Mechanistic Insights into Regulation of Vesicular SOCS3 Secretion by Alveolar Macrophages: Interplay Between Cell Stress and Metabolic Remodeling

Abstract

Extracellular vesicles (EVs) serve as important vectors for intercellular communication by delivering a myriad of packaged cargo molecules (i.e., proteins, lipids, and nucleic acids) from a source cell to a recipient cell. In the lung, alveolar macrophages (AMs) tonically secrete EVs containing suppressor of cytokine signaling 3 (SOCS3) protein. Uptake of SOCS3-containing EVs by alveolar epithelial cells is critical for restraint of cytokine-induced Janus kinase-signal transducer and activator of transcription 3 signaling to promote homeostasis in the distal lung. Changes in vesicular cargo secretion – including but not limited to SOCS3 – have been described in response to various microenvironmental cues, but the cellular and molecular mechanisms that control these changes remain poorly understood. Furthermore, use of quantitative methods to assess alterations in EV cargo packaging remain limited. In this dissertation, we address these gaps in knowledge by studying two perturbations relevant to the lung alveolar microenvironment: oxidative stress and metabolic remodeling.

Firstly, we used cigarette smoke (CS) as a clinically relevant model of oxidative stress to test the effect of reactive oxygen species (ROS) on vesicular SOCS3 release by AMs. Treatment of primary and immortalized AMs with an aqueous extract of CS (CSE) potentiated the secretion of SOCS3 in a ROS-dependent manner. Use of nanoparticle tracking analysis alongside a newly developed carboxyfluorescein succinimidyl ester-based cargo packaging assay demonstrated that CSE augmented AM vesicular SOCS3 release by enhancing both EV biogenesis and the amount of SOCS3 packaged into EVs. Furthermore, use of a 20S proteasome activity assay along with conventional proteasome inhibitors strongly suggested that ROS stimulated SOCS3 secretion via inactivation of the proteasome. These data demonstrate that microenvironmental oxidants tune proteasome activity to modulate vesicular SOCS3 secretion by AMs.

Secondly, as AMs exhibit remarkably low levels of glycolysis at baseline – a metabolic phenotype that supports their homeostatic function – we sought to investigate the effect of glycolytic flux on the release of SOCS3. Primary AMs were treated with the growth and activating factor granulocyte-macrophage colony-stimulating factor (GM-CSF), which promoted increases in glucose metabolism. Consequently, GM-CSF diminished SOCS3 secretion in a glycolysis-dependent manner. Furthermore, inhibition of vesicular SOCS3 release by GM-CSF relied on pyruvate transport into the mitochondria, export of mitochondrial citrate to the cytosol, and the subsequent conversion of cytosolic citrate to acetyl-CoA by ATP citrate lyase (ACLY). Therefore, our data demonstrate that ACLY links increases in glycolysis to diminished release of SOCS3 by metabolically remodeled AMs.

In tandem, our results show that ROS enhance, whereas glycolytic flux inhibits, AM secretion of vesicular SOCS3. These findings significantly advance our understanding of the cellular determinants of vesicular cargo packaging. Although we anticipate these mechanisms to be important for influencing the cargo content of EVs secreted in virtually all tissues, they may be especially meaningful in the lung alveolar milieu characterized by high oxygen tension and low levels of glucose. Future studies are needed to elucidate the molecular chaperones and/or motifs present within SOCS3 that control its vesicular sorting into AM-derived EVs.