
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00281-7
10.1016/j.redox.2024.103303
103303
Research Paper
Vitamin E and GPX4 cooperatively protect treg cells from ferroptosis and alleviate intestinal inflammatory damage in necrotizing enterocolitis
Luo Shunchang ab1
Zeng Yingying cde1
Chen Baozhu ab1
Yan Junjie de
Ma Fei f
Zhuang Guiying g
Hao Hu ab
Cao Guangchao gccao2016@jnu.edu.cn
de⁎
Xiao Xin xiaoxin2@mail.sysu.edu.cn
ab⁎⁎
Li Sitao lisit@mail.sysu.edu.cn
abh⁎⁎⁎
a Department of Pediatrics, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510655, China
b Biomedical Innovation Center, The Sixth Affiliated Hospital, Sun Yat-sen University, 510655, China
c Department of Laboratory Medicine, Nanfang Hospital Baiyun Branch, Southern Medical University, Guangzhou, 510420, China
d State Key Laboratory of Bioactive Molecules and Druggability Assessment, The Biomedical Translational Research Institute, Health Science Center (School of Medicine), Jinan University, Guangzhou, 510632, China
e Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou, 510632, China
f Maternal & Child Health Research Institute, Zhuhai Center for Maternal and Child Health Care, Zhuhai, 519001, China
g The Maternal and Children Health Care Hospital (Huzhong Hospital) of Huadu, Guangzhou, 510800, China
h Department of Pediatrics, Xinyi People's Hospital, Maoming, 525300, China
⁎ Corresponding author. Jinan University, No. 601, Huangpu Avenue West, Guangzhou, 510632, China. gccao2016@jnu.edu.cn
⁎⁎ Corresponding author. The Sixth Affiliated Hospital, Sun Yat-sen University, No. 26, Yuancun Erheng Road, Guangzhou, 510655, China. xiaoxin2@mail.sysu.edu.cn
⁎⁎⁎ Corresponding author. The Sixth Affiliated Hospital, Sun Yat-sen University, No. 26, Yuancun Erheng Road, Guangzhou, 510655, China. lisit@mail.sysu.edu.cn
1 These authors contributed equally to this work.

08 8 2024
9 2024
08 8 2024
75 10330323 7 2024
5 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Background

The notable decline in the number of Tregs within Necrotizing enterocolitis (NEC) intestinal tissues，contribute to excessive inflammation and necrosis, yet the precise underlying factors remain enigmatic. Ferroptosis, a novel cell death stemming from a disrupted lipid redox metabolism, is the focus of this investigation. Specifically, this study delves into the ferroptosis of Treg cells in the context of NEC and observes the protective effects exerted by vitamin E intervention, which aims to mitigate ferroptosis of Treg cells.

Methods

To investigate the reduction of Treg cells in NEC intestine, we analyzed its association with ferroptosis from multiple angles. We constructed a mouse with a specific knockout of Gpx4 in Treg cells, aiming to examine the impact of Treg cell ferroptosis on NEC intestinal injury and localized inflammation. Ultimately, we employed vitamin E treatment to mitigate ferroptosis in NEC intestine's Treg cells, monitoring the subsequent amelioration in intestinal inflammatory damage.

Results

The diminution of Treg cells in NEC is attributed to ferroptosis stemming from diminished GPX4 expression. Gpx4-deficient Treg cells exhibit impaired immunosuppressive function and are susceptible to ferroptosis. This ferroptosis of Treg cells exacerbates intestinal damage and inflammatory response in NEC. Notably, Vitamin E can inhibit the ferroptosis of Treg cells, subsequently alleviating intestinal damage and inflammation in NEC. Additionally, Vitamin E bolsters the anti-lipid peroxidation capability of Treg cells by upregulating the expression of GPX4.

Conclusion

In the context of NEC, the ferroptosis of Treg cells represents a significant factor contributing to intestinal tissue damage and an exaggerated inflammatory response. GPX4 is pivotal for the viability and functionality of Treg cells. Vitamin E exhibits the capability to mitigate the ferroptosis of Treg cells, thereby enhancing their number and function, which plays a crucial role in mitigating intestinal tissue damage and inflammatory response in NEC.

Keywords

Necrotizing enterocolitis
Treg
GPX4
Vitamin E
==== Body
pmc Abbreviations

NEC	Necrotizing enterocolitis	
GPX4	The enzyme glutathione peroxidase 4	
ROS	Reactive oxygen species	
Vit E	Vitamin E	
H&E	Hematoxylin and eosin	
PBS	Phosphate-buffered saline	
HRP	Horseradish peroxidase	
qRT-PCR	Real-time quantitative reverse transcription polymerase chain reaction	
z-VAD	z-VAD-FMK	
Nec-1	Necrostatin-1	
Fer-1	Ferrostatin-1	
α-Toc	α-Tocopherol	
CTV	Cell Trace Violet	
ACSL4	Acyl-CoA synthetase long chain family member 4	
MDA	malondialdehyde	

1 Introduction

During development in the womb, the fetus exist in a state of relative hypoxia [1]. As the fetus develops, its antioxidant defense system undergoes continual enhancement to adjust to the abrupt fluctuations in oxygen concentration following birth. Prior to birth, the fetus's antioxidant defense system reaches its peak readiness to handle the swift alterations in oxygen levels postnatally [2]. However, premature infants, born earlier, possess an immature antioxidant defense system and are more exposed to oxygen free radicals, rendering them highly susceptible to oxidative damage [3]. One such condition resulting from oxidative damage is necrotizing enterocolitis (NEC). NEC has become a significant obstacle in global neonatal medicine. Notably, this condition is prevalent among preterm infants and those with low birth weight, posing a grave risk to their health and survival [4]. NEC is a rapidly advancing disease, necessitating emergency surgical intervention in approximately 50 % of affected infants [5]. Despite surgical treatment, however, mortality rates remain high, with up to 20 %–30 % of children succumbing to the disease [6]. Survivors may also endure long-term health complications, including neurodevelopmental delays and intestinal abnormalities [7,8]. Prematurity, intestinal immaturity, compromised intestinal mucosal barrier, hypoxia-ischemia, and intestinal dysbiosis are recognized as crucial factors in the development of NEC [9]. The confluence of these risk factors triggers an excessive inflammatory response in the NEC intestine, ultimately leading to intestinal avascular necrosis and systemic manifestations.

Ferroptosis represents a distinct form of programmed cell death, differing from apoptosis and necrosis. This process is primarily triggered by an imbalance in the generation and degradation of lipid reactive oxygen species (ROS), stemming from intracellular lipid peroxidation [10]. Ferroptosis is considered a significant contributor to oxidative damage in the intestines of humans and mice, ultimately leading to an excessive inflammatory response [11]. Specifically, inflammatory factors stimulate intestinal epithelial cells, causing an elevation in intracellular iron levels, thereby promoting the accumulation of lipid peroxides and ultimately culminating in ferroptosis [12]. The occurrence of ferroptosis further intensifies intestinal inflammation, thus creating a vicious cycle.

