Dietary Eicosapentaenoic Acid Improves Ozone-Induced Pulmonary Inflammation in C57BL/6 Mice

Abstract

Background: Ambient concentrations of the air pollutant, ozone, are rising with increasing global temperatures. Ozone is known to increase incidence and exacerbation of chronic lung diseases, which will increase as ambient ozone levels rise. Studies have identified diet as a variable that is able to modulate the pulmonary health effects associated with ozone exposure. Eicosapentaenoic acid (EPA) is an n-3 (ω-3) PUFA consumed through diet, which lowers inflammation through conversion to oxylipins including hydroxy-eicosapentaenoic acids (HEPEs). However, the role of dietary EPA in ozone-induced pulmonary inflammation is unknown.

Objective: Therefore, we hypothesized increasing dietary EPA will decrease ozone-induced pulmonary inflammation and injury through the production of HEPEs.

Methods: To test this, male C57BL/6J mice were fed a purified control diet or EPA-supplemented diet for 4 wk and then exposed to filtered air or 1 part per million ozone for 3 h. 24 or 48 h after exposure, bronchoalveolar lavage fluid was collected to assess airspace inflammation/injury and lung tissue was collected for targeted liquid chromatography-mass spectrometry lipidomics.

Results: Following ozone exposure, EPA supplementation did not alter markers of lung injury but decreased ozone-induced airspace neutrophilia. Targeted liquid chromatography-mass spectrometry lipidomics revealed dietary EPA supplementation increased pulmonary EPA-derived metabolites including 5-HEPE and 12-HEPE. Additionally, EPA supplementation decreased pulmonary amounts of proinflammatory arachidonic acid-derived metabolites. To evaluate whether dietary EPA reduces ozone-induced pulmonary inflammation through increased pulmonary HEPEs, C57BL/6J mice were administered 5-HEPEs and 12-HEPEs systemically before filtered air or ozone exposure. Pretreatment with 5-HEPEs and 12-HEPEs reduced ozone-driven increases in airspace macrophages.

Conclusions: Together, these data indicate that an EPA-supplemented diet protects against ozone-induced airspace inflammation which is, in part, due to increasing pulmonary amounts of 5-HEPEs and 12-HEPEs. These findings suggest that dietary EPA and/or increasing EPA-derived metabolites in the lung can reduce ozone-driven incidences and exacerbations of chronic pulmonary diseases.

Keywords: EPA; eicosapentaenoic acid; inflammation; lipid metabolism; lung; nutrition; omega-3; ozone; polyunsaturated fatty acids.

FIGURE 1

An EPA-supplemented diet does not protect against ozone-induced lung injury. Male mice were randomly placed on either a purified lean control diet (control, CON) or a 2% EPA-supplemented diet (EPA) for 4 wk, then exposed to 1-ppm ozone (O₃) or filtered air (FA) for 3 h and necropsied 24 and 48 h following exposure. Bronchoalveolar lavage was analyzed for (A) total protein concentration 24 h postexposure, (B) albumin concentration 24 h postexposure, (C) total protein concentration 48 h postexposure, and (D) albumin concentration 48 h postexposure (n = 6 per group; ∗∗ P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.0001). Circles, control diet (CON) and FA; squares, CON and O₃; triangles, EPA-supplemented diet and FA; and diamonds, EPA and O₃.

FIGURE 2

An EPA-supplemented diet decreased airspace neutrophilia following acute ozone exposure. Male mice were randomly placed on either a purified lean control diet or a 2% EPA-supplemented diet for 4 wk, then exposed to 1-ppm ozone (O₃) or filtered air (FA) for 3 h and necropsied 24 and 48 h following exposure. Bronchoalveolar lavage (BAL) was analyzed for (A) total airspace cells 24 h postexposure, (B) airspace macrophages 24 h postexposure, (C) airspace neutrophils 24 h postexposure, (D) total airspace cells 48 h postexposure, (E) airspace macrophages 48 h postexposure, and (F) airspace neutrophils 48 h postexposure. BAL fluid from 24 h was concentrated for (G) IL-6 and (H) KC cytokine analyses via multiplex ELISA (n = 6 per group; ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗∗P ≤ 0.0001). Circles, control diet (CON) and FA; squares, CON and O₃; triangles, EPA-supplemented diet and FA; and diamonds, EPA and O₃.

