Disruption of pulmonary resolution mediators contribute to exacerbated silver nanoparticle-induced acute inflammation in a metabolic syndrome mouse model

Abstract

Pre-existing conditions modulate sensitivity to numerous xenobiotic exposures such as air pollution. Specifically, individuals suffering from metabolic syndrome (MetS) demonstrate enhanced acute inflammatory responses following particulate matter inhalation. The mechanisms associated with these exacerbated inflammatory responses are unknown, impairing interventional strategies and our understanding of susceptible populations. We hypothesize MetS-associated lipid dysregulation influences mediators of inflammatory resolution signaling contributing to increased acute pulmonary toxicity. To evaluate this hypothesis, healthy and MetS mouse models were treated with either 18-hydroxy eicosapentaenoic acid (18-HEPE), 14-hydroxy docosahexaenoic acid (14-HDHA), 17-hydroxy docosahexaenoic acid (17-HDHA), or saline (control) via intraperitoneal injection prior to oropharyngeal aspiration of silver nanoparticles (AgNP). In mice receiving saline treatment, AgNP exposure resulted in an acute pulmonary inflammatory response that was exacerbated in MetS mice. A targeted lipid assessment demonstrated 18-HEPE, 14-HDHA, and 17-HDHA treatments altered lung levels of specialized pro-resolving lipid mediators (SPMs). 14-HDHA and 17-HDHA treatments more efficiently reduced the exacerbated acute inflammatory response in AgNP exposed MetS mice as compared to 18-HEPE. This included decreased neutrophilic influx, diminished induction of inflammatory cytokines/chemokines, and reduced alterations in SPMs. Examination of SPM receptors determined baseline reductions in MetS mice compared to healthy as well as decreases due to AgNP exposure. Overall, these results demonstrate AgNP exposure disrupts inflammatory resolution, specifically 14-HDHA and 17-HDHA derived SPMs, in MetS contributing to exacerbated acute inflammatory responses. Our findings identify a potential mechanism responsible for enhanced susceptibility in MetS that can be targeted for interventional therapeutic approaches.

Keywords: Inflammatory resolution; Lipid supplementation; Metabolic syndrome; Nanoparticles; Nanotoxicity; Omega-3 polyunsaturated fatty acids; Specialized pro-resolving mediators; Susceptibility.

Fig. 1.

Characterization of body weight and serum lipid levels in healthy and MetS mouse models following 14 weeks on either a healthy or high-fat western diet (HFWD). Thirty minutes prior to oropharyngeal aspiration of pharmaceutical grade sterile water (control) or AgNPs (50 μg) in sterile water, mice were i.p. injected with 1 μg of 14-HDHA, 17-HDHA, or 18-HEPE or vehicle (250 μl of sterile saline). Healthy and MetS mice without or with lipid interventions were characterized by examination of (A) body weight (BW), (B) serum total cholesterol (TC), (C) serum high-density lipoprotein (HDL), (D) serum low-density lipoprotein (LDL), and (E) serum triacylglycerides (TG) levels. Values are expressed as mean ± SEM (n = 6–8/group). # denotes significant differences between healthy and MetS models receiving similar treatment and exposure, (p < 0.05).

Fig. 2.

Acute 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 modulation by lipid interventions. Thirty minutes prior to oropharyngeal aspiration of pharmaceutical grade sterile water (control) or AgNPs (50 μg) in sterile water, mice were i.p. injected with 1 μg of 14-HDHA, 17-HDHA, or 18-HEPE or vehicle (250 μl of sterile saline). Values are expressed as mean ± SEM (n = 6–8/group). * denotes significant differences due to AgNP exposure comparing to model-matched control receiving the same treatment, # denotes significant differences between healthy and MetS mouse models receiving the same treatments and exposure, $ denotes significant differences due to lipid interventions comparing to groups not receiving treatment but the same exposure (p < 0.05).

Fig. 3.

Hyperspectral analysis of AgNPs within macrophages and neutrophils collected from BALF exposed healthy and MetS mouse models. (A) Representative enhanced darkfield images of macrophages and neutrophils 24 h post-exposure to AgNPs at a concentration of 50 μg. White arrows indicate the accumulation of AgNPs within BALF cells. White bar identifies 10 μm scaling. (B) Differences in mean spectra of AgNPs following association with BALF cells. At least 1,000 pixels of AgNPs were collected to form mean spectra. All spectra were normalized based on intensity for comparisons. Gray curve is the spectral profile of the original AgNP sample, the green curve represents AgNPs internalized by neutrophils, and the purple curve represents AgNPs internalized by macrophages. Numbers correspond to peak wavelengths. The top set of curves represent spectra of AgNPs internalized by BALF cells in the healthy model while the bottom set of curves represent spectra of AgNPs internalized by BALF cells in the MetS model. Supplemental Figure 1 includes representative image of original AgNPs as well as mapping results confirming curves represent internalized AgNPs. Representative images of internalized cells and spectral profiles of internalized AgNPs from groups receiving lipid treatments can be found in Supplemental Figure 2 (14-HDHA), Supplemental Figure 3 (17-HDHA), and Supplemental Figure 4 (18-HEPE).

