Embryonal erythropoiesis and aging exploit ferroptosis

Abstract

Ferroptosis is a form of regulated cell necrosis, as a consequence of Fe(II)-dependent lipid peroxidation. Although ferroptosis has been linked to cancer cell death, neurodegeneration and reperfusion injury, physiological roles of ferroptosis have not been elucidated to date mostly due to the lack of appropriate methodologies. Here, we show that 4-hydroxy-2-nonenal (HNE)-modified proteins detected by a HNEJ-1 mouse monoclonal antibody is a robust immunohistochemical technology to locate ferroptosis in tissues in combination with morphological nuclear information, based on various models of ferroptosis, including erastin-induced cysteine-deprivation, conditional Gpx4 knockout and Fe(II)-dependent renal tubular injury, as well as other types of regulated cell death. Specificity of HNEJ-1 with ferroptosis was endorsed by non-selective identification of HNE-modified proteins in an Fe(II)-dependent renal tubular injury model. We further comprehensively searched for signs of ferroptosis in different developmental stages of Fischer-344 rats from E9.5-2.5 years of age. We observed that there was a significant age-dependent increase in ferroptosis in the kidney, spleen, liver, ovary, uterus, cerebellum and bone marrow, which was accompanied by iron accumulation. Not only phagocytic cells but also parenchymal cells were affected. Epidermal ferroptosis in ageing SAMP8 mice was significantly promoted by high-fat or carbohydrate-restricted diets. During embryogenesis of Fischer-344 rats, we found ferroptosis in nucleated erythrocytes at E13.5, which disappeared in enucleated erythrocytes at E18.5. Administration of a ferroptosis inhibitor, liproxstatin-1, significantly delayed erythrocyte enucleation. Therefore, our results demonstrate for the first time the involvement of ferroptosis in physiological processes, such as embryonic erythropoiesis and aging, suggesting the evolutionally acquired mechanism and the inevitable side effects, respectively.

Keywords: 4-Hydroxy-2-nonenal; Aging; Erythropoiesis; Ferroptosis; Iron.

Graphical abstract

Fig. 1

Identification of HNEJ-1 as a ferroptosis-specific antibody among five monoclonal anti-HNE antibodies.(A) Human fibrosarcoma HT1080 cells were treated with erastin (10 μM) in the presence or absence of ferrostatin-1 (Fer-1, 5 μM) for 12 h. Then, they were harvested and fixed with 4% paraformaldehyde (PFA) for cell block. Immunohistochemistry was performed with HNEJ-1∼5, respectively (scale bar = 100 μm). (B) Human fibrosarcoma HT1080 cells were treated with erastin (10 μM) in the presence or absence of Fer-1 (5 μM) or deferoxamine mesylate (DFO, 500 μM) for 12 h. Then, they were fixed with 4% PFA. After blocking, cells were incubated with HNEJ-1 or anti-ACSL4 and finally observed with confocal microscopy (scale bar = 10 μm). (C) Human fibrosarcoma HT1080 cells were treated with RSL3 (0.25 μM) in the presence or absence of Fer-1 (5 μM) or DFO (500 μM) for 3 h. Then, they were fixed with 4% PFA. After blocking, cells were then incubated with HNEJ-1 or anti-ACSL4 antibody and finally observed with confocal microscopy (scale bar = 10 μm). Representative data are shown based on 3 independent experiments and the analysis is shown as means ± SEM (n = 3); ***P < 0.001 vs control (CTRL) unless indicated by bar. Refer to text for details.

Fig. 2

Specificity of HNEJ-1 as a ferroptosis marker using various cell death inducers. (A) Human fibrosarcoma HT1080 cells were treated with staurosporine (STS, 1 μM) for 6 h. Then, they were fixed with 4% PFA. After blocking, cells were then incubated with HNEJ-1 or anti-cleaved caspase 3, and finally observed with confocal microscopy (scale bar = 10 μm). (B) Human fibrosarcoma HT1080 cells were treated with STS (1 μM) for 6 h. Afterwards, they were fixed with 4% PFA. Immunohistochemistry was performed with HNEJ-1, anti-cleaved caspase 3, anti-ACSL4 or anti-PTGS2 antibody (scale bar = 100 μm). (C) HT1080 cells were treated with hydrogen peroxide (500 μM) in the presence or absence of Fer-1 (10 μM) or DFO (500 μM) for 3 h. Subsequently, they were fixed with 4% PFA. Immunohistochemistry was performed with HNEJ-1, anti-ACSL4 or anti-PTGS2 antibody (scale bar = 100 μm). Representative data are shown based on 3 independent experiments and the analysis is shown as means ± SEM (n = 3); ***P < 0.001 vs CTRL unless indicated by bar. Refer to text for details.

