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Neutrophil extracellular trap induced by HMGB1 exacerbates damages in the ischemic brain.

ABSTRACT: It has been reported that neutrophil extracellular traps (NETs) play important roles in non-infectious diseases. In ischemic stroke, neutrophils infiltrate damaged brain tissue soon after injury and aggravate inflammation. Using a rat permanent MCAO model, we showed citrullinated histone H3⁺ (CitH3, a marker of NETosis) induction in neutrophils in leptomeninges and in peripheral blood soon after MCAO. Entry of CitH3⁺ cells occurred through leptomeninges after 6 h of MCAO and these cells were observed in cerebral cortex from 12 h and subsequently in striatum. It is interesting to note that CitH3⁺ induction began in circulating neutrophils before they migrated to brain parenchyma and they were detected as intact or lysed form. High mobility group box 1 (HMGB1), a danger associated molecular pattern (DAMP) molecule, was accumulated massively in serum after permanent MCAO and plays a critical role in CitH3 inductions in neutrophils in brain parenchyma and in peripheral blood. Both the all-thiol and disulfide types of HMGB1 induced CitH3 via their specific receptors, CXCR4 and TLR4, respectively. Importantly, HMGB1 not only induced NETosis but was included as a part of the extruded NETs, and contribute to NETosis-mediated neuronal death. Therefore, it would appear a vicious cycle exists between neuronal cell death and NETosis and HMGB1 mediates detrimental effects exerted by this cycle. When NETosis was suppressed by a PAD inhibitor in MCAO animals, delayed immune cell infiltrations were markedly suppressed and damages in blood vessels were significantly mitigated. The study shows NETosis with the involvement of HMGB1 as a mediator in a vicious cycle aggravates inflammation and subsequent damage in the ischemic brain.

SUBMITTER: Kim SW

PROVIDER: S-EPMC6556959 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Neutrophil extracellular trap induced by HMGB1 exacerbates damages in the ischemic brain.

Kim Seung-Woo SW Lee Hahnbie H Lee Hye-Kyung HK Kim Il-Doo ID Lee Ja-Kyeong JK

Acta neuropathologica communications 20190610 1

It has been reported that neutrophil extracellular traps (NETs) play important roles in non-infectious diseases. In ischemic stroke, neutrophils infiltrate damaged brain tissue soon after injury and aggravate inflammation. Using a rat permanent MCAO model, we showed citrullinated histone H3<sup>+</sup> (CitH3, a marker of NETosis) induction in neutrophils in leptomeninges and in peripheral blood soon after MCAO. Entry of CitH3<sup>+</sup> cells occurred through leptomeninges after 6 h of MCAO an ...[more]

PMID: 31177989

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Project description:RationaleAcute kidney injury (AKI) is a clinical syndrome associated with a multitude of conditions. Although renal replacement therapy (RRT) remains the cornerstone of treatment for advanced AKI, its implementation can potentially pose risks and may not be readily accessible across all healthcare settings and regions. Elevated lactate levels are implicated in sepsis-induced AKI; however, it remains unclear whether increased lactate directly induces AKI or elucidates the underlying mechanisms.MethodsFor human, the measurement of lactate in arterial blood gas is performed using the direct determination of L-lactate through an electrode oxidation method by a blood gas analyzer. For mice, enzyme-linked immunosorbent assay (ELISA) kits were employed to quantify the concentrations of lactate and AKI biomarkers in blood and cell supernatant. The mouse model of AKI was performed with a single intraperitoneal (i.p.) administration of lactate (30 mg/kg) and low-dose LPS (2 mg/kg) for 24 h. Proteomic analysis was conducted to identify lactylated proteins in kidney tissues. Techniques such as, immunoprecipitation, western blotting and immunofluorescence were used to evaluate the levels of HMGB1 lactylation, neutrophil extracellular traps (NETs)and to assess related molecular signaling pathways.Main resultsOur findings indicate that lactate serves as an independent predictor of AKI in patients with acute decompensated heart failure (ADHF). We observed that co-administration of lactate with low-dose lipopolysaccharide (LPS) resulted in lactate overproduction, which subsequently elevated serum levels of creatinine (Cre) and blood urea nitrogen (BUN). Furthermore, the combined application of lactate and low-dose LPS was shown to provoke HMGB1 lactylation within renal tissues. Notably, pretreatment with HMGB1 small interfering RNA (siRNA) effectively diminished lactate-mediated HMGB1 lactylation and alleviated the severity of AKI. Additionally, lactate accumulation was found to enhance the expression levels of NETs in the bloodstream, with circulating NETs levels positively correlating with HMGB1 lactylation. Importantly, pre-administration of HMGB1 inhibitors (glycyrrhizin) or lactate dehydrogenase A (LDH-A) inhibitors (oxamate) reversed the upregulation of NETs induced by lactate and low-dose LPS in both the blood and polymorphonuclear neutrophils (PMNs) cell supernatant, thereby ameliorating AKI associated with lactate accumulation.ConclusionsThese findings illuminate the role of lactate-mediated HMGB1 lactylation in inducing AKI in mice through the activation of the HMGB1-NETs signaling pathway.

