Risks of tumor recurrence after surgical resection have been known for decades, but the mechanisms underlying treatment failures remain poorly understood. Neutrophils, first-line responders after surgical stress, may play an important role in linking inflammation to cancer progression. In response to stress, neutrophils can expel their protein-studded chromatin to form local snares known as neutrophil extracellular traps (NET). In this study, we asked whether as a result of its ability to ensnare moving cells NET formation might promote metastasis after surgical stress. Consistent with this hypothesis, in a cohort of patients undergoing attempted curative liver resection for metastatic colorectal cancer, we observed that increased postoperative NET formation was associated with a >4-fold reduction in disease-free survival. In like manner, in a murine model of surgical stress employing liver ischemia-reperfusion, we observed an increase in NET formation that correlated with an accelerated development and progression of metastatic disease. These effects were abrogated by inhibiting NET formation in mice, through either local treatment with DNAse or inhibition of the enzyme peptidylarginine deaminase (PAD4), which is essential for NET formation. In growing metastatic tumors, we found that intratumoral hypoxia accentuated NET formation. Mechanistic investigations in vitro indicated that mouse neutrophil-derived NET triggered HMGB1 release and activated TLR9-dependent pathways in cancer cells to promote their adhesion, proliferation, migration and invasion. Taken together, our findings implicate NET in the development of liver metastases after surgical stress, suggesting that their elimination may reduce risks of tumor relapse.
Innate immunity plays a crucial role in the response to sterile inflammation such as liver ischemia/reperfusion (I/R) injury. The initiation of liver I/R injury results in the release of damage associated molecular patterns (DAMPs), which trigger innate immune and inflammatory cascade via pattern recognition receptors. Neutrophils are recruited to the liver after I/R and contribute to the organ damage, innate immune and inflammatory responses. Formation of neutrophil extracellular trap (NET) has been recently found in response to various stimuli. However, the role of NETs during liver I/R injury remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo. This was associated with increased NET markers, serum level of myeloperoxidase (MPO)-DNA complexes and tissue level of citrullinated-histone H3 compared to control mice. Treatment with peptidyl-arginine-deiminase (PAD) 4 inhibitor or DNase I significantly protected hepatocytes and reduced inflammation after liver I/R as evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. In vitro, NETs increase hepatocyte death and induce Kupffer cells to release proinflammatory cytokines. DAMPs, such as HMGB1 and histones, released by injured hepatocytes stimulate NET formation through Toll-like receptor (TLR4)- and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout (KO) or TLR9 KO neutrophils confers significant protection from liver I/R injury with significant decrease in NET formation. In addition, we found inhibition of NET formation by PAD4 inhibitor or DNase I reduces HMGB1 and histone-mediated liver I/R injury. Conclusion DAMPs released during liver I/R promotes NET formation through TLRs signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R.
Neutrophils constitute the early innate immune response to perceived infectious and sterile threats. Neutrophil extracellular traps (NETs) are a novel mechanism to counter pathogenic invasion and sequelae of ischemia, including cell death and oxidative stress. Superoxide is a radical intermediate of oxygen metabolism produced by parenchymal and nonparenchymal hepatic cells, and is a hallmark of oxidative stress after liver ischemia-reperfusion (I/R). While extracellular superoxide recruits neutrophils to the liver and initiates sterile inflammatory injury, it is unknown whether superoxide induces the formation of NETs. We hypothesize that superoxide induces NET formation through a signaling cascade involving Toll-like receptor 4 (TLR-4) and neutrophil NADPH oxidase (NOX). We treated neutrophils with extracellular superoxide and observed NET DNA release, histone H3 citrullination and increased levels of MPO-DNA complexes occurring in a TLR-4-dependent manner. Inhibition of superoxide generation by allopurinol and inhibition of NOX by diphenyleneiodonium prevented NET formation. When mice were subjected to warm liver I/R, we found significant NET formation associated with liver necrosis and increased serum ALT in TLR-4 WT but not TLR-4 KO mice. To reduce circulating superoxide, we pretreated mice undergoing I/R with allopurinol and N-acetylcysteine, which resulted in decreased NETs and ameliorated liver injury. Our study demonstrates a requirement for TLR-4 and NOX in superoxide-induced NETs, and suggests involvement of superoxide-induced NETs in pathophysiologic settings.
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