Treg cells occupy a pivotal position in immunomodulation for gut-related diseases, effectively maintaining intestinal immune homeostasis through the inhibition of excessive immune response activation [13]. In the context of NEC, Treg cells are instrumental in its occurrence and progression. Notably, investigations have revealed a significant decrease in the count of Treg cells within the intestinal tissue of NEC-affected children. This reduction in Treg cell population gives rise to an imbalance in immune regulation, thereby hindering the effective suppression of inflammatory response [14]. Further research indicates that Treg cells in NEC patients may exhibit a diminished capacity to suppress inflammatory response and uphold intestinal barrier function, which can exacerbate intestinal inflammation [15]. Unlike effector immune cells, Treg cells heavily rely on lipid β oxidation for energy metabolism [16], a process that is prone to generating high levels of oxygen radicals. In the presence of iron ions, these oxygen radicals oxidize the functional lipid molecules within or on the cell membrane, ultimately leading to ferroptosis [17]. The metabolic preference of Treg cells inherently predisposes them to ferroptosis.

Glutathione peroxidase 4 (GPX4) serves as a pivotal regulatory enzyme in ferroptosis, effectively eliminating excessive lipid peroxidation products and suppressing ferroptosis [18]. Extant research has revealed that inhibiting ferroptosis can ameliorate acute inflammatory damage in the intestine, a finding that has been corroborated in Inflammatory bowel disease [19,20]. By inhibiting ferroptosis in intestinal tissue cells, GPX4 mitigates the acute inflammatory response in the intestine, thereby exerting a protective function [21]. Vitamin E (Vit E), a fat-soluble antioxidant, is essential in maintaining human health. Its distinctive chemical structure enables it to efficiently scavenge free radicals in the body, thereby preventing lipid peroxidation and ferroptosis [22]. Furthermore, pertinent studies have demonstrated that GPX4 can inhibit ferroptosis in T cells, thus preserving their immune functionality [23].

Here, we discovered a significant correlation between the diminished presence of Treg cells in NEC intestinal tissue and ferroptosis, which stemmed from reduced GPX4 expression. Our thorough investigations revealed that Treg cells devoid of Gpx4 in NEC are highly susceptible to ferroptosis, ultimately leading to intestinal damage and exacerbated inflammation. Furthermore, our experiments highlighted vitamin E as a crucial determinant in dictating the fate of Treg cells. In a NEC mouse model where Gpx4 was specifically knocked out in Treg cells, we observed an increase in the number of Treg cells treated with vitamin E, accompanied by a marked reduction in intestinal injury and inflammation associated with NEC. Collectively, our findings indicate that GPX4 synergizes with vitamin E to maintain lipid redox equilibrium, thus preventing ferroptosis of Treg cells and ultimately ameliorating intestinal damage and inflammation in NEC.

2 Material and methods

2.1 Mice and experimental design of NEC

The Gpx4tm1.1Qra/J (Gpx4flox/flox, strain#027964), B6.129(Cg)-Foxp3tm4(YFP/icre)Ayr/J (Foxp3YFP−Cre, strain #016959), and B6. SJL-Ptprca Pepcb/BoyJ (CD45.1, strain #002014) mouse strains were procured from the Jackson Laboratory and housed in SPF conditions in the Laboratory Animal Center of Jinan University. The Gpx4flox/flox and Foxp3YFP−Cre strains were interbred to create Tregs-specific Gpx4-deficient Foxp3YFP−creGpx4f/f (KO) mice, while age and sex-matched Foxp3YFP−Cre mice (WT) served as controls. The newborn mice, aged 6–8 days, were employed for subsequent NEC modeling and associated experiments.

The NEC model was developed based on a previously established methodology, with minor modifications, in 7-day-old mouse pups [24]. These pups were nourished five times daily with a tailored formula [ 2:1 mixture of Similac Advance infant formula (Abbott Nutrition, Columbus, USA) and Esbilac (PetAg, Hampshire, USA) milk replacer for puppies], which incorporated enteric bacteria isolated from a surgical NEC patient (12.5 μl of original stool slurry per 1 ml of formula). Additionally, these mouse pups were subjected to twice-daily exposures to low oxygen conditions (5 % O2) and low temperatures (4 °C), each lasting 10 min, for a total of 4 days.

2.2 Human tissue

We have selected NEC patients, with a gestation age of less than 37 weeks at onset, to constitute the experimental group, while children with full-term congenital intestinal atresia serve as the control group. The human tissue of NEC and congenital intestinal atresia were obtained from the Sixth Affiliated Hospital, Sun Yat-sen University. The relevantcharacteristics of the 10 participants are summarized in Supplementary Table 1. The study was approved by the InstitutionalEthics Committee at the Sixth Affiliated Hospital, Sun Yat-sen University. Informed consent was obtained from all the participants.

2.3 Intestine collection and damage assessment

We initially conducted a precise dissection of the mouse abdomen, and then delicately excised the small intestinal tissue. Following that, we conducted a thorough initial evaluation of the small intestine and promptly excised the terminal 5 cm segment of the ileum. Subsequently, the terminal 0.5 cm segment of each sample was stabilized in 10 % paraformaldehyde and underwent standard hematoxylin and eosin (H&E) staining. To uphold the objectivity and precision of our findings, we enlisted the expertise of two independent pathologists, unaware of the HE staining outcomes, to evaluate the samples. They assessed the severity of mucosal damage on a scale ranging from 0 to 4, adhering to established scoring criteria [25]. According to this evaluation criterion, tissues with a histological score of 2 or above are deemed to display the hallmarks of NEC.

2.4 Immunofluorescence colocalization assays

The paraffin sections, once prepared, undergo crucial steps such as deparaffinization, gradient alcohol dehydration, and ultrapure water immersion to guarantee the stability of their structural integrity. Subsequently, the sections were treated with sodium citrate remediation solution under high pressure and, once cooled to room temperature, rinsed thoroughly with PBS solution. Blocking was then achieved by incubating the sections with 3 %–5 % BSA for 60 min at room temperature. Following this, the samples were incubated with the corresponding primary antibody at 37 °C for 30 min. Subsequently, the samples underwent three washes with PBST solution and were incubated with suitable secondary antibodies for 1 h at 37 °C. The staining process concluded with a 5-min incubation with DAPI at room temperature. Finally, a laser scanning confocal fluorescence microscope was employed to photograph and document the processed sections for further analysis.

