Abstract
Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules—including but not limited to SOCS3—have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs.
Highlights
Tissue homeostasis is maintained by the dynamic regulation of information transfer amongst cells
Treatment of primary alveolar macrophages (AMs) with cigarette smoke extract (CSE) led to an increase in vesicular suppressor of cytokine signaling 3 (SOCS3) release, which was marked at a 7.5% dose (Figure 1A and Figure S8A)
Using CSE treatment as a clinically relevant in vitro model for oxidative stress within the alveolar microenvironment, we demonstrate that reactive oxygen species (ROS) augment vesicular SOCS3 release by primary and immortalized AMs (i.e., MH-S cells), a phenomenon attributable at least in part to catalytic inactivation of the 20S proteasome
Summary
Tissue homeostasis is maintained by the dynamic regulation of information transfer amongst cells. In addition to the contributions of direct cell-cell contact and the release of soluble mediators, the importance of extracellular vesicles (EVs) as vectors for the local and systemic transfer of diverse molecular cargoes between cells has become increasingly appreciated [1,2]. EVs have been classified into exosomes (Exos) and microvesicles (MVs) based on differences in mode of biogenesis, size, and the presence of specific molecular cargoes. Substantial overlap in size and molecular characteristics is recognized to limit the ability to categorically differentiate these two classes of EVs when using traditional isolation methods [3,4]. The pulmonary alveolar space is characterized by uniquely high oxygen tension. Reactive oxygen species (ROS)—whose generation is amplified during
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