AgNP exposure, $ denotes lipids significantly reduced in exposed MetS mice compared to exposed healthy mice, and # denotes significant differences due to statin treatment (p < 0.05). Graphs of other components of the pathway can be found in Supplementary Figure S3.

FIGURE 9

Proposed mechanism by which AgNPs induce exacerbated inflammation within the lungs of MetS mice. AgNP exposure disrupts lipid metabolism, specifically EPA and DHA. Reduced ALOX decreases production of LMIR causing decreased iNOS. This removal of inhibitory inflammatory signaling results in enhanced pro-inflammatory cytokine/chemokine levels leading to exacerbated recruitment of neutrophils into the lung and an enhanced acute inflammatory response. Statin therapy inhibits these exacerbated inflammatory responses and may allow for inflammatory signaling to occur in a similarly manner to healthy mice.