Novel Mechanisms of Ozone-Induced Pulmonary Inflammation and Resolution, and the Potential Protective Role of Scavenger Receptor BI.

Abstract

Introduction: Increases in ambient levels of ozone (O₃), a criteria air pollutant, have been associated with increased susceptibility and exacerbations of chronic pulmonary diseases through lung injury and inflammation. O₃ induces pulmonary inflammation, in part by generating damage-associated molecular patterns (DAMPs), which are recognized by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and scavenger receptors (SRs). This inflammatory response is mediated in part by alveolar macrophages (AMs), which highly express PRRs, including scavenger receptor BI (SR-BI). Once pulmonary inflammation has been induced, an active process of resolution occurs in order to prevent secondary necrosis and to restore tissue homeostasis. The processes known to promote the resolution of inflammation include the clearance by macrophages of apoptotic cells, known as efferocytosis, and the production of specialized pro-resolving mediators (SPMs). Impaired efferocytosis and production of SPMs have been associated with the pathogenesis of chronic lung diseases; however, these impairments have yet to be linked with exposure to air pollutants.

Specific aims: The primary goals of this study were: Aim 1 - to define the role of SR-BI in O₃-derived pulmonary inflammation and resolution of injury; and Aim 2 - to determine if O₃ exposure alters pulmonary production of SPMs and processes known to promote the resolution of pulmonary inflammation and injury.

Methods: To address Aim 1, female wild-type (WT) and SR-BI-deficient, or knock-out (SR-BI KO), mice were exposed to either O₃ or filtered air. In one set of experiments mice were instilled with an oxidized phospholipid (oxPL). Bronchoalveolar lavage fluid (BALF) and lung tissue were collected for the analyses of inflammatory and injury markers and oxPL. To estimate efferocytosis, mice were administered apoptotic cells (derived from the Jurkat T cell line) after O₃ or filtered air exposure.

To address Aim 2, male WT mice were exposed to either O₃ or filtered air, and levels of SPMs were assessed in the lung, as well as markers of inflammation and injury in BALF. In some experiments SPMs were administered before exposure to O₃or filtered air, to determine whether SPMs could mitigate inflammatory or resolution responses. Efferocytosis was measured as in Aim 1.

Results: For Aim 1, SR-BI protein levels increased in the lung tissue of mice exposed to O₃, compared with mice exposed to filtered air. Compared with WT controls, SR-BI KO mice had a significant increase in the number of neutrophils in their airspace 24 hours post O₃ exposure. The oxPL levels increased in the airspace of both WT and SR-BI KO mice after O₃ exposure, compared with filtered air controls. Four hours after instillation of an oxPL, SR-BI KO mice had an increase in BALF neutrophils and total protein, and a nonsignificant increase in macrophages compared with WT controls. O₃ exposure decreased efferocytosis in both WT and SR-BI KO female mice.

For Aim 2, mice given SPM supplementation before O₃ exposure showed significantly increased AM efferocytosis when compared with the O₃exposure control mice and also showed some mitigation of the effects of O₃ on inflammation and injury. Several SPMs and their precursors were measured in lung tissue using reverse-phase high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). At 24 hours after O₃ exposure 14R-hydroxydocosahexaenoic acid (HDHA) and 10,17-dihydroxydocosahexaenoic acid (diHDoHE) were significantly decreased in lung tissue, but at 6 hours after exposure, levels of these SPMs increased.

Conclusions: Our findings identify novel mechanisms by which O₃ may induce pulmonary inflammation and also increase susceptibility to and exacerbations of chronic lung diseases.