View Abstract

A4488 - Reactive Oxygen Species Enhance Suppressor of Cytokine Signaling 3 Packaging into Alveolar Macrophage-Derived Microvesicles
Author Block: M. D. Haggadone1, P. Mancuso2, J. Bazzill3, J. J. Moon3, M. Peters-Golden4; 1Graduate Program in Immunology, University of Michigan, Ann Arbor, MI, United States, 2Department of Nutritional Sciences, University of Michigan, Ann Arbor, MI, United States, 3Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, United States, 4Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, MI, United States.
Rationale: Transfer of extracellular vesicles (EVs) containing diverse protein, nucleic acid, and lipid cargo is an integral form of cell-cell communication. We have published data demonstrating that alveolar macrophages (AMs) secrete microvesicles (MVs), a class of EVs that bud from the plasma membrane, containing suppressor of cytokine signaling 3 (SOCS3). SOCS3 negatively regulates cytokine-induced Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signaling, and we showed that the transcellular acquisition of AM-derived vesicular SOCS3 by neighboring alveolar epithelial cells (AECs) suppresses pro-inflammatory STAT3 signaling in AECs. The lung encounters unusual amounts of reactive oxygen species (ROS) owing to exposures to ambient oxygen, environmental toxins, and the byproducts of inflammation. However, whether ROS modulate EV cargo packaging in any cell type or context remains unknown. We hypothesized that ROS exposure alters SOCS3 packaging into – and secretion in – AM-derived MVs.
Methods: Immortalized mouse AMs (MH-S cells) were treated for 1 h with various doses of the exogenous oxidants hydrogen peroxide (H2O2) and an extract of cigarette smoke in medium (CSE); ROS levels were verified by DCFDA staining. Pre-treatment with the general antioxidant N-acetyl cysteine (NAC) and mitochondrially targeted antioxidant MitoTEMPO was used for scavenging ROS. 20 h following stimulation, MV secretion into conditioned medium (CM) was quantified by flow cytometric analysis of annexin V-stained particles of appropriate size; SOCS3 secretion was determined by western blot of 100 kDa-filtered CM. SOCS3 packaging was calculated by determining the relative ratio of SOCS3 per secreted MV. Data were corroborated using Nanoparticle Tracking Analysis and ELISA to quantify MVs and SOCS3, respectively.
Results: Treatment of MH-S cells with H2O2 (100 μM - 1 M) and CSE (0.25% - 3%) dose-dependently increased ROS levels. However, SOCS3 secretion was only enhanced by treatment with 10 mM H2O2 and 3% CSE. Increased SOCS3 secretion by 10 mM H2O2 did not involve altered MV production and was solely attributable to augmented SOCS3 packaging. However, treatment with 3% CSE led to a ROS-dependent enhancement of SOCS3 secretion caused by increases in both packaging and MV numbers. The stimulatory effects of CSE were abrogated by pre-treatment of MH-S cells with NAC, but not MitoTEMPO, thus excluding involvement of mitochondrial ROS.
Conclusions: Exogenous ROS enhance SOCS3 packaging into AM-generated MVs. These data suggest that AMs might rapidly respond to environmental and secreted sources of ROS by enhancing their release of SOCS3, thereby dampening pro-inflammatory STAT3 signaling in AECs.