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NLRX1 mediated impaired microglial phagocytosis of NETs in cerebral ischemia and reperfusion injury

Cell Death & Differentiation (2025)Cite this article

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Abstract

Ischemic stroke is one of the common causes of disability and death, and subsequent pathological processes consequent to revascularization could promote secondary tissue damage leading to neuronal death, namely cerebral ischemia and reperfusion injury. Neutrophils could invade injured brain parenchyma after vascularization and exert neurotoxicity by forming neutrophil extracellular traps (NETs). However, unwanted NETs were accumulated in the infarcted core of transient middle cerebral artery occlusion (tMCAO) rats and the mechanism is unknown. Efficient microglial phagocytosis is crucial for the homeostasis of cerebral parenchyma after stroke, and dysfunction of microglial phagocytosis of NETs were observed in the infarcted core cortex at tMCAO 1 d and the accumulation of NETs persisted to 7 d, which exerting deleterious neuronal damage after stroke. However, the detailed mechanisms underlying the dysfunction of microglial phagocytosis of NETs remained unclear. Our results further demonstrated that NLRX1 was mainly enhanced in the microglial cells in the infarcted core cortex at tMCAO 1 d and promoted galectin-3 expression on the lysosomes, facilitating the lysosomal dysfunction and impaired microglial phagocytosis via mTOR/TFEB signaling. NLRX1-silencing was able to suppress the galectin-3 intensity, inhibit the phosphorylation of mTOR and facilitate the nuclear localization of TFEB, ameliorating the lysosomal dysfunction and microglial phagocytosis of NETs. Our results uncovered the regulation of NLRX1 in the dysfunctional microglial phagocytosis of NETs and provided insights into the therapeutic potential for targeting at microglial lysosomal function in cerebral ischemia and reperfusion injury.

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Fig. 1: Accumulation of NETs in the infarcted core cortex exerted neurotoxicity at tMCAO 1d.
Fig. 2: Impaired microglial phagocytosis contributed to the accumulation of NETs in the infarcted core cortex at tMCAO 1d.
Fig. 3: NLRX1 might mediate the microglial lysosomal function in the ischemic cortex at tMCAO 1d.
Fig. 4: NLRX1 impaired the microglial phagocytosis of NETs, which exerted neuronal damage.
Fig. 5: Microglial NLRX1 mediated the lysosomal function via mTOR/TFEB after OGD/R.
Fig. 6: NLRX1 promoted Galectin-3 expression, facilitating the activation of mTOR and lysosomal dysfunction.
Fig. 7: Inhibiting microglial mTOR activation improved the phagocytosis of NETs and ameliorated neuronal degeneration in the infarcted core cortex.
Fig. 8: The schematic models of NLRX1 signaling in our models and other disease models.

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Data availability

The data supporting our current research are available form the corresponding author on reasonable request.

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Funding

This work was supported by the Natural Science Foundation of Guangdong Province (No. 2022A1515110616 to Jialing Peng), Plan on enhancing scientific research in GMU (No. 02-410-2302027XM to Jialing Peng), Science and Technology Projects in Guangzhou (No. 2024A04J3703 to Jialing Peng), National Natural Science Foundation of China (No. 82371186 to Jun Liu, No. 82401401 to Jialing Peng) and Key Technologies R&D Program of Guangzhou (No: 202206010007 to Jun Liu). The work was also supported by Guangzhou Medical Key Discipline Construction Project (2025-2027).

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Authors and Affiliations

  1. Department of Neurology and Institute of Neuroscience, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, 510260, China

    Jialing Peng & Jun Liu

  2. The Second Clinical School, Guangzhou Medical University, Guangzhou, 511495, China

    Yuxin Huang & Tengjing He

  3. Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China

    Yang Zhan

  4. Institute of Neurosciences, Department of Neurology and National Core Cognitive Center, The Second Affiliated Hospital of Guangzhou Medical University, No. 250 Changgang East Road, Haizhu District, Guangzhou, 510261, China

    Jun Liu

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  1. Jialing Peng
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Contributions

JLP designed and performed the experiments, wrote the original draft and analyzed the data. YXH analyzed the data and revised the manuscript. TJH performed the data curation and review and editing. YZ provided suggestions, guided the research and revised the manuscript. JL performed the funding acquisition and supervision and review. All authors read and approved the final manuscript.

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Correspondence to Yang Zhan or Jun Liu.

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The authors declare no competing interests.

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All animal experiments were approved by Animals Care and Use Committee of the Second Affiliated Hospital of Guangzhou Medical University (Approval Number, A2022-042). We designed, performed and reported the experiments in accordance with the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines [13].

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Peng, J., Huang, Y., He, T. et al. NLRX1 mediated impaired microglial phagocytosis of NETs in cerebral ischemia and reperfusion injury. Cell Death Differ (2025). https://doi.org/10.1038/s41418-025-01526-3

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