2.5 Preparation of lamina propria lymphocytes

The extraction method for cells from the lamina propria of the small intestine was derived from prior literature and underwent suitable modifications [24]. In detail, the prepared intestinal tissue was immersed in a tissue dissociation solution composed of 5 mM EDTA and 1 mM DTT. This solution was then agitated in a 37 °C shaker for 20–30 min to effectively disintegrate the intestinal epithelial tissue. Following this, the dissociated tissue was transferred to a digestion solution containing specific enzymes (including 0.02 mg/ml DNase I, 1 mg/ml collagenase IV, and 2 % fetal bovine serum) and kept in continuous agitation at 37 °C for an additional 30 min to further enhance digestion. Ultimately, the cells were isolated and refined through density gradient centrifugation, utilizing varying concentrations of Percoll solution.

2.6 Preparation of spleen lymphocytes

Extract the spleen from the mouse's peritoneal cavity and transfer it into pre-cooled phosphate-buffered saline (PBS). Subsequently, gently homogenize the spleen to create a single-cell suspension. Following this, filter the cell suspension through a cell strainer into a centrifuge tube. After centrifuging, discard the supernatant to obtain the desired cell preparation. Resuspend the spleen pellet in erythrocyte lysate and incubate for 5 min at room temperature to achieve lysis, followed by the addition of PBS to halt the lysis process. Centrifuge the mixture, discard the supernatant, and then resuspend the pellet in pre-chilled PBS for further experimentation.

2.7 Western blotting

Ileal tissue or cell samples are incorporated into RIPA lysate, specifically formulated with 1 mM protease and phosphatase inhibitors. Subsequently, the tissue sample undergoes thorough homogenization in a dedicated homogenizer. The homogenized sample is then placed on ice and allowed to lyse for 30 min. Next, a precast 4–15 % polyacrylamide gel is employed to accurately separate the total lysate via electrophoresis. Once the separation is complete, the sample on the gel is transferred to a polyvinylidene fluoride membrane. These membranes are incubated for an extended period at a cryogenic temperature of 4 °C with primary antibodies targeting β-actin (Proteintech, Cat No.:66009-1-Ig) and GPX4 (Proteintech, Cat No.:67,763–1). To further enhance the signal intensity, the membrane is incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Finally, detection is carried out using an enhanced chemiluminescent substrate, and upon completion of the assay, the western blot images are analyzed in detail utilizing Bio-Rad's Image Lab software.

2.8 Real-time quantitative reverse transcription polymerase chain reaction（qRT-PCR）

Total RNA was extracted from ileal tissue utilizing the TRIzol Reagent, and the gDNA was successfully eliminated. Subsequently, the reverse transcription reaction was executed, followed by inactivation of the reverse transcriptase via the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Cat No.:RR047A). The PCR reaction solution was formulated using the SYBR Premix Ex Taq kit (Takara, Cat No.:RR820A), encompassing SYBR Premix Ex Taq, primers, DNA template, and DDH2O. This solution was then inserted into a PCR instrument, where pre-denaturation, PCR cycling, and dissolution curve analysis were conducted in accordance with predefined parameters. The expression disparities between the experimental and control groups were compared (Supplementary Tables 2–3). As referenced in prior research [26], the relative expression level of genes was determined using the 2-△△Ct method, and each gene in each sample was measured thrice, with the average value utilized for statistical analysis.

2.9 Flow cytometry

For intracellular cytokine staining, cells underwent stimulation with PMA and ionomycin for 4–6 h at 37 °C, in the presence of monensin, supplemented with BD Golgi Stop™ protein transport inhibitors. Subsequently, the cells were pretreated with an Fc blocking CD16/CD32 antibody (Invitrogen, Cat No.:14-0161-81) to mitigate non-specific binding. To detect cell surface molecules, we cautiously selected specific surface molecular antibodies, staining them at a 1:200 concentration for 20–30 min at 4 °C. Following surface marker staining, the cells were fixed and permeabilized using Cytofix/Cytoperm (Biosciences, Cat No.:554,714). Subsequently, the cells were stained with appropriate intracellular and intranuclear antibodies at 4 °C, adhering to the manufacturer's instructions. To assess the lipid peroxidation status of cells, we utilized three methods. Firstly, lymphocytes were incubated with BODIPY 581/591C11 (2 μM, invitrogen, Cat No.:D3861) at 37 °C for 30 min. Secondly, malondialdehyde antibody (1:100, abcam, Cat No.:ab27615) was used for staining at 4 °C for 1 h. Thirdly, lymphocytes were incubated with Cell ROX Oxidative Stress Reagents (2 μM, invitrogen, Cat No.:C10422) at 37 °C for 30 min. For GPX4 staining, cells were fixed and permeabilized using Cytofix/Cytoperm, followed by staining with anti-Glutathione Peroxidase 4 antibody (abcam, Cat No.:ab125066) at 4 °C for 1 h. The AlexaFluor 488 secondary antibody (absin, abs20025) was then added for an additional 1-h staining period. After this, surface antibody staining was performed as previously described. Post-staining, fluorescence data was collected using Cytek Aurora/NL and analyzed utilizing Flow Jo software.

2.10 In vitro activation of T cells and apoptosis detection

CD4+ T cells were sorted from spleen cells of Foxp3Cre-YFP or Foxp3Cre-YFP Gpx4fl/fl mice by the Total CD4+ T cell kit (STEMCELL, Cat No.:19,852). CD4+ T cells were diluted to 1 × 106 cells/ml containing anti-CD3ε (10 μg/ml) , anti -CD28 (1 μg/ml), 10 % FBS and fresh RPMI-1640 medium. Furthermore, the inclusion of inhibitors such as z-VAD-FMK (z-VAD, 20 μM, Selleck, Cat No.:S7023), Necrostatin-1 (Nec-1, 20 μM, Selleck, Cat No.:S8037), Ferrostatin-1 (Fer-1, 20 μM, Selleck, Cat No.:S7243), and α-Tocopherol (α-Toc, 20 μM, Selleck, Cat No.:E0191) was executed. The cells were subjected to stimulation durations of 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, and the respective indices of lipid peroxidation in Treg cells were then stained utilizing the aforementioned flow staining method. Additionally, apoptosis assays were conducted using the Annexin V-APC/7-AAD Apoptosis Kit (MultiSciences, Cat No.:70-AT105-100), followed by analysis through flow cytometry.

2.11 In vivo activation of T cells

Foxp3Cre-YFP and Foxp3Cre-YFP Gpx4fl/fl mice were cohoused for a week to facilitate environmental adaptation and physiological stability. Following this, α-CD3ε (1 μg) was administered to induce the desired immune response. Concurrently, in varying groups, Fer-1 (0.8 mg/kg) and α-Toc (0.5 mg/kg) were administered via tail vein injection. After a 24-h period, the mouse spleens were excised and processed into a single-cell suspension. These cells were subsequently stained using a pre-established protocol and analyzed using flow cytometry to assess the activity and extent of lipid peroxidation in Treg cells.