Project description:Neutrophils and liver sinusoidal endothelial cells (LSECs) both contribute to sterile inflammatory injury during ischemia/reperfusion (I/R), a well-known liver surgical stress. Interleukin-33 (IL-33) has been shown to drive neutrophil infiltration during inflammatory responses through its receptor ST2. We recently reported that infiltrating neutrophils form neutrophil extracellular traps (NETs), which exacerbate sterile inflammatory injury in liver I/R. Here, we sought to determine the role of IL-33 in NET formation during liver sterile inflammation. Evaluation of IL-33 forming NETs was investigated using a partial liver I/R model to generate sterile injury in healthy WT, IL-33 and ST2 knockouts. Serum levels of IL-33 and myeloperoxidase (MPO)-DNA complex were measured in both humans and mice after the first surgery. Liver damage was assessed. Mouse neutrophil depletion was performed by intraperitoneal injection of anti-Ly6G antibody before I/R. Patients undergoing liver resection showed a significant increase in serum IL-33 compared to healthy volunteers. This coincided with higher serum MPO-DNA complexes. NET formation was decreased in IL-33 and ST2 knockout mice compared with control mice, after liver I/R. IL-33 or ST2 deficiency protected livers from I/R injury, whereas rIL-33 administration during I/R exacerbated hepatotoxicity and systemic inflammation. In vitro, IL-33 is released from LSECs to promote NET formation. IL-33 deficient LSECs failed to induce NETs. ST2 deficient neutrophils limited their capacity to form NETs in vitro and adoptive transfer of ST2 knockout neutrophils to neutrophil-depleted WT mice significantly decreased NET formation. Data establish that IL-33, mainly released from LSECs, causes excessive sterile inflammation after hepatic I/R by inducing NET formation. Therapeutic targeting of IL-33/ST2 might extend novel strategies to minimize organ damage in various clinical settings associated with sterile inflammation. Liver ischemia and reperfusion injury results in the formation of neutrophil extracellular traps, which contribute to organ damage in liver surgeries. Herein, we show that IL-33 is released from liver sinusoidal endothelial cells to promote NET formation during liver I/R, which exacerbates inflammatory cascades and sterile inflammation.

Project description:UnlabelledInnate 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, which trigger an innate immune and inflammatory cascade through pattern recognition receptors. Neutrophils are recruited to the liver after I/R and contribute to organ damage and innate immune and inflammatory responses. Formation of neutrophil extracellular traps (NETs) 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-DNA complexes, and tissue level of citrullinated-histone H3 compared to control mice. Treatment with peptidyl-arginine-deiminase 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. Damage-associated molecular patterns, such as High Mobility Group Box 1 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 or TLR9 knockout neutrophils confers significant protection from liver I/R injury with a significant decrease in NET formation. In addition, we found inhibition of NET formation by the peptidyl-arginine-deiminase 4 inhibitor and that DNase I reduces High Mobility Group Box 1 and histone-mediated liver I/R injury.ConclusionDamage-associated molecular patterns released during liver I/R promote NET formation through the TLR signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R.

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Neutrophil extracellular trap induced by HMGB1 exacerbates damages in the ischemic brain.

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Neutrophil extracellular trap induced by HMGB1 exacerbates damages in the ischemic brain.

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