A. B. Al-Khafaji1, D. Miller2, H. Huang1, S. Tohme1, A. Tsung1 1University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA 2Beth Israel Deaconess Medical Center,Department Of Medicine,Boston, MA, USA
Introduction: Neutrophils accumulate in the liver after ischemia-reperfusion injury and contribute to inflammation-associated damage. Although intra-hepatocellular sources release reactive oxygen species after I/R, more significant to injury is neutrophils’ oxidative burst observed 6-24h after reperfusion. Neutrophils have recently been shown to extrude select intracellular contents to form a Neutrophil Extracellular Trap (NET). Stimulation of Toll-like receptors (TLRs) initiates a signaling cascade that includes activation of NADPH Oxidase (PHOX), a required step in NET formation. Superoxide has previously been shown to signal for neutrophil activation and increased proinflammatory cytokine production through TLR4, but it is unknown whether it also induces NETs. We hypothesize that in settings of non-infectious inflammation, such as oxidative stress, reactive oxygen species, specifically superoxide, induce NET formation through TLR4.
Methods: WT and TLR4KO neutrophils were treated with xanthine oxidase and its substrate hypoxanthine to generate extracellular superoxide. We inhibited xanthine oxidase by allopurinol and inhibited PHOX by diphenylene iodonium (DPI). We also performed neutrophil depletion and adoptive transfer of TLR4KO neutrophils followed by liver I/R.
Results: In vitro, WT neutrophils exposed to superoxide demonstrated elevated levels of citrullinated Histone H3, a specific NET marker, by western analysis; however, TLR4KO neutrophils expressed only basal cit-H3 despite superoxide treatment. Superoxide-inhibited neutrophils (allopurinol treatment) or PHOX-inhibited (DPI treatment) also expressed only basal cit-H3 compared to treatment with phorbol-myristate-acetate (PMA; positive control) in both WT and TLR4KO neutrophils. Thus, the activity of both TLR4 and PHOX are necessary to complete citrullination of histone H3 and chromatin decondensation. Additionally, superoxide exposure generated characteristic NET fibers in WT neutrophils, but not TLR4KO, as visualized qualitatively by immunofluorescence microscopy and measured quantitatively by mean cytoplasmic spot intensity of extracellular DNA. In vivo, MPO-DNA ELISA, a specific marker for NETs, revealed decreased NET formation in mice adoptively transferred with TLR4KO neutrophils, correlating with protection from liver injury by ALT assay.
Conclusion: In summary, our study demonstrates that superoxide induces NETs, and that WT TLR4 and functional PHOX are required for this process. During liver I/R, ROS can stimulate NETs through TLR4, while its absence precludes NET formation correlating with reduced liver inflammation and injury. Furthermore, our finding that extracellular superoxide induces NETs expands upon previous findings that superoxide activates neutrophils and increases proinflammatory cytokine production. This suggests that products of oxidative stress and damage-associated signals released in liver I/R act via ligand-receptor interactions to promote NET formation.