Macrophage-Derived Mechanisms of Resolution of Environmental Lung Injury

Abstract

Lung inflammation, caused by acute exposure to ozone (O3)– one of the six criteria air pollutants – is a significant source of morbidity in susceptible individuals. The adverse effects of ozone (O3) on respiratory health and its significant impact on global public health are well-established, but the cellular mechanisms that drive these effects remain poorly understood. This study explores mechanisms that regulate resolution of O3-induced lung inflammation, specifically focused on the function of alveolar macrophages. Alveolar macrophages (AMØs) are central regulators of lung immune responses including both the initiation and resolution of inflammation. They regulate inflammation via functions such as production of cytokines, phagocytosis, and efferocytosis. While prior O3 exposure studies have highlighted that exposure leads to an increase in AMØs, the specific role of AMØs in promoting resolution of O3-induced lung inflammation remains unclear.One reason that it is challenging to define the role of AMØs following acute O3 exposure is that within the lung, macrophages can have different origins (ontogeny). This has directed a series of studies focused on determining if differences in AMØ functions are due to their distinct ontogeny. While it has been observed that AMØ derived from circulating monocytes (i.e. monocyte-derived AMØs) play a critical role in regulating chronic/severe injury, the ontogeny of AMØs (i.e. tissue-resident versus monocyte-derived) following acute O3 exposure has been undefined. Using mouse models (lineage labeled, genetic knockouts, and wildtype), we traced the origin of AMØs and found them to be predominantly tissue-resident AMØs following acute O3 exposure, which was then confirmed using data from O3-exposed human volunteers. Depletion of these tissue-resident AMØs resulted in a persistence of neutrophils in the alveolar space after O3 exposure, indicating impaired clearance and persistent inflammation. This impaired clearance was associated with reduced efferocytosis, the clearance of apoptotic cells, a process crucial for resolving inflammation. Mice with a genetic deficiency in MerTK – a key receptor regulating efferocytosis – also resulted in impaired clearance of apoptotic neutrophils following O3 exposure. We thus defined the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis. We then focused on intracellular mechanisms of inflammation resolution that occur within AMØs. We focused on a previously established pathway of inflammation resolution regulation through the metabolism of the amino acid, L-arginine. While L-arginine metabolism by nitric oxide synthase can promote inflammatory responses, L-arginine metabolism by arginase-1 generates metabolites that have the potential to direct inflammation resolution. One such metabolite of L-arginine is spermidine, and it is of interest due to its anti-inflammatory properties observed in many tissues (pulmonary and non-pulmonary) and macrophages. Additionally, prior research suggests that spermidine inhibits N-methyl-d-aspartate (NMDA) receptor activation. We therefore hypothesized that the mechanism by which spermidine leads to resolution of macrophage-derived inflammation is via inhibition of NMDA, and thereby reducing the activation of the pro-inflammatory Nuclear factor kappa B (NF-κB) signaling. Here we expand the understanding of the mechanism for spermidine effect in macrophages via impact on NF-κB signaling via the NMDA receptor. To address this, we initially utilized a mouse model to assess the concentration of L-arginine and its metabolites in BALF following acute O3 exposure. Here, we identified a decrease of L-arginine at 12h post-exposure, with a subsequent increase in spermidine present at 24h post-exposure, a time point critical for resolution of O3-induced lung inflammation. We then conducted a pretreatment exposure in which the mice were treated with spermidine prior to O3 exposure to determine if there was a reduction in inflammation. Mice pretreated with spermidine, when compared to control mice, demonstrated reduced O3-induced lung inflammation. This suggests that spermidine may drive a reduction in pro-inflammatory signaling following acute O3 exposure. We next sought to understand the potential intracellular mechanism driving this response. To test this, we conducted in vitro studies in MH-S cells, AMØ-like immortalized cells. We utilized MH-S cells to define spermidine's effect on pro-inflammatory signaling following Lipopolysaccharide (LPS) exposure. We utilized LPS as a known pro-inflammatory stimulus in AMØ and a canonical activator of NF-κB. Utilizing a rescue model, in which spermidine was given following an initial LPS exposure, we found that spermidine decreased the expression and concentration of NF-κB associated pro-inflammatory cytokines, supporting a role in the resolution of inflammation. Then we pretreated MH-S cells with spermidine and determined that spermidine decreases the activation of NF-κB. We then utilized a known agonist, NMDA, for the NMDA receptor and found that NMDA activates NF-κB. In summary, the research highlights the pivotal role of tissue-resident AMØs and explores the potential of spermidine as a therapeutic agent for resolving environmental-induced lung inflammation.