NLRP3 inflammasome-mediated disruption of mitochondrial homeostasis in alveolar macrophages contributes to ozone-induced acute lung inflammatory injury

Abstract

Ozone (O3), a prevalent atmospheric pollutant, can induce lung injury. However, the molecular mechanisms of O3-induced acute lung inflammatory injury remain unclear. In this study, we investigate the abnormal changes in and molecular mechanism of mitochondrial homeostasis in alveolar macrophages (AMs) in O3-induced acute lung inflammatory injury mice. Mitochondria and mitochondrial reactive oxygen species (mtROS) are labeled with Mito-Tracker® Deep Red and MitoSOX Red, respectively. Mitochondrial DNA (mtDNA) in AMs from the bronchoalveolar lavage fluid (BALF) is detected via real-time PCR, and the expressions of mitochondrial fusion/fission-related and biogenesis-related proteins in AMs are determined via immunofluorescence staining. Our data show that in O3-induced acute lung inflammatory injury mice, the number of AMs and the protein expression of the NLRP3 inflammasome complex in the lung tissue are increased. In AMs from O3-exposed mice, the number of mitochondria, mtROS, and fission-related protein DRP1 are increased, but the levels of Na+-K+-ATPase, fusion-related protein OPA1, biogenesis-related protein NRF1 and mtDNA are significantly decreased. Compared with that in O3-exposed WT mice, lung inflammation is attenuated, especially the indicators of mitochondrial homeostatic imbalance in AMs, which are alleviated in NLRP3‒/‒ and Caspase-1‒/‒ mice after O3 exposure. These findings indicate that the NLRP3 inflammasome-mediated imbalance in mitochondrial homeostasis in AMs contributes to O3-induced acute lung inflammatory injury. This study may provide a new target for the prevention of lung inflammation induced by O3.

1. Introduction

In recent years, ozone (O3) pollution has become increasingly serious, and the rapid expansion of the pollution season has led to severe pollution even in winter and spring [1]. The number of people exposed to O3 will increase significantly in the future under the influence of climate change. As an important air pollutant, O3 has been recognized as an urgent environmental issue, primarily because of its adverse effects on human health [2]. Extensive epidemiological studies have revealed respiratory system damage caused by O3 exposure [3,4]. Short-term inhalation of O3 is associated with an increased risk of hospitalization due to respiratory diseases [5]. The impact of O3 exposure is evident in the induction of airway hyperresponsiveness (AHR) and alveolar degradation, which can occur even within a short period of time (3 to 6 h) at an O3 concentration of 4.28 mg/m3. Additionally, toxicological studies have shown that even low-dose O3 exposure can induce acute lung injury (ALI) in mice [6]. However, the molecular mechanisms of O3-induced acute lung inflammatory injury remain unclear.

Alveolar macrophages (AMs) constitute the primary macrophage population within the lungs, accounting for more than 90% of lung macrophages in a healthy state [7]. These cells serve as the first line of lung defense [8] and are among the few cell types that directly interact with inhaled O3 [9], contributing to the initiation of acute inflammatory responses. Alveolar macrophages are divided into proinflammatory (M1) and anti-inflammatory (M2) phenotypes, and their imbalance is often associated with disease [10]. M1 macrophages may participate in the pathogenesis of asthma by releasing inflammatory factors, thus aggravating lung injury and airway remodeling, while promoting the polarization of M2 macrophages may ameliorate lung injury [11]. Dysregulated activation and cell death of AMs are thought to be central to the progression of lung inflammation [12]. These processes significantly impact the development of ALI by causing the release of diverse inflammatory mediators from AMs in response to both infectious and noninfectious stimuli [13]. NLR family pyrin domain containing 3 (NLRP3), an intracellular innate immune receptor, can bind to apoptosis-associated speck-like protein containing a CARD (ASC) and Caspase1 to form the NLRP3 inflammasome [14], promoting the activation and release of the proinflammatory cytokines IL-1β and IL-18 [15]. NLRP3 and Caspase1, as the initiating and effector proteins of the NLRP3 inflammasome, may be more valuable to study. Increasing evidences indicated that NLRP3 can alleviate ALI by regulating the AMs pyroptosis [16,17]. A previous report demonstrated that the NLRP3 inflammasome is predominantly localized in AMs, which is essential for t