2.12 Treg cell inhibition assay in vitro

Treg cells from Foxp3Cre-YFP and Foxp3Cre-YFP Gpx4fl/fl mice were sorted by a BD FACS Aria flow sorter. Meanwhile, naive CD4+ T cells were sorted from CD45.1 mice using the Naive CD4+ T cells sorting kit (STEMCELL, Cat No.:19,765). The naive CD4+ T cells were labeled with Cell Trace Violet (CTV, (Invitrogen, Cat No.:C34571) according to the reagent instructions. Medium containing CD3/CD28 beads and IL-2 was then mixed and diluted in a ratio of Treg to Naive CD4+ T cells at a ratio of 0:1, 1:1, 1:2, 1:4, 1:8, and 1:16. After 72 h, the CTV fluorescence intensity of CD45.1+CD4+ T cells was detected by Cytek Aurora/NL flow cytometer to assess cell proliferation.

2.13 Statistical analysis

We present all data in the format of mean ± standard error (s.e.m.). To determine the P-value, we primarily utilize statistical methods such as one-way ANOVA, multi-way ANOVA, two-tailed unpaired t-test, and two-tailed paired t-test, all executed through the GraphPad Prism software, version 9.0. In the event of any specific calculation requirements, we adhere to the prescribed instructions accordingly. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) indicate statistically significant changes. NS, not significant.

3 Results

3.1 GPX4 reduction leads to ferroptosis of treg cells in NEC

To investigate the significance of ferroptosis in NEC, we employed electron microscopy to scrutinize the intestinal tissues of NEC patients (Supplementary Table 1) and mouse models of NEC. The findings revealed prominent alterations in mitochondria within the intestinal tissue of NEC, characterized by mitochondrial condensation, heterogeneous size distribution, reduced cristae, and cristae breakage (Fig. 1A). These alterations are quintessential indicators of ferroptosis. Furthermore, the transcriptional level of GPX4 in NEC intestinal tissue was observed to be downregulated, whereas the transcriptional level of Acyl-CoA synthetase long chain family member 4 (ACSL4) was upregulated (Fig. 1B), further validating the presence of ferroptosis in NEC intestinal tissue. Treg cells play a pivotal role in upholding immune balance and forestalling excessive inflammatory reactions. It was noted that the proportion and count of Treg cells in NEC were diminished (Fig. 1C–D). To elucidate whether the decline in Treg cells is associated with ferroptosis, we conducted immunofluorescence co-localization experiments in the intestinal tissues of NEC patients. Our results revealed that the decreased expression of Foxp3 in NEC intestinal tissues concurred with a reduction in GPX4 expression (Fig. 1E). Additionally, we measured the lipid peroxidation levels in Treg cells, demonstrating an elevation in Bodipy C11 levels in NEC intestinal tissue (Fig. 1F). Similarly, Malondialdehyde (MDA) levels in Treg cells were also augmented in NEC intestinal tissue (Fig. 1G). These suggest that the depletion of Treg cells in NEC intestine is associated with ferroptosis, likely stemming from the decrease in GPX4.Fig. 1 A decrease in intestinal Treg cells in NEC is associated with ferroptosis due to decreased GPX4.

A Transmission electron microscopy (TEM) images, featuring a scale of 1 μm, offer insights into the intestinal tract of NEC patients and NEC mice. The red arrow highlights the mitochondria, providing a precise visual representation of their location and structure. B Intestinal tissues from NEC patients (n = 5) and NEC mice (n = 8) were examined for the transcription levels of ferroptosis-related regulatory proteins. C-D Flow cytometry and statistical diagram of intestinal lamina proprina Treg cells in the mouse model of NEC and the control group. E Flow cytometry detection and statistical histogram of lipid peroxidation level (Bodipy C11) of Treg cells in the intestinal lamina propria of NEC mice (n = 5). F Flow cytometry detection and statistical histogram of malondialdehyde (MDA) expression level of Treg cells in the intestinal lamina propria of NEC mice (n = 5). G Expression and colocalization of GPX4, a key regulatory protein of ferroptosis on Treg in the intestinal lamina propria of NEC patients, and the colocalization of GPX4 and Foxp3 in situ indicated by the red arrow (the scale is 2 μm). p < 0.05 (*),p < 0.001 (***). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1

3.2 Gpx4 -deficient treg cells are susceptible to ferroptosis upon activation in vitro or in vivo

To further explore the effect of Gpx4 deletion on Treg cells, we successfully constructed genetically engineered mice with specific Gpx4 knockout on Treg cells (Supplementary Fig. 1). Under normal physiological conditions, the immune homeostasis of the spleen remains unaffected (Supplementary Fig. 2). CD4+ T cells isolated from the spleens of Foxp3Cre-YFP (WT) and Foxp3Cre-YFP Gpx4fl/fl (KO) mice were stimulated with anti-CD3ε and anti-CD28 antibodies, enabling us to observe the survival of Treg cells at various time points. As depicted in Supplementary Fig. 3, a gradual increase in the mortality rate of Gpx4-deficient Treg cells was observed. Furthermore, when we analyzed the apoptosis of Treg cells after 3 h of TCR activation in vitro, we noted a significant rise in late apoptotic or necrotic cells (Annexin+, 7AAD+) among Gpx4-deficient Treg cells upon TCR activation (Fig. 2A). This finding indicates that Gpx4-deficient Treg cells exhibit a predisposition towards cell death. To gain further insights, splenic CD4+ T cells from Foxp3Cre-YFP and Foxp3Cre-YFP Gpx4fl/fl mice were activated via TCR stimulation in the presence of Fer-1, z-VAD, or Nec-1 for 3 h. Subsequently, the activity and apoptosis of Treg cells were assessed using flow cytometry. The results revealed a reduction in the death of Gpx4-deficient Treg cells when ferroptosis inhibitors were added (Fig. 2A). Additionally, we evaluated the level of lipid peroxidation in Treg cells under TCR activation conditions. Notably, a significant elevation in lipid peroxidation (Bodipy C11) was observed in Gpx4-deficient Treg cells (Fig. 2B). Moreover, the production of malondialdehyde (MDA), a key indicator of ferroptosis, was also increased in Gpx4-deficient Treg cells (Fig. 2C). To further validate our findings, we introduced ferroptosis inhibitor (Fer-1), apoptosis inhibitor (z-VAD), or cell necrosis inhibitor (Nec-1) into Treg cells during the TCR activation process. The results demonstrated a decrease in lipid peroxidation and MDA production in Gpx4-deficient Treg cells following the addition of Fer-1 (Fig. 2D–E).Fig. 2 Gpx4 -deficient Treg cells are susceptible to ferroptosis upon activation in vitro or in vivo.