Fig. 4.

AgNP-induced pulmonary inflammatory gene expression including (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 healthy and MetS lung tissue. Thirty minutes prior to oropharyngeal aspiration of pharmaceutical grade sterile water (control) or AgNPs (50 μg) in sterile water, mice were i.p. injected with 1μg of 14-HDHA, 17-HDHA, or 18-HEPE or vehicle (250 μl of sterile saline). Values are expressed as mean ± SEM (n = 6–8/group). * denotes significant differences due to AgNP exposure comparing to model-matched control receiving the same treatment, # denotes significant differences between healthy and MetS mouse models receiving the same treatments and exposure, $ denotes significant differences due to lipid interventions comparing to groups not receiving treatment but the same exposure (p < 0.05).

Fig. 5.

Alterations in BALF cytokine/chemokines proteins 24 h after AgNP exposure in healthy and MetS mice and the influence of lipid interventions. Proteins levels of (A) MIP-2, (B) MCP-1, (C) IL-6, and (D) CXCL1 were examined in BALF to determine disease-related differences in AgNP-induced inflammatory response and the influence of lipid treatments. Thirty minutes prior to oropharyngeal aspiration of pharmaceutical grade sterile water (control) or AgNPs (50 μg) in sterile water, mice were i.p. injected with 1μg 14-HDHA, 17-HDHA, or 18-HEPE or vehicle (250 μl of sterile saline). Values are expressed as mean ± SEM (n = 6–8/group). * denotes significant differences due to AgNP exposure comparing to model-matched control receiving the same treatment, # denotes significant differences between healthy and MetS mouse models receiving the same treatments and exposure, $ denotes significant differences due to lipid interventions comparing to groups not receiving treatment but the same exposure (p < 0.05).

Fig. 6.

Alterations in genes involved in lipid metabolism 24 h following AgNP exposure in lung tissue from healthy and MetS mice and the influence of lipid interventions. (A) arachidonate-5 lipoxygenase (ALOX-5), and (B) arachidonate 15-lipoxygenase (ALOX-15) were evaluated in lung tissue. Thirty minutes prior to oropharyngeal aspiration of pharmaceutical grade sterile water (control) or AgNPs (50 μg) in sterile water, mice were i.p. injected with 1μg 14-HDHA, 17-HDHA, or 18-HEPE or vehicle (250 μl of sterile saline). Values are expressed as mean ± SEM (n = 6–8/group) fold change compared with control. * denotes significant differences due to AgNP exposure comparing to model-matched control receiving the same treatment, # denotes significant differences between healthy and MetS mouse models receiving the same treatments and exposure, $ denotes significant differences due to lipid interventions comparing to groups not receiving treatment but the same exposure (p < 0.05).

Fig. 7.

Assessment of docosahexaenoic acid (DHA)-derived SPMs in the lung tissue of healthy and MetS mice 24 h following AgNP exposure and the influence of 14-HDHA intervention. A targeted LC/MS/MS approach determined quantitative differences in (A) 14-HDHA, (B) the 14-HDHA-derived SPM Maresin-1 (MaR1), (C) 17-HDHA, (D) the 17-HDHA-derived SPM resolvin D1 (RvD1), and (E) the 17-HDHA-derived SPM protectin-D1 (PD1). Thirty minutes prior to oropharyngeal aspiration of pharmaceutical grade sterile water (control) or AgNPs (50 μg) in sterile water, mice were i.p. injected with 1μg 14-HDHA, 17-HDHA, or 18-HEPE or vehicle (250 μl of sterile saline). Additional lipids (AA, DHA, EPA) evaluated in pulmonary tissue can be found in Supplemental Figure 6. * denotes significant differences due to AgNP exposure comparing to model-matched control receiving the same treatment, # denotes significant differences between healthy and MetS mouse models receiving the same treatments and exposure, $ denotes significant differences due to lipid interventions comparing to groups not receiving treatment but the same exposure (p < 0.05).

Fig. 8.

Alterations in pulmonary resolution receptors 24 h following AgNP exposure in healthy and MetS mouse models. AgNP-induced alterations in the gene expression of (A) leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6), (B) Chemokine like receptor 1 (ChemR23), and (C) G protein-coupled receptor 18 (GPCR18) were evaluated in lung tissue. Alterations in receptors associated with lipid mediator of inflammatory resolution were assessed by western blot in healthy and MetS. Receptor levels of (D) LgR6, a receptor for Mar1, (E) ChemR23, a receptor for RvE1, and (F) GCPR18, a receptor for RvD2 were examined in lung tissue to determine disease-related differences in AgNP-induced inflammatory response. (G-I) Representative western blot images of LgR6, ChemR23, and GCPR18. Values are expressed as mean ± SEM (n = 6–8/group). * denotes significant differences due to AgNP exposure, # denotes significant differences between healthy and MetS mouse models (p < 0.05).