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Microglia and macrophage exhibit attenuated inflammatory response and ferroptosis resistance after RSL3 stimulation
Yu Cui1, Zhaolong Zhang2, Xin Zhou1
1Institute of Neuroregeneration & Neurorehabilitation, Department of Pathophysiology, Qingdao University, Ningxia Road 308, Qingdao, 266071, China.
Background:
Many neurological diseases involve neuroinflammation, during which overproduction of cytokines by immune cells, especially microglia, can aggregate neuronal death. Ferroptosis is a recently discovered cell metabolism-related form of cell death and RSL3 is a well-known inducer of cell ferroptosis. Here, we aimed to investigate the effects of RSL3 in neuroinflammation and sensitivity of different type of microglia and macrophage to ferroptosis.
Methods:
Here, we used quantitative RT-PCR analysis and ELISA analysis to analyze the production of proinflammatory cytokine production of microglia and macrophages after lipopolysaccharides (LPS) stimulation. We used CCK8, LDH, and flow cytometry analysis to evaluate the sensitivity of different microglia and macrophages to RSL3-induced ferroptosis. Western blot was used to test the activation of inflammatory signaling pathway and knockdown efficiency. SiRNA-mediated interference was conducted to knockdown GPX4 or Nrf2 in BV2 microglia. Intraperitoneal injection of LPS was performed to evaluate systemic inflammation and neuroinflammation severity in in vivo conditions.
Results:
We found that ferroptosis inducer RSL3 inhibited lipopolysaccharides (LPS)-induced inflammation of microglia and peritoneal macrophages (PMs) in a cell ferroptosis-independent manner, whereas cell ferroptosis-conditioned medium significantly triggered inflammation of microglia and PMs. Different type of microglia and macrophages showed varied sensitivity to RSL3-induced ferroptosis. Mechanistically, RSL3 induced Nrf2 protein expression to inhibit RNA Polymerase II recruitment to transcription start site of proinflammatory cytokine genes to repress cytokine transcription, and protect cells from ferroptosis. Furthermore, simultaneously injection of RSL3 and Fer-1 ameliorated LPS-induced neuroinflammation in in vivo conditions.
Conclusions:
These data revealed the proinflammatory role of ferroptosis in microglia and macrophages, identified RSL3 as a novel inhibitor of LPS-induced inflammation, and uncovered the molecular regulation of microglia and macrophage sensitivity to ferroptosis. Thus, targeting ferroptosis in diseases by using RSL3 should consider both the pro-ferroptosis effect and the anti-inflammation effect to achieve optimal outcome.
Insights
Ferroptosis inducer RSL3 inhibits neuroinflammation by repressing cytokine production, independent of cell death. Targeting ferroptosis with RSL3 offers dual benefits for neurological diseases, balancing cell death and anti-inflammatory effects.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neuroinflammation, characterized by cytokine overproduction from microglia, contributes to neuronal death in neurological diseases.
- Ferroptosis is a distinct cell death pathway regulated by cellular metabolism.
- RSL3 is a known inducer of ferroptosis, prompting investigation into its role in neuroinflammation.
Purpose of the Study:
- To investigate the effects of ferroptosis inducer RSL3 on neuroinflammation.
- To assess the sensitivity of different microglia and macrophage types to RSL3-induced ferroptosis.
- To elucidate the molecular mechanisms underlying RSL3's effects on inflammation and ferroptosis.
Main Methods:
- Quantitative RT-PCR and ELISA were used to measure cytokine production in microglia and macrophages post-lipopolysaccharide (LPS) stimulation.
- Cell viability assays (CCK8, LDH) and flow cytometry evaluated sensitivity to RSL3-induced ferroptosis.
- Western blot assessed inflammatory signaling pathways, and siRNA targeted GPX4 or Nrf2 in BV2 microglia; in vivo studies used LPS injection.
Main Results:
- RSL3 inhibited LPS-induced inflammation in microglia and macrophages independently of ferroptosis induction; conversely, ferroptosis-conditioned medium exacerbated inflammation.
- Varied sensitivities to RSL3-induced ferroptosis were observed across different microglia and macrophage types.
- RSL3 upregulated Nrf2, which inhibited RNA Polymerase II recruitment to cytokine gene promoters, thereby repressing transcription and protecting cells.
Conclusions:
- Ferroptosis plays a pro-inflammatory role in microglia and macrophages.
- RSL3 acts as a novel inhibitor of LPS-induced inflammation, with potential therapeutic applications in neurological diseases.
- Understanding the dual pro-ferroptosis and anti-inflammatory effects of RSL3 is crucial for optimizing its use in treating diseases.
