Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.

Endothelial cells synthesize biochemical signals to coordinate a response to insults, resolve inflammation and restore barrier integrity. Vascular cells release a variety of vasoactive bioactive lipid metabolites during the inflammatory response and produce pro-resolving mediators (e.g., Lipoxin A4, LXA4) in cooperation with leukocytes and platelets to bring a halt to inflammation. Aspirin, used in a variety of cardiovascular and pro-thrombotic disorders (e.g., atherosclerosis, angina, preeclampsia), potently inhibits proinflammatory eicosanoid formation. Moreover, aspirin stimulates the synthesis of pro-resolving lipid mediators (SPM), so-called Aspirin-Triggered Lipoxins (ATL). We demonstrate that cytokines stimulated a time- and dose-dependent increase in PGI2 (6-ketoPGF1α) and PGE2 formation that is blocked by aspirin. Eicosanoid production was caused by cytokine-induced expression of cyclooxygenase-2 (COX-2). We also detected increased production of pro-resolving LXA4 in cytokine-stimulated endothelial cells. The R-enantiomer of LXA4, 15-epi-LXA4, was enhanced by aspirin, but only in the presence of cytokine challenge, indicating dependence on COX-2 expression. In contrast to previous reports, we detected arachidonate 5-lipoxygenase (ALOX5) mRNA expression and its cognate protein (5-lipoxygenase, 5-LOX), suggesting that endothelial cells possess the enzymatic machinery necessary to synthesize both pro-inflammatory and pro-resolving lipid mediators independent of added leukocytes or platelets. Finally, we observed that, endothelial cells produced LTB4 in the absence of leukocytes. These results indicate that endothelial cells produce both pro-inflammatory and pro-resolving lipid mediators in the absence of other cell types and aspirin exerts pleiotropic actions influencing both COX and LOX pathways.

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The inappropriate survival and persistent activation of myofibroblasts impedes the normal resolution of wound repair and promotes fibrosis. Myofibroblasts from patients with fibrotic lung disease have increased expression of Inhibitor of Apoptosis family proteins (IAPs), and pro-fibrotic stimuli, including Transforming Growth Factor Beta-1 (TGF-β1) induce IAP family protein expression. Pharmacologic inhibition of the "cellular" IAPs (cIAP-1, cIAP-2 and XIAP) diminishes lung fibrosis in murine models, and knockdown of X-linked IAP (XIAP) sensitizes fibroblasts to undergo apoptosis. TGF-β1 also induces "metabolic reprogramming", with aerobic glycolysis in normal fibroblasts. Moreover, glutaminase-dependent alpha-ketoglutarate production maintains XIAP expression in fibroblasts while glutaminase deficiency leads to decreased XIAP and enhanced apoptosis. The mechanisms that underlie aerobic glycolysis in fibroblasts, and how metabolic reprogramming affects fibroblast phenotypes, have not been determined. We hypothesized that cellular IAPs, and XIAP specifically, would regulate the metabolic reprogramming of lung fibroblasts by TGF-β1. Methods: Normal human lung fibroblasts (IMR-90) were cultured to 60% confluence and growth arrested for 24 hours prior to treatment with TGF-β1 (2 ng/ml) for 24-48 hours. As indicated, cells were transfected with siRNA targeting individual IAPs. IAP expression was assessed by quantitative real-time rtPCR. Mitochondrial Respiration and glycolysis were assessed using Seahorse xfe96 analyzer. Results: cIAP-1 and XIAP increased rapidly following TGF-β1 treatment and the increased levels persisted for 24-48 hours. In contrast, cIAP-2 expression decreased at 24 and 48 hours. Using siRNA targeting each of the cellular IAPs, we examined their role in mitochondrial respiration and glycolysis following TGF-β1 exposure. As expected, TGF-β1 increased respiration and ATP production in parallel with increased glycolysis. Silencing XIAP or cIAP-2 enhanced TGF-β1-stimulated mitochondrial respiration. In contrast, silencing cIAP-1 diminished basal and TGF-β1-stimulated respiration. Silencing cIAP-1 also prevented the increased glycolysis following TGF-β1. However, silencing of XIAP or cIAP-2 had no effect on glycolysis. Conclusions: Metabolic reprogramming has emerged as a characteristic phenotype of TGF-β1-activated myofibroblasts, and our studies demonstrate a novel role for cellular IAP family proteins in this process. Specifically, we identify a critical role for cIAP-1 in TGF-β1-induced aerobic glycolysis. Surprisingly, these studies also suggest that XIAP and cIAP-2 suppress TGF-β1-stimulated mitochondrial respiration and have no role in TGF-β1 stimulated glycolysis. Taken together, these findings indicate complex counter-regulatory roles for the structurally homologous cellular IAP family members in TGF-β1-stimulated aerobic glycolysis in lung fibroblasts.