Fig. 3

Intracellular targets of HNEJ-1 in ferroptotic cells. (A) Human fibrosarcoma HT1080 cells were treated with erastin (10 μM) for 12 h. Cells were then stained for 30 min/37 °C with MitoTracker™ Deep Red FM. They were then fixed with 4% PFA. After blocking, cells were then incubated with HNEJ-1 for 1 h at room temperature and finally observed with confocal microscopy (1 unit for each axis = 5 μm for 3D rendering; scale bar = 5 μm for orthogonal projection). (B) Human fibrosarcoma HT1080 cells were treated with RSL3 (0.25 μM) for 3 h; or erastin (10 μM) for 12 h. Cells were then washed and fixed with 4% PFA. For the detection of HNE modification on the plasma membrane, cells were treated without permeabilization. After blocking, cells were incubated with HNEJ-1 antibody for 1 h at room temperature and finally observed with confocal microscopy (scale bar = 10 μm). Representative data are shown from 3 experiments and the analysis is shown as means ± SEM (n = 3); ***P < 0.001 vs CTRL unless indicated by bar. Refer to text for details. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

HNEJ-1 as a ferroptosis marker for immunohistochemistry in tissue. (A) Immunohistochemical positivity on Gpx4 conditional knockout kidney in comparison to wild-type kidney of C57BL/6 mouse was evaluated with HNEJ-1, anti-ACSL4 or anti-PTGS2 antibody (scale bar = 100 μm). Representative data are shown based on 3 independent experiments. (B) Rats (Wistar strain, male, 6 weeks of age) were euthanized 3 h after administration of 15 mg iron/kg of Fe-NTA and a portion of the kidney was fixed with 10% neutral buffered formalin for immunohistochemistry. Immunopositivity in the renal proximal tubules 3 h after Fe-NTA administration was assessed with immunohistochemistry using HNEJ-1, anti-ACSL4 and anti-PTGS2 antibody (scale bar = 100 μm).

Fig. 5

HNEJ-1 as a ferroptosis marker for the detection of physiological ferroptosis in kidney, spleen and liver. (A) HE staining, Berlin blue staining and immunostaining with HNEJ-1 for kidney, liver and spleen in different developmental stages of Fischer-344 rats (scale bar = 50 μm). (B) Quantification of iron deposits in kidney, liver and spleen in different developmental stages of Fischer-344 rats. (C) Quantification of HNE-modified proteins in kidney, liver and spleen in different developmental stages of Fischer-344 rats. (D) Serial immunostaining of Berlin blue staining, HNEJ-1 and CD68 in the aged kidney, liver and spleen (scale bar = 50 μm). Representative data are shown based on 3 independent experiments and the analysis is shown as means ± SEM (n = 3–6); *P < 0.05, **P < 0.01, ***P < 0.001 vs new born (NB). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

HNEJ-1 as a ferroptosis marker for the detection of physiological ferroptosis in ovary and uterus. (A) HE staining, Berlin blue staining and immunostaining with HNEJ-1 for ovary and uterus in different developmental stages of Fischer-344 rats (scale bar = 50 μm). (B) Quantification of iron deposits in ovary and uterus in different developmental stages of Fischer-344 rats. (C) Quantification of HNE-modified proteins in ovary and uterus in different developmental stages of Fischer-344 rats. (D) Serial immunostaining of Berlin blue staining, HNEJ-1 and CD68 in the aged ovary and uterus (scale bar = 50 μm). Representative data are shown based on 3 independent experiments and the analysis is shown as means ± SEM (n = 3–6); *P < 0.05, **P < 0.01, ***P < 0.001 vs 3-4 weeks. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

HNE modification in skin senescence. (A) Masson's trichrome staining of CTRL group (SAMP8 mice; normal diet); HF group (SAMP8 mice; high-fat diet), and CHR group (SAMP8 mice; carbohydrate-restricted diet; scale bar = 100 μm). (B) Immunostaining and quantification with 8-OHdG for CTRL group, HF group and CHR group (scale bar = 100 μm). (C) Immunostaining and quantification with HNEJ-1 for CTRL group, HF group and CHR group (scale bar = 100 μm). Representative data are shown from 3 independent experiments and the analysis is shown as means ± SEM (n = 6); *P < 0.05, **P < 0.01, ***P < 0.001 vs CTRL group. Refer to text for details.

Fig. 8

Ferroptotic events are involved in embryonic hematopoiesis. (A) HNE modification in extra-embryonic endodermal component of visceral yolk sac at the stage of E9.5 (n = 5; scale bar = 100 μm). (B) HNE modification in trophoblast giant cells at the stage of E9.5 (n = 5; scale bar = 50 μm). (C) Rat embryos (Fischer-344) in different stages (E13.5, E15.5 and E18.5) were collected and fixed with 10% neutral buffered formalin for HE staining and immunostaining with HNEJ-1. HNE modification of nucleated red blood cells (RBC) was quantified as percentage of positive cells and positive pixel count (A.U.) per cell (n = 4–8; scale bar = 50 μm). (D) Percentage of RBC in embryos from control group and Lipro-1 group (n = 3) were calculated as number of nucleated RBC/total number of RBC from at least 4 embryos per pregnant rat (scale bar = 50 μm). (E) Immunoblot analysis of Lamin B and Transferrin receptor 1 (TfR1) in embryos from control group and Lipro-1 group. Representative data are shown based on 3 independent experiments and the analysis is shown as means ± SEM, ***P < 0.001 vs E13 or CTRL unless indicated by bar. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)