A Splenic CD4+ T cells from Foxp3YFP−creGpx4f/f and Foxp3YFP−Cre mice were stimulated with anti-CD3ε and anti-CD28 antibodies, at the time Ferrostatin 1(Fer-1,20 μM), z-VAD-FMK (z-VAD, 20 μM) or Necrostatin-1 (Nec-1, 20 μM) were added to the cells and cultured for 3 h. Apoptosis of Treg cells was detected by flow cytometry. B-C Splenic CD4+ T cells from Foxp3YFP−creGpx4f/f and Foxp3YFP−Cre mice were activated via TCR stimulation. The oxidation of lipids of Treg cells were detected via Bodipy (n = 3) and MDA (n = 3). D-E Splenic CD4+ T cells from Foxp3YFP−creGpx4f/f and Foxp3YFP−Cre mice were activated via TCR stimulation in the presence of Ferrostatin 1(Fer-1,20 μM), z-VAD-FMK (z-VAD, 20 μM) or Necrostatin-1 (Nec-1, 20 μM) for 3 h. The oxidation of lipids of Treg cells detected via Bodipy (n = 3) and MDA (n = 3). F–I Treg cells in vivo are activated by injecting anti-CD3ε (1 μg) into Foxp3Cre-YFP (WT) and Foxp3Cre-YFPGpx4fl/fl (KO) mice, and then the activity of Treg cells and their lipid peroxidation (BODIPY C11, MDA and ROS) are detected by flow cytometry. J CD45.1+ naïve CD4+ T cells (Teff) were pre-stained with Cell Trace Violet and cocultured with CD45.2+ WT or KO Treg cells at different ratios for 72 h. The proliferation of CD45.1+CD4+T cells was analyzed via Flow Cytometry and the suppression ratios were calculated (n = 3). p < 0.05 (*),p < 0.001 (***). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Our prior research revealed that Treg cells lacking the Gpx4 gene tend to undergo ferroptosis upon activation in vitro. Given the substantial disparity in oxygen concentration between the body's interior and exterior, we delved deeper into whether Treg cells devoid of Gpx4 are equally susceptible to ferroptosis when activated within a biological environment. To initiate the desired immune response, we administered α-CD3ε via the tail vein into Foxp3Cre-YFP (WT) and Foxp3Cre-YFP Gpx4fl/fl (KO) mice. Our findings indicate that Gpx4-deficient Treg cells exhibit an increased mortality rate when TCR activation occurs in vivo (Fig. 2F). Additionally, we observed a significant elevation in the lipid peroxidation level (Bodipy C11) of Treg cells (Fig. 2G), as well as a marked increase in ROS (Fig. 2H) and malondialdehyde (MDA) production (Fig. 2I). These findings suggest that GPX4 plays a pivotal role in preventing Treg cells from undergoing ferroptosis.

Furthermore, we conducted in vitro cell experiments to examine whether the suppressive effect of Treg cells on T cells is altered in the absence of the Gpx4 gene. Our results demonstrate that, in comparison to Treg cells derived from WT mice, Gpx4-deficient Treg cells are unable to effectively inhibit the proliferation of CD4+ T cells (Fig. 2J). Based on these findings, we conclude that Treg cells lacking the Gpx4 are prone to ferroptosis, both in vitro and in vivo, upon activation by TCR, and their immunosuppressive function is consequently compromised.

3.3 Treg ferroptosis aggravates intestinal injury and inflammatory response in NEC

The aforementioned studies have established a correlation between the decline in Treg cells in NEC intestinal tissue and the ferroptosis of these cells. GPX4, a pivotal antioxidant protein, possesses the capability to suppress ferroptosis in cells. In terms of normal physiological conditions, the targeted knockdown of Gpx4 in Treg cells does not exacerbate intestinal injury in newborn mice (Supplementary Figs. 4A–C). To determine whether the specific knockout of Gpx4 in Treg cells exacerbates NEC intestinal damage, weinduced NEC models in Foxp3Cre-YFP (WT) and Foxp3Cre-YFP Gpx4fl/fl (KO) mice. We then observed and compared the morphology and length of the small intestine in both groups of modeled mice. Our findings revealed that NEC mice in the KO group exhibited pronounced intestinal distension, thinning, and a marked reduction in intestinal length (Fig. 3A–C). To gain visual insights into the intestinal damage, we conducted intestinal HE staining. The findings revealed a marked exacerbation of NEC-induced intestinal injury, manifesting in disrupted integrity of intestinal epithelial cells and heightened infiltration of inflammatory cells in KO mice (Fig. 3D). Concurrently, we assessed the transcriptional levels of proteins associated with intestinal permeability and observed a significant downregulation of Claudin 1, Occludin, MUC-2, and ZO-1 in the KO group (Fig. 3E). Such reductions in these proteins can potentially lead to elevated intestinal permeability, thereby intensifying intestinal damage. These outcomes indicate a substantial aggravation of NEC-induced intestinal injury following the specific knockout of Gpx4 in Treg cells.Fig. 3 Treg ferroptosis aggravates intestinal injury and inflammatory response in NEC.

A–D The NEC modeling constructs were performed for the Foxp3Cre-YFP (WT) and Foxp3Cre-YFPGpx4fl/fl (KO) mice. Then the appearance, pathological status and the length of small intestine were observed. E The mRNA expression of the gut-permeable proteins Claudin 1, MUC-2, Occludin, and ZO-1 in NEC modeling mice was counted by fluorescence quantitative PCR. F–I The NEC model construction was performed on Foxp3Cre-YFP (WT) and Foxp3Cre-YFPGpx4fl/fl(KO) mice. Then, the proportion and number changes and the lipid peroxidation (BODIPY C11,MDA) of Treg and neutrophils in intestinal lamina propria were measured by flow cytometry (n = 6). J The mRNA expression of NEC intestinal tissue-related inflammatory factors IL-17, INF- γ, TNF- γ, TNF- α, TGF- β, IL-6, and IL-4 (n = 6–8) after ferroptosis in Treg cells. p < 0.05 (*),p < 0.01 (**) and p < 0.001 (***). NS, not significant.

