IFP35, a novel DAMP, aggravates neuroinflammation following acute ischemic stroke via TLR4/NF-κB/NLRP3 signaling

J Neuroinflammation. 2025 Jun 25;22(1):164. doi: 10.1186/s12974-025-03492-6.

Abstract

Background: Acute ischemic stroke is a disastrous disease characterized by damaging blood flow in the brain, leading to acute brain injury. Acute brain ischemia elicits severe inflammation, thus in turn, aggravates neural injury. Interferon-Induced Protein 35 (IFP35), is a 35 kDa protein, a novel type of DAMP that trigger inflammatory responses, exacerbating acute and chronic inflammatory disease. This study aimed to investigate the potential neuroinflammation role of IFP35 in acute ischemic stroke in a mouse model of MCAO.

Methods: C57BL/6 male mice were subjected to middle cerebral artery occlusion (MCAO) to establish an animal model of acute ischemic stroke. Leveraging serum from stroke patients, serum and brain tissue after MCAO mice, IFP35 was released. Immunofluorescence assay was used to investigated the cell sources of IFP35 expression after MCAO. The impact of IFP35 on neuroinflammation and neural injury was assessed by siRNA-mediated cerebral IFP35 knockdown. Behavioral tests, and brain tissues were harvested for histological analysis and biochemical assays. TUNEL assays were used to evaluate neuronal damage. TTC staining was performed to assess infarction volumes. Additionally, using western blotting and immunofluorescence assays, we further assessed the contribution of TLR4/NF-κB/NLRP3 signaling in MCAO mice and BV2 cells.

Results: IFP35 was accumulated in peripheral blood of cerebral ischaemia patients, ischemia mice serum, as well as peri-infarct regions in focal cerebral ischemia mice. Although endothelial cells, microglia, and astrocytes are capable of expressing IFP35, cerebral neural cells seem to express and release more IFP35 compare to other cell types. Knockdown of IFP35 alleviated the production of neuroinflammatory cytokines, decreased neuronal death, and minimized infarct volumes, ultimately leading to improved neurological outcomes. Importantly, IFP35 triggered the activation of NF-κΒ and NLRP3 signaling, exacerbating neuroinflammation and brain injury by binding its receptor TLR4.

Conclusions: This study revealed IFP35 as a novel DAMP released during cerebral ischemia that promotes neuroinflammation and injury, expanding the current understanding of inflammatory networks following stroke.

Keywords: Acute ischemic stroke; Damage-associated molecular patterns (DAMPs); Interferon-Induced protein 35 (IFP35); Neuroinflammation.

MeSH terms

  • Alarmins* / metabolism
  • Animals
  • Humans
  • Infarction, Middle Cerebral Artery / metabolism
  • Infarction, Middle Cerebral Artery / pathology
  • Ischemic Stroke* / metabolism
  • Ischemic Stroke* / pathology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • NF-kappa B* / metabolism
  • NLR Family, Pyrin Domain-Containing 3 Protein* / metabolism
  • Neuroinflammatory Diseases* / etiology
  • Neuroinflammatory Diseases* / metabolism
  • Neuroinflammatory Diseases* / pathology
  • Signal Transduction / physiology
  • Toll-Like Receptor 4* / metabolism

Substances

  • Toll-Like Receptor 4
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • NF-kappa B
  • Tlr4 protein, mouse
  • Nlrp3 protein, mouse
  • Alarmins