FIGURE 3

An EPA-supplemented diet increases airspace EPA, n–3 DPA, and n–6 DPA while decreasing AA. Male mice were randomly placed on either a purified lean control diet or a 2% EPA-supplemented diet for 4 wk, then exposed to 1-ppm ozone (O₃) or filtered air (FA) for 3 h and necropsied 24 h following exposure. Bronchoalveolar lavage (BAL0 fluid was concentrated for LC-MS/MS–targeted fatty acid lipidomics including, (A) α-linolenic acid (ALA), (B) EPA, (C) n–3 docosapentaenoic acid (DPA), (D) DHA, (E) linoleic acid (LA), (F) arachidonic acid (AA), and (G) n–6 DPA (n = 6 per group; ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001). Circles, control diet (CON) and FA; squares, CON and O₃; triangles, EPA-supplemented diet and FA; and diamonds, EPA and O₃.

FIGURE 4

An EPA-supplemented diet increases pulmonary production of EPA-derived oxylipins. Male mice were randomly placed on either a purified lean control diet or a 2% EPA-supplemented diet for 4 weeks, then exposed to 1-ppm ozone (O₃) or filtered air (FA) for 3 h and necropsied 24 h following exposure. Lung tissue was collected for LC-MS/MS–targeted lipidomics including (A) 5-HEPE, (B) 12-HEPE, (C) 15-HEPE, (D) 17,18-EpETE, and (E) 17,18-DiHETE (n = 6 per group; ∗ P ≤ 0.05, ∗∗∗∗ P ≤ 0.0001). Circles, control diet (CON) and FA; squares, CON and O₃; triangles, EPA-supplemented diet and FA; and diamonds, EPA and O₃. EpETE, epoxy-eicosatetraenoic acid; HEPE, hydroxy-eicosapentaenoic acid; HETE, hydroxyeicosatetraenoic acid.

FIGURE 5

An EPA-supplemented diet decreases the pulmonary production of arachidonic acid (AA)-derived eicosanoids. Male mice were randomly placed on either a purified lean control diet or a 2% EPA-supplemented diet for 4 wk, then exposed to 1-ppm ozone (O₃) or filtered air (FA) for 3 h and necropsied 24 h following exposure. Lung tissue was collected for LC-MS/MS–targeted lipidomics including (A) LTB₄, (B) PGE₂, (C) TXB₂, (D) 6-keto-PGF_1α, (E) PGF_2α isomers, (F) 11-HETE, (G) 12-HETE, and (H) 15-HETE (n = 6 per group; ∗ P ≤ 0.05, ∗∗ P ≤ 0.01) Circles, control diet (CON) and FA; squares, CON and O₃; triangles, EPA-supplemented diet and FA; and diamonds, EPA and O₃. HETE, hydroxyeicosatetraenoic acid; LT, leukotriene; PG, prostaglandin; TX, thromboxane.

FIGURE 6

5-hydroxy-eicosapentaenoic acid (HEPE) and 12-HEPE oxylipin treatment does not alter ozone-induced lung injury. Male mice were administered via intraperitoneal injections either 150 ng of 5-HEPE and 150 ng of 12-HEPE or vehicle control 1 h before 1-ppm ozone (O₃) or filtered air (FA) exposure for 3 h and necropsied 24 h following exposure. Bronchoalveolar lavage was analyzed for (A) total protein concentration 24 h postexposure and (B) airspace serum albumin 24 h postexposure (n = 7–13 per group; ∗P ≤ 0.05, ∗∗∗∗P ≤ 0.0001). Circles, phosphate-buffered saline (PBS) vehicle control (PBS) and FA; squares, PBS and O₃; triangles, 5- and 12-HEPE cocktail (5/12-HEPEs) and FA; and diamonds, 5/12-HEPEs and O₃.

FIGURE 7

5-Hydroxy-eicosapentaenoic acid (HEPE) and 12-HEPE oxylipin treatment decreases ozone-induced immune cell concentrations in the airspace. Male mice were administered either 300 ng total of 5-HEPE and 12-HEPE or vehicle control 1 h before 1-ppm ozone (O₃) or filtered air (FA) exposure for 3 h and necropsied 24 h following exposure. Bronchoalveolar lavage (BAL) was analyzed for (A) total airspace cell counts, (B) macrophages 24 h postexposure, and (C) neutrophils in the airspace 24 h postexposure. BAL fluid was analyzed for (D) MCP-1 and (E) KC via ELISA (n = 7–13 per group; ∗P ≤ 0.05, ∗∗∗P ≤ 0.001). Circles, phosphate-buffered saline (PBS) vehicle control (PBS) and FA; squares, PBS and O₃; triangles, 5- and 12-HEPE cocktail (5/12-HEPEs) and FA; and diamonds, 5/12-HEPEs and O₃.