Fig. 3

Our studies have revealed that Treg cells, when deprived of the Gpx4, exhibit heightened susceptibility to ferroptosis in their activated state. Under normal physiological conditions, the targeted knockdown of Gpx4 in Treg cells does not perturb the immune homeostasis within the intestines of newborn mice (Supplementary Figs. 4D–H). Subsequently, we conducted a comparative analysis of Treg cells in the intestinal lamina propria of NEC mice belonging to the KO group and the WT group. Our findings indicate that the proportion and quantity of Treg cells in the intestinal lamina propria of NEC mice in the KO group were conspicuously diminished compared to those in the WT group (Fig. 3F–G). This preliminary observation suggests that the ablation of the Gpx4 may adversely impact the normal functioning of Treg cells in the intestine. To further investigate this phenomenon, we examined ferroptosis in Treg cells. As depicted in Fig. 3H–I, lipid peroxidation in Treg cells residing in the intestinal lamina propria of NEC mice in the KO group was notably augmented, accompanied by a significant increase in MDA production. Our experimental data unequivocally demonstrates that the knockout of the Gpx4 gene in Treg cells is indeed associated with the aggravation of intestinal injury in NEC, which is attributed to ferroptosis of these cells.

As essential immune regulators, Treg cells play a pivotal role in preserving intestinal immune homeostasis. To delve into the consequences of local inflammatory damage in NEC intestine following Treg cell ferroptosis, we initially compared the neutrophil proportion in the intestinal lamina of NEC between the WT and KO groups. Remarkably, the neutrophil proportion in the intestinal lamina propria of NEC in the KO group was conspicuously elevated (Fig. 3J). Furthermore, we evaluated the transcriptional levels of local inflammatory factors in the NEC intestine. Compared to the WT group, the expressions of pro-inflammatory factors IL-17, INF-γ, and TNFα in the intestinal tract of NEC in the KO group were significantly upregulated, whereas the expression of the anti-inflammatory cytokine TGF-β was notably downregulated (Fig. 3K). These findings indicate that Treg cell ferroptosis in NEC intensifies the inflammatory response in the intestine.

3.4 Vitamin E inhibits treg ferroptosis and improves intestinal inflammatory response in NEC

Vitamin E possesses the ability to scavenge free radicals within the body, effectively preventing the emergence of lipid peroxidation [27]. Now, the question arises: Can vitamin E inhibit ferroptosis in Treg cells that have undergone Gpx4 gene knockout? To address this, we isolated CD4+ T cells from the spleens of Foxp3Cre-YFPGpx4fl/fl mice and subjected them to a stimulatory environment in 1640 medium supplemented with 10 % serum, anti-CD3ε, and anti-CD28 for a duration of 6 h. At the onset of stimulation, we administered ferroptosis inhibitors (Fer-1) and vitamin E analogues (α-tocopherol, α-Toc) separately, while maintaining a DMSO group as a comparative control. Post-stimulation, we assessed the activity and lipid peroxidation levels of the Treg cells through flow cytometry. Our findings indicated that Treg cells supplemented with vitamin E exhibited a comparable trend to those treated with ferroptosis inhibitors, in contrast to the control group. Specifically, a notable decrease in cell mortality (Supplementary Fig. 5A) and lipid peroxidation levels (Supplementary Fig. 5B) was observed in the vitamin E-treated Treg cells. This experimental outcome underscores the pivotal role of vitamin E in suppressing ferroptosis in Treg cells.

We delve deeper into the impact of vitamin E treatment on intestinal injury in NEC. Foxp3Cre-YFP and Foxp3Cre-YFPGpx4fl/fl mice were induced with NEC and administered 10 mg/kg·d vitamin E for prophylactic purposes, while breastfed mice without vitamin E treatment served as controls. Following the establishment of the model, we carefully observed and evaluated the appearance, length, and pathological damage of the mice's small intestine. Notably, the symptoms of intestinal flatulence, thinning, and shortened length in NEC mice treated with vitamin E exhibited significant improvement. Through pathological scoring of the small intestine, we discovered a marked reduction in intestinal damage among NEC mice treated with vitamin E (Fig. 4A–C). Additionally, we detect the transcription levels of NEC's intestinal mucosal permeability-related proteins. The results revealed a significant increase in the transcription levels of Claudin 1, Occludin, MUC-2, and ZO-1, intestinal mucosal permeability-related proteins, in NEC mice treated with vitamin E (Fig. 4D). These findings indicate that vitamin E treatment can alleviate intestinal injury in NEC, induced by ferroptosis of Treg cells.Fig. 4 Vitamin E inhibits Treg ferroptosis and improves intestinal inflammatory response in NEC.

A–C The NEC modeling constructs were performed for the Foxp3Cre-YFP (WT) and Foxp3Cre-YFPGpx4fl/fl (KO) mice. At the same time, preventive treatment is carried out by adding vitamin E (10 mg/kg·d). Then, the changes in intestinal appearance, length and intestinal damage of different groups of NEC were evaluated. D Changes in the transcript levels of NEC intestinal permeability proteins Claudin 1, Occludin, MUC-2 and ZO-1 after vitamin E treatment. E–HFoxp3Cre-YFP (WT) and Foxp3Cre-YFPGpx4fl/fl (KO) mice were induced into NEC models, and vitamin E was used for prophylactic treatment during modeling. After successful modeling, the changes in the proportion and number of intestinal Treg cells, lipid peroxidation（BODIPY C11）and malondialdehyde (MDA) of Treg cells and neutrophils in NEC were detected. I The mRNA expression of Foxp3Cre-YFPGpx4fl/fl intestinal tissue-related inflammatory factors IL-17, INF- γ, TNF- α, and TGF- β, IL-6, IL-6, IL-4 modeled by NEC after vitamin E treatment. p < 0.05 (*),p < 0.01 (**) and p < 0.001 (***).

Fig. 4

We subsequently delved into the influence of vitamin E supplementation on the ferroptosis of Treg cells within the intestinal tissue of NEC. Specifically, we quantified the expression of Treg cells in the lamina propria of the small intestine. Our findings revealed a notable elevation in both the proportion and count of Treg cells in the small intestine of NEC subjects administered with vitamin E (Fig. 4E–F). Furthermore, we assessed ferroptosis-related markers in Treg cells. Remarkably, following vitamin E treatment, the lipid peroxidation levels (BODIPY C11) in Treg cells of NEC mice's small intestine underwent a significant decline (Fig. 4G), along with a marked reduction in malondialdehyde (MDA) production (Fig. 4H). These compelling results firmly indicate that vitamin E is efficacious in mitigating ferroptosis in Treg cells residing in the NEC intestine.

Our study reveals that vitamin E possesses distinctive therapeutic benefits in the management of NEC, especially in mitigating the ferroptosis phenomenon observed in intestinal Treg cells. Does vitamin E potentially exhibit a therapeutic influence on the hyperinflammatory response triggered by ferroptosis in Treg cells in NEC? Our experimental findings indicate a significant reduction in the proportion of neutrophils within the intestinal lamina of Foxp3Cre-YFPGpx4fl/fl mice following vitamin E treatment (Fig. 4I). Furthermore, we delved into the transcriptional levels of local inflammatory factors within the intestine. The results indicate that vitamin E therapy notably diminishes the expression of pro-inflammatory factors such as INF-γ, TNF-α, IL-1β, and IL-6 in NEC, while enhancing the expression of the anti-inflammatory cytokine TGF-β (Fig. 4J). In summary, vitamin E treatment effectively mitigates ferroptosis in intestinal Treg cells in NEC, thereby alleviating intestinal damage and inflammatory response associated with this condition.

