Microglia and macrophage exhibit attenuated inflammatory response and ferroptosis resistance after RSL3 stimulation via increasing Nrf2 expression

Abstract

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.

Keywords: Ferroptosis; LPS; Macrophage; Microglia; Neuroinflammation; RSL3.

Fig. 1

RSL3 inhibits LPS-induced proinflammatory cytokine production of microglia and PMs. a qRT-PCR analysis of TNF, IL-6, and IL-1b mRNA levels in LPS (100 ng/ML)-stimulated PMs subjected to different doses of RSL3 treatment for 4 h. b ELISA of TNFα, IL-6, and IL-1β in supernatants of LPS-stimulated PMs subjected to different doses of RSL3 treatment for 8 h. c qRT-PCR analysis of TNF, IL-6, and IL-1b mRNA levels in LPS (100 ng/ML)-stimulated BV2 cells subjected to different doses of RSL3 treatment for 4 h. d ELISA of TNFα, IL-6, and IL-1β in supernatants of LPS-stimulated BV2 cells subjected to different doses of RSL3 treatment for 8 h. The data are means ± SD; for all panels, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way-ANOVA analysis followed by Dunnett test. n.s., no significant. The data are combined from three independent experiments

Fig. 2

Cell ferroptosis-conditioned medium induces proinflammatory cytokine production. a Flow cytometry analysis of C11-BODIPY oxidation in PC12 cells treated with RSL3 (1 μM) or RSL3 plus Fer (1 μM) for 10 h. b Cell viability or c LDH release was assessed and quantified for PC12 cells subjected to RSL3 (1 μM) treatment for 10 h. qRT-PCR analysis of TNF, IL-6, and IL-1b mRNA levels in primary microglia (d) or PMs (e) incubated with DMSO-treated cell culture supernatant or RSL3-treated cell culture supernatant for 8 h from PC12 cells. The data are means ± SD; for all panels, *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test. The data are combined from three independent experiments unless otherwise indicated

Fig. 3

The sensitivity of microglia and macrophages to RSL3-induced ferroptosis. a Cell viability of primary microglia, BV2 cells, PMs, and PC12 cells subjected to different doses of RSL3 treatment for 10 h. b LDH release assay of microglia, BV2 cells, PMs, and PC12 cells subjected to different doses of RSL3 treatment for 10 h. c Flow cytometry analysis of C11-BODIPY oxidation in BV2 cells treated with different doses of RSL3 for 9 h. d Flow cytometry analysis of C11-BODIPY oxidation in PMs treated with different doses of RSL3 for 10 h. e Flow cytometry analysis of C11-BODIPY oxidation in BV2 cells treated with RSL3 (1 μM) or RSL3 plus Fer-1 (1 μM) for 10 h. The data are means ± SD. All data are representative of or combined from at least three independent experiments

Fig. 4

RSL3 inhibits LPS-induced inflammation in a ferroptosis-independent manner. a The qRT-PCR analysis of IL-6 and IL-1b mRNA levels in LPS-stimulated microglia subjected to RSL3 (500 nM), RSL3 plus Fer-1 (1 μM) treatment for 4 h. b ELISA of IL-6 and IL-1β in supernatants of LPS (100 ng/ML) stimulated microglia subjected to RSL3, RSL3 plus Fer treatment for 8 h. c The qRT-PCR analysis of IL-6 and IL-1b mRNA levels in LPS-stimulated PMs subjected to RSL3, RSL3 plus Fer treatment for 4 h. d ELISA of IL-6 and IL-1β in supernatants of PMs subjected to RSL3, RSL3 plus Fer treatment for 8 h loading control. e Cell viability of primary microglia subjected to RSL3 (1 μM), RSL3 plus Fer treatment in the presence of LPS for 10 h. f Representative immunoblot analysis of GPX4 in BV2 microglia transfected with siNC or siGPX4 for 48 h. g The qRT-PCR analysis of TNF, IL-6, and IL-1b mRNA levels in LPS-stimulated BV2 microglia transfected with siNC or siGPX4 for 48 h and stimulated with LPS for 4 h. The data are means ± SD, for all panels: *P < 0.05, **P < 0.01, ***P < 0.001. n.s., no significant. a–e One-way-ANOVA analysis was performed. g Two-way-ANOVA analysis followed by Bonferroni post hoc test was performed. All data are representative of or combined from at least three independent experiments

Fig. 5

RSL3 inhibits cytokine gene transcription. a The qRT-PCR analysis of IL6 and IL-1b mRNA levels in RSL3 or vehicle-treated BV2 microglia subjected to ActD (1 μg/ml) treatment for the indicated times after LPS pre-treated for 2 h. b Representative immunoblot analysis of the phosphorylated (p-) or total proteins in lysates of BV2 microglia subjected to RSL3 or DMSO in the presence of LPS (100 ng/ML) for indicated times. ChIP-qRT-PCR analysis of P65 (c) and RNA Pol II (d) binding in IL-6 and IL-1b loci in BV2 microglia lysates. BV2 microglia were stimulated with LPS (100 ng/ML) or LPS plus RSL3 (500 nM) for 4 h and CHIP assay was then performed. pro, promoter; TSS, transcription start site. The data are means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way-ANOVA analysis followed by Turkey test. n.s., no significant. All data are representative of or combined from at least three independent experiments

Fig. 6

Knockdown of Nrf2 reverses ferroptosis resistance and inflammation suppression in response to RSL3 stimulation. Representative immunoblot analysis of Nrf2 in lysates of BV2 microglia (a) or PMs (b) subjected to RSL3 (500 nM) or DMSO treatment in the presence of LPS (100 ng/ML) for indicated times. Cell viability of BV2 microglia (c) or PMs (d) was assessed and quantified transfected with siNC or siNrf2 subjected to different doses of RSL3 treatment for 10 h. The qRT-PCR analysis of IL-6 and IL-1b in BV2 microglia (e) or PMs (f) transfected with siNC or siNrf2 in the presence or absence of RSL3. One-way-ANOVA analysis was used. The data are means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way-ANOVA analysis followed by Turkey test. n.s., no significant. All data are representative of or combined from at least three independent experiments

Fig. 7

RSL3 in combination with Fer-1 mitigates LPS-induced inflammation. a Survival of C57BL/6 mice (injected with RSL3 plus Fer-1 or vehicle; n = 9 for each treatment) after lethal challenge with LPS (10 mg/kg body weight). b ELISA of TNFα, IL-6, and IL-1β in serum from C57BL/6 mice (injected with RSL3 plus Fer-1 or vehicle; n = 5 per group) after challenging with PBS or LPS for 6 h. c Hematoxylin and eosin staining of lungs from mice (injected with RSL3 plus Fer-1 or vehicle; n = 5 per group) for different treatment after challenging with PBS or LPS. Scale bar, 50 μm. d The number of microglia (CD45^intCD11b^high) in brain tissues after indicated treatment (LPS, 5 mg/kg body weight) based on flow cytometry analysis. e The qRT-PCR analysis of TNF, IL-6, and IL-1b in brain tissues under different treatment (LPS, 5mg/kg body weight). The data are means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way-ANOVA analysis followed by Turkey test. n.s., no significant. All data are representative of or combined from at least three independent experiments