Dataset Information

Brain border-derived CXCL2⁺ neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.

ABSTRACT:

Background

The brain border compartments harbor a diverse population of immune cells and serve as invasion sites for leukocyte influx into the brain following CNS injury. However, how brain-border myeloid cells affect stroke pathology remains poorly characterized.

Methods and results

Here, we showed that ischemic stroke-induced expansion of CXCL2⁺ neutrophils, which exhibit highly proinflammatory features. We tracked CXCL2⁺ neutrophils in vivo by utilizing a photoconvertible Kik-GR mouse (fluorescent proteins Kikume Green Red, Kik-GR) and found that brain-infiltrating CXCL2⁺ neutrophils following ischemic stroke were mainly derived from the brain border rather than the periphery. We demonstrated that CXCL2 neutralization inhibited the formation and releasing of neutrophil extracellular traps (NETs) from in vitro cultured primary neutrophils. Furthermore, CXCL2-neutralizing antibody treatment reduced brain infarcts and improved vascular reperfusion at day 3 postischemic stroke.

Conclusions

Collectively, brain border-derived CXCL2⁺ neutrophil expansion may impair vascular reperfusion by releasing NETs following ischemic stroke.

SUBMITTER: Huang T

PROVIDER: S-EPMC11319398 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Publications

Brain border-derived CXCL2<sup>+</sup> neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.

Huang Tingting T Guo Yunlu Y Xie Wanqing W Yin Jiemin J Zhang Yueman Y Chen Weijie W Huang Dan D Li Peiying P

CNS neuroscience & therapeutics 20240801 8

<h4>Background</h4>The brain border compartments harbor a diverse population of immune cells and serve as invasion sites for leukocyte influx into the brain following CNS injury. However, how brain-border myeloid cells affect stroke pathology remains poorly characterized.<h4>Methods and results</h4>Here, we showed that ischemic stroke-induced expansion of CXCL2<sup>+</sup> neutrophils, which exhibit highly proinflammatory features. We tracked CXCL2<sup>+</sup> neutrophils in vivo by utilizing a ...[more]

PMID: 39135337

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Project description:BackgroundNeutrophil extracellular traps (NETs)-as double-edged swords of innate immunity-are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs.MethodsWe analysed the prolongation of neutrophil survival in vitro under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO).ResultsUntreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α.ConclusionsG-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions.

Project description:BackgroundRenal ischemic reperfusion injury (RIRI) is the most hackneyed cause of acute renal injury with high incidence. As a slit diaphragm (SD), TRPC6 (transient receptor potential channel 6) can maintain the structure and function of glomerular podocytes, and its activation has been reported to prominently alleviate ischemia reperfusion (I/R). However, the specific mechanism of TRPC6 in RIRI is uncertain.MethodsThe TRPC6 specific shRNA or overexpressing plasmid was used to decrease or increase TRPC6 level in HK-2 cells, respectively. Subsequently, the OGD/R (oxygen-glucose deprivation and re-oxygenation) HK-2 cells and RIRI model rats was established to examine the effect of TRPC6 in RIRI in vitro. After processing, viability was confirmed with MTT; cell necrosis was analyzed with flow cytometry; necrosis and autophagy-related proteins were verified with Western blot; free Zn2+ was tested with an Zn2+ fluorescent probe; and cell autophagy was monitored with MDC (monodansylcadaverine) method. Furthermore, TRPC6 agonist (OGA) or TRPC6 inhibitor (SKF96365) were introduced to increase or inhibit the activity of TRPC6 in RIRI model rats, and the kidney injury was assessed with H&E staining and RIP1 and PARP-1 expressions were examined with IHC (immunohistochemistry) staining.ResultsOur results verified TRPC6 could markedly enhance viability, Zn2+ influx, and autophagy, and suppressed necrosis in OGD/R HK-2 cells. In addition, increase of Zn2+ or autophagy activation produced similar results to TRPC6 overexpression in viability, autophagy, and necrosis of OGD/R HK-2 cells. Rescue experiment results also showed TRPC6 could prevent necrosis and facilitate Zn2+ influx and autophagy of OGD/R HK-2 cells by inducing Zn2+ influx and autophagy. Moreover, TRPC6 could ameliorate kidney injury, block necrosis, and enhance autophagy in RIRI model rats by promoting Zn2+ influx and autophagy.ConclusionsTRPC6 could prevent necrosis and induce autophagy to alleviate RIRI by accelerating Zn2+ influx and autophagy. This shows TRPC6/Zn2+ influx/autophagy might be a novel therapeutic strategy for RIRI.

Dataset Information

Brain border-derived CXCL2⁺ neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.

Background

Methods and results

Conclusions

Publications

Brain border-derived CXCL2<sup>+</sup> neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Brain border-derived CXCL2+ neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.

Background

Methods and results

Conclusions

Publications

Brain border-derived CXCL2&lt;sup&gt;+&lt;/sup&gt; neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Brain border-derived CXCL2⁺ neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.

Brain border-derived CXCL2<sup>+</sup> neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.