3.5 Vitamin E can increase GPX4 expression and thus inhibit ferroptosis

Prior studies have revealed that vitamin E effectively ameliorates ferroptosis in Treg cells, thereby mitigating intestinal damage in NEC. GPX4, a pivotal molecule in lipid peroxidation regulation, plays a crucial role in this process. Does vitamin E's inhibition of ferroptosis correlate with the expression of GPX4? We induced ferroptosis in Treg cells using Erastin in vitro and treated them with DMSO, Fer-1, and α-Toc for 24 h. Our findings indicate that α-Toc exhibited similar effects to Fer-1, significantly reducing lipid peroxidation (BODIPY C11) (Fig. 5A) and oxidative stress (ROS) (Fig. 5B), while enhancing GPX4 expression (Fig. 5C). To further explore the relationship between vitamin E and GPX4, we evaluated their interaction under in vivo conditions of activated Treg cells. Specifically, we activated Treg cells using TCR and intervened by administering Fer-1 and α-Toc via tail vein injection. The results demonstrated a decrease in lipid peroxidation and ROS levels in Treg cells following treatment with α-Toc and Fer-1 (Fig. 5D–E), accompanied by an increase in GPX4 expression (Fig. 5F–G). These observations suggest that vitamin E not only directly alleviates lipid peroxidation but also upregulates GPX4 expression. Therefore, we questioned whether vitamin E exhibits similar effects in an NEC model. To address this, we induced NEC in Foxp3Cre-YFP mice and administered vitamin E prophylactically. Subsequently, we measured the protein expression level of GPX4 in NEC intestinal tissues. Notably, the protein expression level of GPX4 was elevated in the intestinal tissues of NEC mice treated with vitamin E (Fig. 5H). These findings indicate that vitamin E not only directly reduces lipid peroxidation and oxidative stress to mitigate ferroptosis, but also promotes GPX4 expression, further inhibiting ferroptosis.Fig. 5 Vitamin E can increase GPX4 expression and thus inhibit ferroptosis.

A–C Treg cells were induced ferroptosis by Erastin in vitro, and DMSO, Fer-1 and α-Toc were added to the cells and cultured for 24 h. Then the lipid peroxidation level, ROS level and Gpx4 expression level of Treg cells were detected by flow cytometry. D–F Inject anti-CD3ε into Foxp3Cre-YFPGpx4fl/fl(KO) mice to activate Treg cells in the body. At the same time, Fer-1 and α-Toc are injected through the tail vein. After 24 h, detect the lipid peroxidation index Bodipy C11, ROS and GPX4 expression of Treg cells. G Treg cells from spleen were sorted and induced ferroptosis by Erastin, and DMSO, Fer-1 and α-Toc were added to the cells and cultured for 24 h. Then the expression of GPX4 protein in Treg cells was detected by western blot. HFoxp3Cre-YFP mice were induced into the NEC model and treated with vitamin E, and the protein expression of GPX4 in NEC intestinal tissues was detected by Western blot. p < 0.05 (*),p < 0.01 (**) and p < 0.001 (***). NS, not significant.

Fig. 5

4 Discussion

Ferroptosis, a highly noteworthy form of cell death in recent years, has garnered considerable attention. Distinct from common cell death modalities like apoptosis and necrosis, its unique characteristics and intricate mechanisms have been extensively examined [28]. The fundamental mechanism underlying ferroptosis revolves around the abnormal accumulation of iron ions and reactive oxygen species (ROS) within cells, leading to severe damage to cell membranes and organelles, thereby inducing cell death [22]. Extensive research has established a strong correlation between ferroptosis and the emergence and progression of numerous tissue-related disorders [29]. Analogously, ferroptosis has been observed in the intestinal tissue affected by necrotizing enterocolitis [30].

Treg cells, crucial immune regulators of NEC pathogenesis, play a pivotal role in mitigating intestinal injury among neonates. Their reduced number, however, intensifies the injury, thereby heightening the risk of NEC [31]. Nonetheless, the precise mechanism underlying the Treg cell decrement in NEC intestinal tracts remains elusive. Our investigation uncovered a link between the Treg cell decrease and ferroptosis in NEC, highlighting the critical role of GPX4 expression reduction in inducing ferroptosis in these cells. Further inquiries have demonstrated that the genetic deletion of Gpx4 in Treg cells intensifies lipid peroxidation, thereby fostering ferroptosis. Comparatively, vitamin E prophylaxis effectively inhibits the lipid peroxidation process. Additionally, it enhances the expression of GPX4 in Treg cells, subsequently preventing the occurrence of ferroptosis. Furthermore, vitamin E mitigates intestinal damage and inflammation in NEC that is induced by GPX4 depletion. Our research uncovers the correlation between the reduction in Treg cells and ferroptosis in the NEC intestine, highlighting the crucial role of vitamin E and GPX4 in sustaining intestinal homeostasis in NEC.

GPX4, as an indispensable intracellular lipid peroxidation regulator, plays a central role in the regulation of ferroptosis. GPX4 also plays a vital role in ensuring the functional integrity and steady-state maintenance of all types of organizations [32]. Previous studies have reported that GPX4 can inhibit ferroptosis of intestinal epithelial cells to improve the intestinal barrier and thus improve intestinal damage in ulcerative colitis [33]. In addition, it can also reduce the inflammatory damage of the intestinal tract in inflammatory bowel disease by promoting the expression of GPX4 [34]. Hyperinflammatory reaction is currently acknowledged as the primary pathological process of NEC, with the diminished count of Treg cells being a key contributor to its hyperinflammatory state. Our research reveals that the decline in Treg cells within the NEC intestine is linked to ferroptosis stemming from reduced GPX4 expression. Aligning with previous studies [35], Treg cells in tumors with a specific Gpx4 knockout are prone to ferroptosis. However, our investigation specifically centers on Treg cells in NEC. Our findings indicate that Treg cells deficient in Gpx4 tend to undergo ferroptosis, further aggravating intestinal damage and inflammation. This underscores the diverse consequences of Gpx4 deficiency in Treg cells across various diseases.

For a considerable period, the central challenge in NEC treatment has been inhibiting the inflammatory response and mitigating intestinal damage within the NEC tract. Treg cells play a key role in inhibiting intestinal inflammatory response of NEC. Our research reveals the indispensable role of GPX4 in the survival of these cells in vivo or in vitro. This underscores the significance of our lipid peroxidation clearance for Treg cell maintenance. Consequently, we pose the question: can lipophilic antioxidants, such as vitamin E, enhance the survival rate of Treg cells, thereby inhibiting intestinal damage and inflammatory response in NEC?

