Abstract

• Ozone exposure decreases pulmonary and systemic SPM production and pulmonary SPM receptors

• Supplementation may improve inflammation in the lungs and downregulates cytokine production

• SPM supplementation increases WBC and monocyte production in the blood which may indicate a decrease of translocation of immune cells into the lungs.

• Overall Conclusion: Alterations in SPM production in O3 induced mice may be involved in the regulatory process in O3 induced pulmonary and systemic inflammation.

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Methods & Results

Alterations in Pulmonary and Systemic Specializing Pro-Resolving Lipid Mediator Production Post-Ozone Exposure

Introduction

Figure 1. Ozone exposure reduces SPMs in the lung and spleen..

Conclusions and Future Directions

Background. Ozone (O3) exposure is associated with an increase in cardiopulmonary-induced morbidity and mortality. O3 reacts with lipids in the lung to generate oxidized products that can induce pulmonary and systemic inflammation. Specialized pro-resolving mediators (SPMs) are produced in tissue to support resolution of the immune response. The role of SPMs in O3-induced cardiopulmonary inflammation is unknown. We hypothesize that O3 exposure induces pulmonary and systemic inflammation causing a reduction in SPMs which are critical to resolve the O3-induced pulmonary and systemic inflammation. Methods. C57Bl/6J male mice were exposed to filtered air (FA) or 1 ppm ozone (O3) for 3h and necropsied 24h post-exposure. Pulmonary and systemic inflammation was determined by bronchoalveolar lavage fluid, cytokine production, and cellular differentials. Lung mRNA was analyzed using rtPCR to determine changes in pulmonary SPM receptors. Lung and spleen SPM concentrations were quantified by liquid chromatography-mass spectrometry (LC-MS/MS). SPMs 14-HDHA, 17-HDHA, and PDX were intraperitoneal (i.p.) injected 1h prior to FA or O3exposure and analyzed for BAL cytokine concentrations, complete blood counts (CBCs), and cell differentials. Results. 24h post-O3 exposure pulmonary inflammation was significantly increased with pulmonary neutrophilia and protein production in BAL. There were significant decreases in lipid mediators in O3 exposed mice. In the lungs and in spleens, there were significant decreases in the SPMs 14-HDHA and PDX. SPM receptors BLT-1 and ALX/FPR2 were significantly downregulated. Post-SPM injection results indicated suppression of pulmonary neutrophilic inflammation and a recovery of peripheral white blood cells (WBC). Conclusions. These data demonstrate that O3 exposure modulates the lipidome. O3 induced decreases of SPM and lipid mediator levels may contribute to pulmonary and cardiovascular inflammation and dysfunction. Future studies will elucidate if there are significant changes in docosahexaenoic acid (DHA) in the lung and determine if SPMs regulate immune cell infiltration in the lungs.

• Pulmonary exposure to air pollutants, such as ozone (O3), leads to enhanced pulmonary and cardiovascular morbidity and mortality (Alfaro-Moreno et al., 2007; Nawrot et al., 2006; Pope et al., 2002).

• O3 exposure has been reported to induce pulmonary inflammation and vascular dysfunction in rodent and human models (Robertson et al., 2013; Peter et al., 2004).

• Specialized Pro-Resolving Mediators (SPMs) return inflammation in effected tissue to homeostasis but the effect of O3 on SPM concentrations is unknown.

• SPMs have been shown to suppress production of pro-inflammatory mediators in the context of cigarette smoke inflammation (Hsiao et al 2013).

• Hypothesis: O3 exposure decreases SPM concentrations in the lung increasing pulmonary and systemic inflammation.

WT (C57BL/6J)

Murine model of ozone exposure

A) B)

Figure 2. Ozone decreases pulmonary SPM receptors.

K.M. Gowdy1, B.J. Kilburg-Basnyat1, S.W. Reece1, M. Hodge1, M.L. Armstrong2, S. Davies3, and S.R. Shaikh4

1Department of Pharmacology and Toxicology, East Carolina University, Greenville, NC; 2School of Pharmacy, University of Colorado, Denver, Denver, CO; Vanderbilt University, Nashville, TN; 4Department of Biochemistry, East Carolina University, Greenville, NC

Filtered Air

1ppm Ozone

BAL analysisBlood analysis

Lung and Spleen for lipid mediator

analysis

Figure 3. SPM supplementation improves lung inflammation.

Specialized Pro-Resolving Mediators (SPMs). C57Bl/6J (WT) male mice were exposed to 1ppm of ozone (O3) or filtered air (FA). Lungs and Spleens were removed 24h post FA or O3 exposure and analyzed using LC/MS. Concentrations of the SPMs 14 HDHA,17 HDHA, and Protectin DX (PDX) in spleen and lungs. (n=6 per group; *p<0.05).

A) B)

Acute inflammation

Specialized Pro-Resolving Mediators (SPMs)

↑Lipid MediatorsResolution of Inflammation

Tissue Injury

This research was supported by the Health Effects Institute Walter A. Rosenblith Award (to K.M.G.) and the National Center for Complementaryand Integrative Health NIH R01AT008375 (SRS). This research will be presented at 2017 Society of Toxicology meeting courtesy of CHHE travel

support. We would also like to thank Anita Coburn and Courtney Silliman for help with CBCs.

Acknowledgements

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* 14- HDHA can be converted to maresin(macrophage mediator in resolving inflammation)

ω-3 Polyunsaturated Fatty Acids

Murine model of ozone exposure

RvE1 and RvE2 are agonists for ChemR23 and antagonists for BLT-1 (LTB4 receptor)

Lipoxin A4 (LXA4) and D series resolvins, RvD1, RvD3, and RvD5 are agonists for GPR32/DRV1 and ALX/FPR2

Spite et al., 2014

BLT-1 and ALX/FPR2 mRNA expression is upregulated in lung tissue 24h post-O3 exposure. C57Bl/6J (WT) male mice were exposed to 1ppm O3 or FA. RNA was isolated from lungs with BLT-1, ALX/FPR2, and CHEMR23 expression measured by real time PCR. Data is presented as ddCt compared to GAPDH and β-actin expression. (n=5 per group; *p<0.05, **p<0.01)

SPM supplementation decreases O3 induced lung inflammation. C57Bl/6J (WT) male mice were exposed to 1ppm of O3 or FA. A) Lungs were lavaged and differential cell counts were recorded 24hrs after O3 exposure (n=10 per group) B). RNA was isolated from lungs with cytokine expression measured by real time PCR. Data is presented as ddCt compared to GAPDH (n=5 per group; #p<0.10 *p<0.05, **p<0.01).

SPM supplementation increases peripheral inflammatory blood cells. C57Bl/6J (WT) male mice were exposed to 1ppm of O3 or FA. A) O3suppressed white blood cells (WBCs) and SPM supplementation rescued this suppression (n=10 per group) B) 24hrs after O3 exposure, complete blood differentials were analyzed (n=5 per group).

Figure 4. SPM supplementation improves O3-induced systemic immune suppression

WT (C57BL/6J)

A) B)

Lung Spleen