Vitamin E is a fat-soluble antioxidant. Its unique chemical structure enables it to efficiently neutralize free radicals in the body, thereby preventing lipid peroxidation [36]. Furthermore, vitamin E mitigates lipid peroxidation induced by GPX4 deficiency in various cells, including hematopoietic stem cells, hepatocytes, and red blood cells [[37], [38], [39]]. Our research underscores the pivotal role of vitamin E in not only directly safeguarding Treg cells against lipid peroxidation and ferroptosis but also upregulating Gpx4 expression, which subsequently halts lipid peroxidation in Treg cells. Consequently, it ameliorates intestinal damage and inflammatory responses in the NEC intestine.

This study is subject to several limitations. Firstly, we refrained from utilizing GPX4 over-expression mice to elucidate the protective influence of GPX4 on Treg cells. Furthermore, despite the ability of vitamin E to up-regulate GPX4 expression and suppress lipid peroxidation, the underlying mechanism remains undetermined.

5 Conclusion

In the context of NEC, ferroptosis of Treg cells emerges as a pivotal factor contributing significantly to intestinal tissue damage and an exaggerated inflammatory response. The survival and functionality of Treg cells are heavily reliant on GPX4. Vitamin E, as a fat-soluble antioxidant, effectively mitigates the ferroptosis of Treg cells, thereby enhancing their numbers and functionality. This, in turn, aids in alleviating intestinal tissue damage and inflammation associated with NEC. Our study aims to offer novel insights and potential therapeutic strategies for the treatment of NEC.

Disclosure of interest

The authors have indicated they have no potential conflicts of interest to disclose.

Funding

This work is supported by the National Natural Science Foundation of China (grant 82271736 to Sitao Li, grant 82371713 to Fei Ma, grant 82071680 to Hu Hao); the Guangdong Basic and Applied Basic Research Foundation (grant 2024A1515010520 to Sitao Li, grant 2022A1515010388 to Fei Ma; grant 2023A1515012569 to Hu Hao; grant 2023B1515020018  and 2023B0303000004  to Guangchao Cao); Science and Technology Projects of Social Development in Zhuhai (Grant 2320004000001 and 2220004000299 to Fei Ma); Huadu District Basic and Applied Basic Research Joint Funded Project (Grant 23HDQYLH25 to Guiying Zhuang).

Data statement

Data will be made available on request.

Data availability

Data will be made available on request.

CRediT authorship contribution statement

Shunchang Luo: Data curation, Formal analysis, Validation, Writing – original draft. Yingying Zeng: Formal analysis, Validation. Baozhu Chen: Formal analysis, Investigation, Methodology. Junjie Yan: Formal analysis, Methodology. Fei Ma: Funding acquisition, Methodology. Guiying Zhuang: Funding acquisition. Hu Hao: Funding acquisition. Guangchao Cao: Funding acquisition, Resources, Writing – review & editing. Xin Xiao: Project administration, Resources, Supervision. Sitao Li: Funding acquisition, Resources, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Multimedia component 1

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Multimedia component 2

Supplementary Fig. 1. Successful specific knockout of Gpx4 in Treg cells.

A-BGpx4flox/flox and Foxp3YFP−Cre strains were crossed to generate Tregs-specific GPX4 deficient Foxp3YFP−creGpx4f/f (KO) mice. Genetic identification was performed on the obtained knockout mice, Only mice with both Gpx4flox / flox and Foxp3YFP−Cre gene bands were identified as Foxp3YFP−cre Gpx4f/f (KO) mice. C Treg cells were sorted from the spleens of Foxp3YFP−Cre (WT) and Foxp3YFP−creGpx4f/f (KO) mice. The protein of Treg cells was taken and the expression of GPX4 in Treg cells was detected by western blot.

Supplementary Fig. 2. Treg-specific deficient Gpx4 does not affect the immune homeostasis of the spleen.

A Remove the spleen of the newborn (10 days) and adult (6 weeks) of Foxp3YFP-Cre (WT) and Foxp3YFP-creGpx4f/f (KO). The changes in the appearance of the spleen in the KO group and WT group were evaluated respectively. B The proportion and number changes of CD4 + T cells and CD8 + T cells in the spleen of Foxp3YFP-Cre(WT) and Foxp3YFP-creGpx4f / f (KO) mice are shown. C Cytokine expression of CD4+ T cells and CD8 + T cells in the spleen of Foxp3YFP-Cre (WT) and Foxp3YFP-creGpx4f / f (KO) mice is shown.NS, not significant.

Supplementary Fig. 3. Gpx4 -deficient Treg are susceptible to death after TCR stimulation.

A Splenic CD4+ T cells from Foxp3YFP-creGpx4f/f and Foxp3YFP-Cre mice were stimulated with anti-CD3εand anti-CD28 antibodies. Subsequently, the activity of CD4+FOXP3+Treg cells at different time points (0h,1h,2h,3h.4h.5h and 6h) was measured.

Supplementary Fig. 4. Treg-specific deficient Gpx4 does not affect the immune homeostasis of the intestine.

A-C The overall appearance, small intestine length, and pathological changes of Foxp3YFP-Cre (WT) and Foxp3YFP-creGpx4f / f (KO) neonatal mice are shown. D-F Shows the changes of CD4+ T cells, CD8 + T cells, Treg and neutrophils in the lamina propria of Foxp3YFP-Cre (WT) and Foxp3YFP-creGpx4f / f (KO) neonatal mice. G-H Cytokine expression of CD8+ T cells from CD4+ T cells in the lamina propria of the small intestine of Foxp3YFP-Cre (WT) and Foxp3YFP-creGpx4f / f (KO) neonatal mice is shown.p < 0.05 (*),NS, not significant.

Supplementary Fig. 5. Treg-specific deficient Gpx4 does not affect the immune homeostasis of the intestine.

A-B Splenic CD4+ T cells from Foxp3Cre-YFPGpx4fl/fl mice were activated via TCR stimulation for 6 h with or without Fer-1 (20μM) andα-Toc (20μM). The death rates (n=4,A) and the levels of Bodipy C11 (n=4,B) of CD4+ FOXP3+ Treg cells were detected and shown.

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Acknowledgments

We are profoundly grateful to the National Natural Science Foundation of China, the Guangdong Basic and Applied Basic Research Foundation, the Science and Technology Projects of Social Development in Zhuhai, and the Huadu District Basic and Applied Basic Research District Institute for their invaluable support. Furthermore, We are also extremely thankful for the strong backing of the Sixth Affiliated Hospital, Sun Yat-sen University. Lastly, we extend our heartfelt gratitude to the volunteers for their dedicated participation.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103303.
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